RT-PCR was used to determine if *recA* is co-transcribed with one or more of its neighboring genes (Fig. 5B-D). Antisense primers (F1 and F3; see Fig. 5) were used to generate cDNA from points originating in the downstream gene BB0129 and within the *recA* coding region. The cDNA was amplified in PCR reactions using the antisense primer and various sense primers to determine if mRNA transcripts spanned the junctions between adjacent genes. To determine if *recA* is co-transcribed with downstream genes, cDNA generated using primer F1 was PCR amplified using the same antisense primer and primers R1, R1A and R2B (Fig. 5B). All primer pairs produced products of the correct size when compared to positive controls, indicating the presence of an RNA transcript spanning genes BB0129-BB0132 and suggesting *recA* is co-transcribed with at least two downstream genes. In addition, the positive RT product obtained with primers F1 and R2B (lane 9; Fig. 5B) suggested that *recA* may be part of a larger operon encompassing one or both upstream genes, as the R2B primer was located 1.2 kb upstream of the *recA* translational start site. To confirm that *recA* is cotranscribed with BB0132, cDNA was generated using antisense primer F3 and PCR amplified using the same antisense primer and sense primers R1A, R2, R2A, R2B and R3

kb ladder was used as a size standard (lane 1 of B, C and D).

transcriptional unit and identifying the *recA* promoter(s).

this strain exhibited a marked reduction in transformation frequency compared to the wildtype strain, RecA function was restored as JR1 demonstrated a 2-fold greater recombination frequency as compared to B31-A (Table 1). The reduction in transformation frequency observed in JR1 is likely due to the incompatibility of pRecA.3 and ErmC/pCE320 as they are both derivatives of the pCE320 shuttle vector.

#### **2.4 Expression of** *recA* **determined by quantitative RT-PCR (qRT-PCR)**

One possible explanation for the lack of recombination observed in the JA15 strain is that *recA* transcription was reduced, leading to decreased levels of functional RecA protein. To test this, we evaluated *recA* expression in B31-A, JA10, JA15 and JR1 using qRT-PCR (Table 2). RNA was extracted from late-log phase cells (~2.0 107 cells ml-1) and the amount of *recA* transcript present in 10 ng of total RNA was determined. As expected, *recA* transcript levels in the JA15 complemented mutant were 61.5-fold lower than those measured in wild-type cells, possibly accounting for the lack of recombination observed in this strain. In contrast, *recA* transcript levels in the JR1 complemented mutant, in which *recA* expression was driven by a constitutive promoter, were 15.7-fold higher than the wild type. No *recA* transcription was detected in JA10 cells. These results may explain the 2-fold increase observed in recombination frequency in JR1 compared to the wild type. It is important to note that despite the differences in *recA* levels in these different strains, the growth rate between strains did not vary and the growth curves in complete BSK-II were indistinguishable (data not shown).

a Average *recA* copy number in 10 ng of total RNA from three biological replicates b SD, standard deviation

c Fold change in average copy number from wild-type levels d ND, Not detected

Table 2. *recA* transcript levels.

#### **2.5 Characterization of the** *recA* **transcriptional unit**

Analysis of the *B. burgdorferi* genome reveals two genes (BB0132 and BB0133) immediately upstream of *recA* (BB0131) that are oriented in the same direction (Fig. 5A). The reduced expression of *recA* in the JA15 complemented mutant may be the result of regulatory elements encoded outside of the 512 bp of upstream DNA amplified to generate the complementation construct pBB0131comp.2. It is also possible, given the organization of the flanking genes, that *recA* is co-transcribed with one or both of the upstream genes as part of

this strain exhibited a marked reduction in transformation frequency compared to the wildtype strain, RecA function was restored as JR1 demonstrated a 2-fold greater recombination frequency as compared to B31-A (Table 1). The reduction in transformation frequency observed in JR1 is likely due to the incompatibility of pRecA.3 and ErmC/pCE320 as they

One possible explanation for the lack of recombination observed in the JA15 strain is that *recA* transcription was reduced, leading to decreased levels of functional RecA protein. To test this, we evaluated *recA* expression in B31-A, JA10, JA15 and JR1 using qRT-PCR (Table 2). RNA was extracted from late-log phase cells (~2.0 107 cells ml-1) and the amount of *recA* transcript present in 10 ng of total RNA was determined. As expected, *recA* transcript levels in the JA15 complemented mutant were 61.5-fold lower than those measured in wild-type cells, possibly accounting for the lack of recombination observed in this strain. In contrast, *recA* transcript levels in the JR1 complemented mutant, in which *recA* expression was driven by a constitutive promoter, were 15.7-fold higher than the wild type. No *recA* transcription was detected in JA10 cells. These results may explain the 2-fold increase observed in recombination frequency in JR1 compared to the wild type. It is important to note that despite the differences in *recA* levels in these different strains, the growth rate between strains did not vary and the growth curves in complete BSK-II were indistinguishable (data

Strain *recA* Copy Number Fold Differencec

Analysis of the *B. burgdorferi* genome reveals two genes (BB0132 and BB0133) immediately upstream of *recA* (BB0131) that are oriented in the same direction (Fig. 5A). The reduced expression of *recA* in the JA15 complemented mutant may be the result of regulatory elements encoded outside of the 512 bp of upstream DNA amplified to generate the complementation construct pBB0131comp.2. It is also possible, given the organization of the flanking genes, that *recA* is co-transcribed with one or both of the upstream genes as part of

Averagea (SDb)

B31-A 8.49 105 (1.57 105) 1 JA10 NDd ND JA15 1.38 104 (2.03 103) 0.016 JR1 1.33 107 (2.69 106) 15.7

a Average *recA* copy number in 10 ng of total RNA from three biological replicates b SD, standard deviation

c Fold change in average copy number from wild-type levels d ND, Not detected

**2.5 Characterization of the** *recA* **transcriptional unit**

**2.4 Expression of** *recA* **determined by quantitative RT-PCR (qRT-PCR)**

are both derivatives of the pCE320 shuttle vector.

not shown).

Table 2. *recA* transcript levels.

Fig. 5. Organization of *recA* and flanking genes. (A) Arrangement of the chromosomal region spanning genes BB0129 to BB0134 (7628 bp). Labeled arrows indicate the antisense and sense primers used for RT-PCR. (B) RT-PCR of B31-A RNA using a BB0129 antisense strand primer (F1) and sense strand primers R1 (lane 3), R1A (lanes 6) and R2B (lane 9). Positive control PCR reactions using B31-A genomic DNA as a template (lanes 2, 5 and 8) and no RT controls (lanes 4, 7 and 10) were also performed for each primer pair. (C) RT-PCR of B31-A RNA using a BB0131 (*recA*) antisense strand primer (F3) and sense strand primers R1A (lane 3), R2 (lane 6), R2A (lane 9), R2B (lane 12) and R3 (lane 15). Positive control PCR reactions using B31-A genomic DNA as a template (lanes 2, 5, 8, 11 and 14) and no RT controls (lanes 4, 7, 10, 13 and 16) were performed for each primer pair. (D) RT-PCR of B31- A RNA using a BB0131 (*recA*) antisense strand primer F3 with sense strand primers R4 (lane 3) and R5 (lane 6). Positive control PCR reactions using B31-A genomic DNA as a template (lanes 2 and 5) and no RT controls (lanes 4 and 7) were performed for each primer pair. A 1 kb ladder was used as a size standard (lane 1 of B, C and D).

an operon, and that the low level of expression observed in JA15 was due to a weak internal promoter. As previous studies have not evaluated the organization of the *recA* transcript in *B. burgdorferi*, we attempted to shed light on these questions by characterizing the *recA* transcriptional unit and identifying the *recA* promoter(s).

RT-PCR was used to determine if *recA* is co-transcribed with one or more of its neighboring genes (Fig. 5B-D). Antisense primers (F1 and F3; see Fig. 5) were used to generate cDNA from points originating in the downstream gene BB0129 and within the *recA* coding region. The cDNA was amplified in PCR reactions using the antisense primer and various sense primers to determine if mRNA transcripts spanned the junctions between adjacent genes. To determine if *recA* is co-transcribed with downstream genes, cDNA generated using primer F1 was PCR amplified using the same antisense primer and primers R1, R1A and R2B (Fig. 5B). All primer pairs produced products of the correct size when compared to positive controls, indicating the presence of an RNA transcript spanning genes BB0129-BB0132 and suggesting *recA* is co-transcribed with at least two downstream genes. In addition, the positive RT product obtained with primers F1 and R2B (lane 9; Fig. 5B) suggested that *recA* may be part of a larger operon encompassing one or both upstream genes, as the R2B primer was located 1.2 kb upstream of the *recA* translational start site. To confirm that *recA* is cotranscribed with BB0132, cDNA was generated using antisense primer F3 and PCR amplified using the same antisense primer and sense primers R1A, R2, R2A, R2B and R3

Adaptation to Glucosamine Starvation in *Borrelia burgdorferi* is Mediated by *recA* 123

Fig. 6. Identification of the transcriptional start sites of the *recA* transcript. 5' RACE was used to determine the approximate transcriptional start sites of the *recA* transcript. A-tailed PCR products obtained using primer F4 (A; lane 2) or primer F6 (B; lane 2) with the oligo dT-anchor primer were separated by gel electrophoresis on a 1% TAE agarose gel. A 1 kb ladder was used as a size standard (lane 1). (C) DNA sequence of the dA-tailed 5' RACE product obtained with primer F4 showing the approximate transcriptional start site (enlarged font). Underlined guanosines indicate site of mispriming with the oligo dC-anchor primer and dG-tailed 5' RACE product. (D) DNA sequence of the dA-tailed 5' RACE product obtained with primer F6

Fig. 7. Identification of the putative *recA* promoters. Comparison of the putative *recA* promoters with the RpoD, RpoS and RpoN-dependent promoters in *B. burgdorferi*. Nucleotide positions in the predicted *recA* promoters that match the consensus RpoD-

amplification with the nested primer F6 and the complementary-tailed anchor primers (C or G, respectively) did not produce a PCR product (data not shown). Therefore, we used the approximate transcriptional start site determined from the A-tailed cDNA to identify the putative -10 and -35 promoter regions. Comparison to *B. burgdorferi* consensus promoters suggests this operon is under the control of an RpoD-dependent promoter as homology was observed between five of the six bases within the -35 region and ten of eleven bases within the

showing the approximate transcriptional start site (enlarged font).

dependent promoter sequence are highlighted.

extended -10 region (Fig. 7).

(Fig. 5C). An RT product of the correct size was obtained with all primer pairs when compared to the positive controls, confirming the results obtained with antisense primer F1 (Fig. 5B) and strongly suggesting that *recA* is co-transcribed with BB0132. To determine if BB0133 is also part of this transcriptional unit, cDNA generated with antisense primer F3 was PCR amplified using the same antisense primer and sense primers R4 and R5 (Fig. 5D). An RT product of the correct size was obtained with both primer pairs when compared to the positive controls, providing evidence that BB0133 is also part of this operon. Taken together, these RT results strongly suggest *recA* is co-transcribed with the upstream genes BB0132 and BB0133 and at least two downstream genes (BB0130 and BB0129).

#### **2.6 5' RACE and promoter analysis**

The RT results presented above strongly suggest that *recA* is co-transcribed with the upstream genes BB0132 and BB0133 (Fig. 5B-D); however, we did observe reduced expression of *recA* in our mutant complemented with the wild-type gene and 512 bp of upstream DNA (Table 2). Together, these results suggest two *recA* transcripts may exist, one beginning upstream of BB0133 and the other beginning within the 512 bp immediately upstream of the *recA* gene. Therefore, we used 5' RACE to identify the two *recA* transcritptional start sites, and compared the promoter regions upstream of the start sites to previously identified RpoD, RpoS and RpoN-dependent promoter sequences in *B. burgdorferi* (Studholme & Buck 2000; Caimano et al. 2007; Rhodes et al. 2009).

To identify the transcriptional start site immediately upstream of the *recA* gene, total RNA was extracted from B31-A cells cultured to late log phase (~2.0 107 cells ml-1) in complete BSK-II and cDNA was generated using antisense primer F3 (Fig. 5A) located within the *recA* coding region. The cDNA was column purified and a homopolymeric A-tail was added using terminal transferase. PCR amplification of the A-tailed cDNA with nested gene specific primer F4 and an oligo dT-anchor primer resulted in a product of approximately 250 bp (lane 2; Fig. 6A). The DNA band was extracted from the gel and sequenced with primer F4, and the transcriptional start site was determined to be 44 or 45 bp upstream of the *recA* translational start (Fig. 6C). We attempted to clarify the start site using G-tailed cDNA, but were unable to obtain reliable sequence data for this PCR product due to mispriming of the oligo dC-anchor primer to three guanosines (underlined in Fig. 6C) located 29-31 bp upstream of the *recA* translational start site (data not shown). Based on the sequence data obtained from the A-tailed reaction we identified the putative -10 and -35 promoter regions immediately upstream of the *recA* gene. Comparison of these putative promoter regions with previously described *B. burgdorferi* promoter consensus sequences revealed similarity to the RpoD-dependent consensus promoter, as homology was observed in five of the six bases within the -35 region and seven of the eleven bases within the extended -10 region (Fig. 7).

To determine the transcriptional start site upstream of BB0133, total RNA was extracted from B31-A cells cultured to late log phase (~2.0 107 cells ml-1) in complete BSK-II, and cDNA was generated using antisense primer F5 (Fig. 5A) located within BB0132. A homopolymeric A-tail was added and PCR of the A-tailed cDNA with nested gene specific primer F6 and the oligo dT-anchor primer resulted in a 1.2 kb product (Fig. 6B). The DNA band was gel extracted and sequenced with nested primer F8, and the transcriptional start site was determined to be 45 or 46 bp upstream of the BB0133 translational start site (Fig. 6D). Both G-tailed and C-tailed cDNA were used to try and resolve this ambiguity, but PCR

(Fig. 5C). An RT product of the correct size was obtained with all primer pairs when compared to the positive controls, confirming the results obtained with antisense primer F1 (Fig. 5B) and strongly suggesting that *recA* is co-transcribed with BB0132. To determine if BB0133 is also part of this transcriptional unit, cDNA generated with antisense primer F3 was PCR amplified using the same antisense primer and sense primers R4 and R5 (Fig. 5D). An RT product of the correct size was obtained with both primer pairs when compared to the positive controls, providing evidence that BB0133 is also part of this operon. Taken together, these RT results strongly suggest *recA* is co-transcribed with the upstream genes

The RT results presented above strongly suggest that *recA* is co-transcribed with the upstream genes BB0132 and BB0133 (Fig. 5B-D); however, we did observe reduced expression of *recA* in our mutant complemented with the wild-type gene and 512 bp of upstream DNA (Table 2). Together, these results suggest two *recA* transcripts may exist, one beginning upstream of BB0133 and the other beginning within the 512 bp immediately upstream of the *recA* gene. Therefore, we used 5' RACE to identify the two *recA* transcritptional start sites, and compared the promoter regions upstream of the start sites to previously identified RpoD, RpoS and RpoN-dependent promoter sequences in *B.*

To identify the transcriptional start site immediately upstream of the *recA* gene, total RNA was extracted from B31-A cells cultured to late log phase (~2.0 107 cells ml-1) in complete BSK-II and cDNA was generated using antisense primer F3 (Fig. 5A) located within the *recA* coding region. The cDNA was column purified and a homopolymeric A-tail was added using terminal transferase. PCR amplification of the A-tailed cDNA with nested gene specific primer F4 and an oligo dT-anchor primer resulted in a product of approximately 250 bp (lane 2; Fig. 6A). The DNA band was extracted from the gel and sequenced with primer F4, and the transcriptional start site was determined to be 44 or 45 bp upstream of the *recA* translational start (Fig. 6C). We attempted to clarify the start site using G-tailed cDNA, but were unable to obtain reliable sequence data for this PCR product due to mispriming of the oligo dC-anchor primer to three guanosines (underlined in Fig. 6C) located 29-31 bp upstream of the *recA* translational start site (data not shown). Based on the sequence data obtained from the A-tailed reaction we identified the putative -10 and -35 promoter regions immediately upstream of the *recA* gene. Comparison of these putative promoter regions with previously described *B. burgdorferi* promoter consensus sequences revealed similarity to the RpoD-dependent consensus promoter, as homology was observed in five of the six bases within the -35 region and seven of the

To determine the transcriptional start site upstream of BB0133, total RNA was extracted from B31-A cells cultured to late log phase (~2.0 107 cells ml-1) in complete BSK-II, and cDNA was generated using antisense primer F5 (Fig. 5A) located within BB0132. A homopolymeric A-tail was added and PCR of the A-tailed cDNA with nested gene specific primer F6 and the oligo dT-anchor primer resulted in a 1.2 kb product (Fig. 6B). The DNA band was gel extracted and sequenced with nested primer F8, and the transcriptional start site was determined to be 45 or 46 bp upstream of the BB0133 translational start site (Fig. 6D). Both G-tailed and C-tailed cDNA were used to try and resolve this ambiguity, but PCR

BB0132 and BB0133 and at least two downstream genes (BB0130 and BB0129).

*burgdorferi* (Studholme & Buck 2000; Caimano et al. 2007; Rhodes et al. 2009).

eleven bases within the extended -10 region (Fig. 7).

**2.6 5' RACE and promoter analysis**

Fig. 6. Identification of the transcriptional start sites of the *recA* transcript. 5' RACE was used to determine the approximate transcriptional start sites of the *recA* transcript. A-tailed PCR products obtained using primer F4 (A; lane 2) or primer F6 (B; lane 2) with the oligo dT-anchor primer were separated by gel electrophoresis on a 1% TAE agarose gel. A 1 kb ladder was used as a size standard (lane 1). (C) DNA sequence of the dA-tailed 5' RACE product obtained with primer F4 showing the approximate transcriptional start site (enlarged font). Underlined guanosines indicate site of mispriming with the oligo dC-anchor primer and dG-tailed 5' RACE product. (D) DNA sequence of the dA-tailed 5' RACE product obtained with primer F6 showing the approximate transcriptional start site (enlarged font).

Fig. 7. Identification of the putative *recA* promoters. Comparison of the putative *recA* promoters with the RpoD, RpoS and RpoN-dependent promoters in *B. burgdorferi*. Nucleotide positions in the predicted *recA* promoters that match the consensus RpoDdependent promoter sequence are highlighted.

amplification with the nested primer F6 and the complementary-tailed anchor primers (C or G, respectively) did not produce a PCR product (data not shown). Therefore, we used the approximate transcriptional start site determined from the A-tailed cDNA to identify the putative -10 and -35 promoter regions. Comparison to *B. burgdorferi* consensus promoters suggests this operon is under the control of an RpoD-dependent promoter as homology was observed between five of the six bases within the -35 region and ten of eleven bases within the extended -10 region (Fig. 7).

Adaptation to Glucosamine Starvation in *Borrelia burgdorferi* is Mediated by *recA* 125

Exp.a Adaptedb Not

B31-A 33 30 3 91 JA10 15 0 15 0 JA15 17 5 12 29 JR1 13 3 10 23

a Number of independent growth experiments conducted in which cells were diluted from BSK-II

d Percent Adapted calculated as the number adapted (b) divided by the total number of experiments (a)

To provide further evidence that RecA plays a role in the adaptation to GlcNAc starvation, we evaluated growth after GlcNAc starvation in the *recA* complemented mutants. Growth of the JA15 complemented mutant was followed in 17 separate experiments and single exponential phase growth to high cell densities in the absence of free GlcNAc was observed 5 times. Similarly, the JR1 complemented mutant was evaluated in 13 separate growth experiments and exhibited a single exponential phase in BSK-II lacking GlcNAc 3 times. Taken together, these results suggest RecA plays a role in adaptation to GlcNAc starvation

*B. burgdorferi* exhibits a unique biphasic growth pattern when cultured in the absence of free GlcNAc, with cells reaching optimal density in the second exponential phase. However, it had not been conclusively determined whether a physiologic or genetic adaptation mechanism is responsible for second exponential phase growth. This study was conducted to address this question as well as identify genes important to the process. We provide evidence that adaptation to GlcNAc starvation in *B. burgdorferi* is the result of a genetic event, and suggest a role for the homologous recombination protein RecA. In addition, we characterized the *recA* transcript and showed that transcription occurs from two different

In a previous report, Tilly et al (Tilly et al. 2001) suggested that the biphasic growth observed during *B. burgdorferi* cultivation in the absence of free GlcNAc was the result of a physiologic adaptation, as cells diluted from the second exponential phase into fresh medium lacking GlcNAc still exhibited biphasic growth. However, careful inspection of the growth curve from that study revealed that starvation-adapted cells grew nearly 10-fold higher in the first exponential phase after re-introduction into BSK-II lacking GlcNAc, than cells that were not previously exposed to the starvation condition (Tilly et al. 2001). To us, these results suggested that the adaptation that permitted second exponential phase growth was maintained by cells after passage in the same medium. We followed the growth of cells transferred from the second exponential phase into BSK-II lacking GlcNAc and found that

b Adapted – the number of times cells reached high cell density (1-2 107 cells ml-1) in a single

Adaptedc

Percent Adaptedd

Strain Number of Growth

c Not Adapted – the number of times biphasic growth was observed

and that the level of RecA expression may be important in the process.

exponential phase

multiplied by 100

**3. Discussion**

promoter sites.

lacking GlcNAc into the same medium

Table 3. Adaptation to GlcNAc starvation.

#### **2.7 Growth of the** *recA* **mutant in BSK-II without GlcNAc**

Growth analysis of wild-type *B. burgdorferi* adapted to GlcNAc starvation suggests this organism undergoes a mutational event that allows growth to high cell densities in a single exponential phase in the absence of free GlcNAc (Figs. 2-4). Previous reports have demonstrated that DSBR proteins play a role in stress-induced mutations observed in other organisms (for reviews see (Galhardo et al. 2007; Hastings 2007)). Therefore, we evaluated the ability of a *recA* mutant in *B. burgdorferi* (JA10) to grow and adapt to GlcNAc starvation (Fig. 8). Late log phase *recA* mutant (JA10) cells cultured in complete BSK-II were diluted to 1.0 105 cells ml-1 in BSK-II lacking GlcNAc. Growth of the *recA* mutant prior to adaptation to GlcNAc starvation resulted in biphasic growth similar to wild-type cells that were not previously adapted to GlcNAc starvation (compare Fig. 8 open circles to Fig. 1A open circles). Similarly, when the *recA* mutant was diluted from the second exponential phase of a GlcNAc-starved culture (Fig. 8; 300 h) into the same medium biphasic growth was still observed (Fig. 8 open and closed triangles). This is in contrast to the single exponential phase typically observed in starvation-adapted wild-type cells that were cultured in the absence of GlcNAc (Fig. 1C).

Fig. 8. Growth of JA10 (*recA* mutant) after adaptation to GlcNAc starvation. Growth of JA10 in BSK-II with and without 1.5 mM GlcNAc, and growth of JA10 cells after adaptation to GlcNAc starvation. Late log phase cells were diluted to 1.0 105 cells ml-1 in complete BSK-II (open circle) or BSK-II lacking GlcNAc (closed circle), and cells were enumerated daily by darkfield microscopy. Cells adapted to GlcNAc starvation were diluted (arrows) to 1.0 105 cells ml-1 at Day 12 (closed triangle) and Day 13 (open triangle) in BSK-II without GlcNAc, and growth was followed daily by darkfield microscopy.

Multiple growth experiments evaluating adaptation to GlcNAc starvation (i.e. dilution from the second exponential phase) were performed for each strain (Table 3). The growth experiment was repeated 15 times with *recA* mutant cells, and biphasic growth was observed each time cells were diluted from the second exponential phase of a culture starved for GlcNAc. This is in contrast to wild-type cells which exhibited biphasic growth only 3 times in 33 separate growth experiments, and reached high cell densities in a single exponential phase in 30 separate experiments.

a Number of independent growth experiments conducted in which cells were diluted from BSK-II lacking GlcNAc into the same medium

b Adapted – the number of times cells reached high cell density (1-2 107 cells ml-1) in a single exponential phase

c Not Adapted – the number of times biphasic growth was observed

d Percent Adapted calculated as the number adapted (b) divided by the total number of experiments (a) multiplied by 100

Table 3. Adaptation to GlcNAc starvation.

To provide further evidence that RecA plays a role in the adaptation to GlcNAc starvation, we evaluated growth after GlcNAc starvation in the *recA* complemented mutants. Growth of the JA15 complemented mutant was followed in 17 separate experiments and single exponential phase growth to high cell densities in the absence of free GlcNAc was observed 5 times. Similarly, the JR1 complemented mutant was evaluated in 13 separate growth experiments and exhibited a single exponential phase in BSK-II lacking GlcNAc 3 times. Taken together, these results suggest RecA plays a role in adaptation to GlcNAc starvation and that the level of RecA expression may be important in the process.

### **3. Discussion**

124 Lyme Disease

Growth analysis of wild-type *B. burgdorferi* adapted to GlcNAc starvation suggests this organism undergoes a mutational event that allows growth to high cell densities in a single exponential phase in the absence of free GlcNAc (Figs. 2-4). Previous reports have demonstrated that DSBR proteins play a role in stress-induced mutations observed in other organisms (for reviews see (Galhardo et al. 2007; Hastings 2007)). Therefore, we evaluated the ability of a *recA* mutant in *B. burgdorferi* (JA10) to grow and adapt to GlcNAc starvation (Fig. 8). Late log phase *recA* mutant (JA10) cells cultured in complete BSK-II were diluted to 1.0 105 cells ml-1 in BSK-II lacking GlcNAc. Growth of the *recA* mutant prior to adaptation to GlcNAc starvation resulted in biphasic growth similar to wild-type cells that were not previously adapted to GlcNAc starvation (compare Fig. 8 open circles to Fig. 1A open circles). Similarly, when the *recA* mutant was diluted from the second exponential phase of a GlcNAc-starved culture (Fig. 8; 300 h) into the same medium biphasic growth was still observed (Fig. 8 open and closed triangles). This is in contrast to the single exponential phase typically observed in starvation-adapted wild-type cells that were cultured in the

Fig. 8. Growth of JA10 (*recA* mutant) after adaptation to GlcNAc starvation. Growth of JA10 in BSK-II with and without 1.5 mM GlcNAc, and growth of JA10 cells after adaptation to GlcNAc starvation. Late log phase cells were diluted to 1.0 105 cells ml-1 in complete BSK-II (open circle) or BSK-II lacking GlcNAc (closed circle), and cells were enumerated daily by darkfield microscopy. Cells adapted to GlcNAc starvation were diluted (arrows) to 1.0 105 cells ml-1 at Day 12 (closed triangle) and Day 13 (open triangle) in BSK-II without GlcNAc,

Multiple growth experiments evaluating adaptation to GlcNAc starvation (i.e. dilution from the second exponential phase) were performed for each strain (Table 3). The growth experiment was repeated 15 times with *recA* mutant cells, and biphasic growth was observed each time cells were diluted from the second exponential phase of a culture starved for GlcNAc. This is in contrast to wild-type cells which exhibited biphasic growth only 3 times in 33 separate growth experiments, and reached high cell densities in a single

and growth was followed daily by darkfield microscopy.

exponential phase in 30 separate experiments.

**2.7 Growth of the** *recA* **mutant in BSK-II without GlcNAc**

absence of GlcNAc (Fig. 1C).

*B. burgdorferi* exhibits a unique biphasic growth pattern when cultured in the absence of free GlcNAc, with cells reaching optimal density in the second exponential phase. However, it had not been conclusively determined whether a physiologic or genetic adaptation mechanism is responsible for second exponential phase growth. This study was conducted to address this question as well as identify genes important to the process. We provide evidence that adaptation to GlcNAc starvation in *B. burgdorferi* is the result of a genetic event, and suggest a role for the homologous recombination protein RecA. In addition, we characterized the *recA* transcript and showed that transcription occurs from two different promoter sites.

In a previous report, Tilly et al (Tilly et al. 2001) suggested that the biphasic growth observed during *B. burgdorferi* cultivation in the absence of free GlcNAc was the result of a physiologic adaptation, as cells diluted from the second exponential phase into fresh medium lacking GlcNAc still exhibited biphasic growth. However, careful inspection of the growth curve from that study revealed that starvation-adapted cells grew nearly 10-fold higher in the first exponential phase after re-introduction into BSK-II lacking GlcNAc, than cells that were not previously exposed to the starvation condition (Tilly et al. 2001). To us, these results suggested that the adaptation that permitted second exponential phase growth was maintained by cells after passage in the same medium. We followed the growth of cells transferred from the second exponential phase into BSK-II lacking GlcNAc and found that

Adaptation to Glucosamine Starvation in *Borrelia burgdorferi* is Mediated by *recA* 127

stresses (Feng et al. 1996; Harris et al. 1999). In addition, inactivation of MMR genes results in a mutator phenotype that has been observed in natural bacterial populations (LeClerc et al. 1996; Matic et al. 1997; Oliver et al. 2002). We evaluated our GlcNAc starvation-adapted clones for a mutation in *mutS* or *mutL* to determine if adaptation occurred due to inactivation of the MMR system. Genomic DNA was extracted from five starvation-adapted clones (Fig. 4, clones 1-5) and the *mutS* and *mutL* genes were sequenced along with 1 kb of flanking DNA on either side of the gene (data not shown). No mutations were observed in any of the clones, suggesting these clones have functional MMR systems and that adaptive mutation did not occur due to the absence of this important DNA repair pathway. It is unknown if MutS and MutL levels were reduced during GlcNAc starvation, but it may be of

The point mutants observed in the *E. coli* Lac assay require the presence of RpoS and the general stress response (Lombardo et al. 2004). It is unclear exactly how RpoS or components of the RpoS regulon regulate the formation of point mutations that confer the Lac+ phenotype, but it is known that RpoS promotes the use of the error-prone DNA polymerase DinB in DSBR (Ponder et al. 2005). As *B. burgdorferi* does not possess a DinB homologue or other error-prone DNA polymerases, it is unlikely this mechanism plays a role in adaptation of this spirochete to GlcNAc starvation. However, the RpoS regulon has also been shown to be necessary for adaptive amplification in the *E. coli* Lac assay (Lombardo et al. 2004), and previous studies conducted in our lab have demonstrated the importance of RpoS in GlcNAc starvation and biphasic growth (Rhodes et al. 2009). We reported a role for RpoS in the regulation of chitobiose utilization, and also demonstrated that an *rpoS* mutant was unable to initiate second exponential phase growth in the absence of GlcNAc until after 400 hours. In contrast, wild-type cells initiate a second exponential phase by 125 hours and reach optimal cell densities by 300 hours. It is possible the long delay observed in the *rpoS* mutant reflects the inability of these cells to amplify the gene(s)

The DSBR protein RecA is also required for stress-induced point mutation and amplification in the *E. coli* Lac assay. With regard to point mutants, RecA most likely plays a role in increased mutagenesis through the up-regulation of the SOS response. As discussed above, *B. burgdorferi* does not possess the components for a functional SOS pathway, and so adaptive mutation in response to GlcNAc starvation by this mechanism is unlikely. However, homologous recombination and DSBR proteins such as RecA are also required for adaptation via amplification. Therefore, we evaluated the ability of a *recA* mutant to adapt to GlcNAc starvation by culturing cells in BSK-II without GlcNAc, and then diluting cells from the second exponential phase into the same medium. While the *recA* mutant exhibited biphasic growth similar to the wild type, these cells were unable to permanently adapt to GlcNAc starvation (Fig. 8) in any of the 15 independent experiments conducted (Table 3). These results suggest a role for RecA in the adaptation of wild-type cells to GlcNAc starvation, possibly through amplification of target gene(s) by homologous recombination. Complementation of the *recA* mutant with two different complementation plasmids resulted in *recA* expression that was either 62.5-fold lower (JA15) or 15.7-fold higher (JR1) than wildtype expression (Table 2), and only partial restoration of the adaptive phenotype (Table 3). The partial restoration of the adaptive phenotype in the JA15 complemented mutant may be due to reduced levels of RecA protein resulting in less amplification of the target genes necessary for

interest to determine these levels in future experiments.

needed to obtain sequestered GlcNAc in the second exponential phase.

wild-type cells grew to a high cell density in a single exponential phase (Fig. 1C) in 30 out of 33 independent growth experiments (Table 3). This indicated that adaptation to the starvation condition was retained by the cells in nearly all of the experiments. However, it was not clear whether the adaptation was physiologic or genetic, as the starvation-stress condition was never removed (i.e. cells were only transferred from the second exponential phase directly into media lacking GlcNAc). Since other microorganisms exhibit stressinduced mutagenesis in response to nutrient deprivation and other stresses (for reviews see (Finkel 2006; Galhardo et al. 2007; Hastings 2007)), we hypothesized that *B. burgdorferi* may use similar mechanisms to adapt to GlcNAc starvation.

To determine if adaptation to GlcNAc starvation was physiologic or genetic, starvationadapted cells were serially passaged in complete BSK-II to relieve the starvation-stress, then re-introduced to the starvation-stress condition (i.e. BSK-II lacking GlcNAc). If adaptation was physiologic then cells should exhibit biphasic growth; however, we found that after as many as twelve serial passages in a complete medium, cells still reached high cell density in a single exponential phase when re-introduced to a medium lacking free GlcNAc (Fig. 2). In addition, starvation-adapted cells were plated on BSK-II agar after seven serial passages in complete BSK-II and the growth of ten clones was followed in BSK-II broth lacking GlcNAc. Eight of the ten clones grew to optimal cell densities in a single exponential phase (Fig. 3B). It is possible that the two clones exhibiting biphasic growth were never adapted and only able to persist due to the release of GlcNAc from sequestered sources by the cells that had undergone adaptation. It is also possible that these two clones were originally adapted to GlcNAc starvation, but during serial passage in complete media became de-adapted, exhibiting the original wild-type phenotype (discussed below). In any case, re-plating of one of the adapted clones (RR45) resulted in all ten clones growing to a high cell density in a single exponential phase (Fig. 4B). These results strongly suggest that *B. burgdorferi* undergoes stress-induced mutagenesis that allows cells to adapt to GlcNAc starvation.

As discussed above, stress-induced adaptive mutagenesis in the *E. coli* Lac assay results in either point mutants or amplification of the "leaky" *lac* region that relieves the stress and allows cells to utilize lactose (Cairns & Foster 1991). This adaptive mutagenesis occurs by a number of different pathways (Cairns & Foster 1991; Harris et al. 1994; Harris et al. 1999; McKenzie et al. 2000; McKenzie et al. 2001; Lombardo et al. 2004), three of which are present in *B. burgdorferi*: i) MMR, ii) RpoS starvation-stress-response and iii) homologous recombination by DSBR proteins. The gene or genes directly involved in the utilization of sequestered GlcNAc in the second exponential phase remain unknown; therefore, it is difficult to determine if the genetic adaptation observed in *B. burgdorferi* is the result of a point mutation, amplification or both. However, data collected under GlcNAc starvation conditions relating to these three pathways in *B. burgdorferi* may shed some light on this question.

MMR is a highly conserved process that repairs mismatched bases that arise during replication, increasing the fidelity of DNA replication 100 to 1000-fold (reviewed in (Kunkel& Erie 2005)). DNA mismatches are recognized by the MutS and MutL proteins, and the mismatched base is removed from the unmethylated strand by the endonuclease activity of MutH. A reduction in MMR activity during stationary phase has been described and is one mechanism by which mutation frequency is increased, possibly resulting in advantageous point mutations (not amplifications) that allow for adaptation to certain

wild-type cells grew to a high cell density in a single exponential phase (Fig. 1C) in 30 out of 33 independent growth experiments (Table 3). This indicated that adaptation to the starvation condition was retained by the cells in nearly all of the experiments. However, it was not clear whether the adaptation was physiologic or genetic, as the starvation-stress condition was never removed (i.e. cells were only transferred from the second exponential phase directly into media lacking GlcNAc). Since other microorganisms exhibit stressinduced mutagenesis in response to nutrient deprivation and other stresses (for reviews see (Finkel 2006; Galhardo et al. 2007; Hastings 2007)), we hypothesized that *B. burgdorferi* may

To determine if adaptation to GlcNAc starvation was physiologic or genetic, starvationadapted cells were serially passaged in complete BSK-II to relieve the starvation-stress, then re-introduced to the starvation-stress condition (i.e. BSK-II lacking GlcNAc). If adaptation was physiologic then cells should exhibit biphasic growth; however, we found that after as many as twelve serial passages in a complete medium, cells still reached high cell density in a single exponential phase when re-introduced to a medium lacking free GlcNAc (Fig. 2). In addition, starvation-adapted cells were plated on BSK-II agar after seven serial passages in complete BSK-II and the growth of ten clones was followed in BSK-II broth lacking GlcNAc. Eight of the ten clones grew to optimal cell densities in a single exponential phase (Fig. 3B). It is possible that the two clones exhibiting biphasic growth were never adapted and only able to persist due to the release of GlcNAc from sequestered sources by the cells that had undergone adaptation. It is also possible that these two clones were originally adapted to GlcNAc starvation, but during serial passage in complete media became de-adapted, exhibiting the original wild-type phenotype (discussed below). In any case, re-plating of one of the adapted clones (RR45) resulted in all ten clones growing to a high cell density in a single exponential phase (Fig. 4B). These results strongly suggest that *B. burgdorferi* undergoes stress-induced mutagenesis that allows cells to adapt to GlcNAc starvation.

As discussed above, stress-induced adaptive mutagenesis in the *E. coli* Lac assay results in either point mutants or amplification of the "leaky" *lac* region that relieves the stress and allows cells to utilize lactose (Cairns & Foster 1991). This adaptive mutagenesis occurs by a number of different pathways (Cairns & Foster 1991; Harris et al. 1994; Harris et al. 1999; McKenzie et al. 2000; McKenzie et al. 2001; Lombardo et al. 2004), three of which are present in *B. burgdorferi*: i) MMR, ii) RpoS starvation-stress-response and iii) homologous recombination by DSBR proteins. The gene or genes directly involved in the utilization of sequestered GlcNAc in the second exponential phase remain unknown; therefore, it is difficult to determine if the genetic adaptation observed in *B. burgdorferi* is the result of a point mutation, amplification or both. However, data collected under GlcNAc starvation conditions relating to these three pathways in *B. burgdorferi* may shed some light on this

MMR is a highly conserved process that repairs mismatched bases that arise during replication, increasing the fidelity of DNA replication 100 to 1000-fold (reviewed in (Kunkel& Erie 2005)). DNA mismatches are recognized by the MutS and MutL proteins, and the mismatched base is removed from the unmethylated strand by the endonuclease activity of MutH. A reduction in MMR activity during stationary phase has been described and is one mechanism by which mutation frequency is increased, possibly resulting in advantageous point mutations (not amplifications) that allow for adaptation to certain

use similar mechanisms to adapt to GlcNAc starvation.

question.

stresses (Feng et al. 1996; Harris et al. 1999). In addition, inactivation of MMR genes results in a mutator phenotype that has been observed in natural bacterial populations (LeClerc et al. 1996; Matic et al. 1997; Oliver et al. 2002). We evaluated our GlcNAc starvation-adapted clones for a mutation in *mutS* or *mutL* to determine if adaptation occurred due to inactivation of the MMR system. Genomic DNA was extracted from five starvation-adapted clones (Fig. 4, clones 1-5) and the *mutS* and *mutL* genes were sequenced along with 1 kb of flanking DNA on either side of the gene (data not shown). No mutations were observed in any of the clones, suggesting these clones have functional MMR systems and that adaptive mutation did not occur due to the absence of this important DNA repair pathway. It is unknown if MutS and MutL levels were reduced during GlcNAc starvation, but it may be of interest to determine these levels in future experiments.

The point mutants observed in the *E. coli* Lac assay require the presence of RpoS and the general stress response (Lombardo et al. 2004). It is unclear exactly how RpoS or components of the RpoS regulon regulate the formation of point mutations that confer the Lac+ phenotype, but it is known that RpoS promotes the use of the error-prone DNA polymerase DinB in DSBR (Ponder et al. 2005). As *B. burgdorferi* does not possess a DinB homologue or other error-prone DNA polymerases, it is unlikely this mechanism plays a role in adaptation of this spirochete to GlcNAc starvation. However, the RpoS regulon has also been shown to be necessary for adaptive amplification in the *E. coli* Lac assay (Lombardo et al. 2004), and previous studies conducted in our lab have demonstrated the importance of RpoS in GlcNAc starvation and biphasic growth (Rhodes et al. 2009). We reported a role for RpoS in the regulation of chitobiose utilization, and also demonstrated that an *rpoS* mutant was unable to initiate second exponential phase growth in the absence of GlcNAc until after 400 hours. In contrast, wild-type cells initiate a second exponential phase by 125 hours and reach optimal cell densities by 300 hours. It is possible the long delay observed in the *rpoS* mutant reflects the inability of these cells to amplify the gene(s) needed to obtain sequestered GlcNAc in the second exponential phase.

The DSBR protein RecA is also required for stress-induced point mutation and amplification in the *E. coli* Lac assay. With regard to point mutants, RecA most likely plays a role in increased mutagenesis through the up-regulation of the SOS response. As discussed above, *B. burgdorferi* does not possess the components for a functional SOS pathway, and so adaptive mutation in response to GlcNAc starvation by this mechanism is unlikely. However, homologous recombination and DSBR proteins such as RecA are also required for adaptation via amplification. Therefore, we evaluated the ability of a *recA* mutant to adapt to GlcNAc starvation by culturing cells in BSK-II without GlcNAc, and then diluting cells from the second exponential phase into the same medium. While the *recA* mutant exhibited biphasic growth similar to the wild type, these cells were unable to permanently adapt to GlcNAc starvation (Fig. 8) in any of the 15 independent experiments conducted (Table 3). These results suggest a role for RecA in the adaptation of wild-type cells to GlcNAc starvation, possibly through amplification of target gene(s) by homologous recombination.

Complementation of the *recA* mutant with two different complementation plasmids resulted in *recA* expression that was either 62.5-fold lower (JA15) or 15.7-fold higher (JR1) than wildtype expression (Table 2), and only partial restoration of the adaptive phenotype (Table 3). The partial restoration of the adaptive phenotype in the JA15 complemented mutant may be due to reduced levels of RecA protein resulting in less amplification of the target genes necessary for

Adaptation to Glucosamine Starvation in *Borrelia burgdorferi* is Mediated by *recA* 129

For analysis of growth in liquid cultures, late-log phase cells (5.0 107 cells ml-1 to 1.0 108 cells ml-1) were diluted to 1.0 105 cells ml-1 in BSK-II with or without GlcNAc. In general, 6- 12 l of culture was used to inoculate 6 ml of fresh culture medium; therefore, negligible amounts of free GlcNAc were transferred with the inoculum when analyzing growth in medium lacking GlcNAc. Cells were enumerated daily by darkfield microscopy using a Petroff-Hauser (Hauser Scientific; Horsham, PA) counting chamber. Specifically, 2.5 l of liquid culture was transferred to the counting chamber, and cells in all 25 squares were counted. Cells were diluted 1:10 in BSK-II prior to counting when the culture density was greater than 1.0 107 cells ml-1. All growth experiments were carried out at 33C in the presence of 3% CO2. Unless otherwise stated, each growth curve presented in this study is representative of at least three independent trials, as growth data could not be pooled due to the length of experiments and the different times at which bacteria were enumerated.

The construct used to generate a *recA* (BB0131) mutation in the B31-A background was created as follows: i) A 2.1 kb fragment of the 3' end of BB0131 and flanking DNA was PCR amplified with engineered restriction sites using primers (Table 5) 5' BB0131 mutF1 (KpnI) and 5' BB0131 mutR2 (XbaI); ii) the PCR fragment was TA cloned into pCR2.1 (Invitrogen) to generate plasmid pBB0131.1; iii) plasmids pBB0131.1 and pKFSS1 (Frank et al. 2003) (a *B. burgdorferi* shuttle vector conferring streptomycin resistance; Table 4) were digested with KpnI and XbaI and separated by gel electrophoresis; iv) the 2.1 kb band from pBB0131.1 was gel extracted and cloned into the gel extracted pKFSS1 fragment to generate pBB0131.2; v) the 2.1 kb fragment and flanking streptomycin resistance gene were PCR amplified from pBB0131.2 using primers 5' BB0131 mutF1 (KpnI) and pKFSS1 R1; vi) the 3.6 kb amplicon was TA cloned into pGEM T-Easy (Promega, Corp.; Madison, WI) to generate pBB0131.3A or B (based on the orientation of the PCR product insertion); vii) plasmid pBB0131.3B was identified by restriction digest in which the 3' end of the streptomycin resistance gene was adjacent to the XmaI site in the pGEM T-Easy vector; viii) the 5' end of BB0131 and flanking DNA (1.8 kb) was PCR amplified with engineered restriction sites using primers 3' BB0131 mutF1 (XmaI) and 3' BB0131 mutR1 (SacII) and TA cloned into pCR2.1 to generate pBB0131.4; ix) plasmids pBB0131.3B and pBB0131.4 were digested with XmaI and SacII and separated by gel electrophoresis; x) the 1.8 kb fragment from pBB0131.4 was gel extracted and cloned into the gel extracted fragment from pBB0131.3B to generate the final construct pBB0131.5. All plasmids were confirmed by restriction digest and DNA sequencing. In summary, 877 of the 1098 bp constituting the *recA* coding region were deleted and replaced with the streptomycin resistance gene transcribed in the opposite orientation and under the control of the constitutive *B. burgdorferi flgB* promoter. Plasmid pBB0131.5 was confirmed by PCR using primers flanking the antibiotic insertion site (BB0131 mut confirm F1 and 5' recA SalI) that resulted in a 1.7 kb fragment (Fig. 9, lane 3) compared to a 1.2 kb fragment generated with

To generate the *recA* deletion mutant, competent B31-A cells were prepared and transformed with 15 µg of pBB0131.5 as previously described (Samuels et al. 1994). Briefly, transformed cells were resuspended in 10 ml of complete BSK-II and allowed to recover

**4.2 Growth curve analysis**

**4.3 Construction of a** *recA* **mutant in** *B. burgdorferi*

the wild-type genomic DNA as a template (Fig. 9, lane 2).

obtaining sequestered GlcNAc. While only partial restoration of the adaptive phenotype in the JR1 complemented mutant may be due to a deamplification of the repeated target gene(s) because of abnormally high levels of RecA. It has been observed in the *E. coli* Lac assay that *lac*amplified clones (Lac+) plated on rich medium containing a color indicator for -galactosidase give rise to both white (Lac-) and sectored colonies (blue colonies with white sectors) due to deamplification of the repeated *lac* region, and that deamplification is mediated by RecA (Tlsty et al. 1984). This deamplification mechanism could also account for the two clones that exhibited biphasic growth in BSK-II lacking GlcNAc after repeated passage in complete BSK-II (Fig. 3B), and the three GlcNAc starvation experiments in wild-type cells that did not result in the adaptive phenotype (Table 3).

As a result of the reduced *recA* expression in our complemented mutant with the wild-type *recA* gene and 512 bp of upstream DNA (Table 2; JA15) we characterized the *recA* transcriptional unit (Fig. 5) and identified two putative promoters for this gene (Fig. 6 and 7). RT-PCR analysis confirmed that *recA* was co-transcribed with both upstream genes (BB0132 encoding the transcription elongation factor GreA; and BB0133 encoding a protein with a tetratricopeptide repeat domain) and at least two of the downstream genes (BB0130 encoding a conserved hypothetical protein; and BB0129 encoding an RNA-uridine isomerase). The combination of our RT-PCR data with our expression data from JA15 suggested the possibility of two promoters, one just upstream of the *recA* coding region and the other upstream of BB0133. We confirmed this using 5' RACE (Fig. 6), and identified two putative promoters based on comparison to previously identified *B. burgdorferi* RpoD, RpoS and RpoN-dependent promoters (Fig. 7) (Studholme & Buck 2000; Caimano et al. 2007; Rhodes et al. 2009). The promoter identified upstream of BB0133 is nearly identical to the RpoD consensus promoter indicating constitutive expression under normal conditions, while the putative promoter immediately upstream of the *recA* coding region shows the most, but only limited, homology to the RpoD consensus sequence. In light of these results and the role *recA* plays in adaptation to GlcNAc starvation, it is interesting to speculate that the promoter immediately upstream of *recA* may be involved in the expression of this gene under specific cellular conditions or stresses.

#### **4. Methods**

#### **4.1 Bacterial strains and culture conditions**

The bacterial strains and plasmids used in this study are described in Table 4. Wild-type and mutant *B. burgdorferi* strains were routinely cultured in modified BSK-II (Barbour 1984) medium supplemented with 7% rabbit serum (Invitrogen, Corp.; Carlsbad, CA) and any necessary antibiotics. BSK-II was modified from the original recipe by replacing 10x CMRL-1066 with 10x Media 199 (Invitrogen). In addition, free GlcNAc was left out of BSK-II for certain growth experiments. For selection and maintenance of antibiotic resistance in *B. burgdorferi*, streptomycin, kanamycin and erythromycin were used at concentrations of 100 g ml-1, 340 g ml-1 and 1 g ml-1 respectively.

*E. coli* DH5 was used to maintain plasmids during cloning procedures and was cultured in lysogeny broth (LB; 1% tryptone, 0.5% yeast extract, 1% NaCl) with the appropriate antibiotic(s). Antibiotics were used at the following concentrations for *E. coli* DH5: streptomycin, 100 g ml-1; kanamycin, 50 g ml-1; ampicillin, 200 g ml-1.

#### **4.2 Growth curve analysis**

128 Lyme Disease

obtaining sequestered GlcNAc. While only partial restoration of the adaptive phenotype in the JR1 complemented mutant may be due to a deamplification of the repeated target gene(s) because of abnormally high levels of RecA. It has been observed in the *E. coli* Lac assay that *lac*amplified clones (Lac+) plated on rich medium containing a color indicator for -galactosidase give rise to both white (Lac-) and sectored colonies (blue colonies with white sectors) due to deamplification of the repeated *lac* region, and that deamplification is mediated by RecA (Tlsty et al. 1984). This deamplification mechanism could also account for the two clones that exhibited biphasic growth in BSK-II lacking GlcNAc after repeated passage in complete BSK-II (Fig. 3B), and the three GlcNAc starvation experiments in wild-type cells that did not result in

As a result of the reduced *recA* expression in our complemented mutant with the wild-type *recA* gene and 512 bp of upstream DNA (Table 2; JA15) we characterized the *recA* transcriptional unit (Fig. 5) and identified two putative promoters for this gene (Fig. 6 and 7). RT-PCR analysis confirmed that *recA* was co-transcribed with both upstream genes (BB0132 encoding the transcription elongation factor GreA; and BB0133 encoding a protein with a tetratricopeptide repeat domain) and at least two of the downstream genes (BB0130 encoding a conserved hypothetical protein; and BB0129 encoding an RNA-uridine isomerase). The combination of our RT-PCR data with our expression data from JA15 suggested the possibility of two promoters, one just upstream of the *recA* coding region and the other upstream of BB0133. We confirmed this using 5' RACE (Fig. 6), and identified two putative promoters based on comparison to previously identified *B. burgdorferi* RpoD, RpoS and RpoN-dependent promoters (Fig. 7) (Studholme & Buck 2000; Caimano et al. 2007; Rhodes et al. 2009). The promoter identified upstream of BB0133 is nearly identical to the RpoD consensus promoter indicating constitutive expression under normal conditions, while the putative promoter immediately upstream of the *recA* coding region shows the most, but only limited, homology to the RpoD consensus sequence. In light of these results and the role *recA* plays in adaptation to GlcNAc starvation, it is interesting to speculate that the promoter immediately upstream of *recA* may be involved in the expression of this gene

The bacterial strains and plasmids used in this study are described in Table 4. Wild-type and mutant *B. burgdorferi* strains were routinely cultured in modified BSK-II (Barbour 1984) medium supplemented with 7% rabbit serum (Invitrogen, Corp.; Carlsbad, CA) and any necessary antibiotics. BSK-II was modified from the original recipe by replacing 10x CMRL-1066 with 10x Media 199 (Invitrogen). In addition, free GlcNAc was left out of BSK-II for certain growth experiments. For selection and maintenance of antibiotic resistance in *B. burgdorferi*, streptomycin, kanamycin and erythromycin were used at concentrations of 100

*E. coli* DH5 was used to maintain plasmids during cloning procedures and was cultured in lysogeny broth (LB; 1% tryptone, 0.5% yeast extract, 1% NaCl) with the appropriate antibiotic(s). Antibiotics were used at the following concentrations for *E. coli* DH5:

streptomycin, 100 g ml-1; kanamycin, 50 g ml-1; ampicillin, 200 g ml-1.

the adaptive phenotype (Table 3).

under specific cellular conditions or stresses.

**4.1 Bacterial strains and culture conditions**

g ml-1, 340 g ml-1 and 1 g ml-1 respectively.

**4. Methods**

For analysis of growth in liquid cultures, late-log phase cells (5.0 107 cells ml-1 to 1.0 108 cells ml-1) were diluted to 1.0 105 cells ml-1 in BSK-II with or without GlcNAc. In general, 6- 12 l of culture was used to inoculate 6 ml of fresh culture medium; therefore, negligible amounts of free GlcNAc were transferred with the inoculum when analyzing growth in medium lacking GlcNAc. Cells were enumerated daily by darkfield microscopy using a Petroff-Hauser (Hauser Scientific; Horsham, PA) counting chamber. Specifically, 2.5 l of liquid culture was transferred to the counting chamber, and cells in all 25 squares were counted. Cells were diluted 1:10 in BSK-II prior to counting when the culture density was greater than 1.0 107 cells ml-1. All growth experiments were carried out at 33C in the presence of 3% CO2. Unless otherwise stated, each growth curve presented in this study is representative of at least three independent trials, as growth data could not be pooled due to the length of experiments and the different times at which bacteria were enumerated.

#### **4.3 Construction of a** *recA* **mutant in** *B. burgdorferi*

The construct used to generate a *recA* (BB0131) mutation in the B31-A background was created as follows: i) A 2.1 kb fragment of the 3' end of BB0131 and flanking DNA was PCR amplified with engineered restriction sites using primers (Table 5) 5' BB0131 mutF1 (KpnI) and 5' BB0131 mutR2 (XbaI); ii) the PCR fragment was TA cloned into pCR2.1 (Invitrogen) to generate plasmid pBB0131.1; iii) plasmids pBB0131.1 and pKFSS1 (Frank et al. 2003) (a *B. burgdorferi* shuttle vector conferring streptomycin resistance; Table 4) were digested with KpnI and XbaI and separated by gel electrophoresis; iv) the 2.1 kb band from pBB0131.1 was gel extracted and cloned into the gel extracted pKFSS1 fragment to generate pBB0131.2; v) the 2.1 kb fragment and flanking streptomycin resistance gene were PCR amplified from pBB0131.2 using primers 5' BB0131 mutF1 (KpnI) and pKFSS1 R1; vi) the 3.6 kb amplicon was TA cloned into pGEM T-Easy (Promega, Corp.; Madison, WI) to generate pBB0131.3A or B (based on the orientation of the PCR product insertion); vii) plasmid pBB0131.3B was identified by restriction digest in which the 3' end of the streptomycin resistance gene was adjacent to the XmaI site in the pGEM T-Easy vector; viii) the 5' end of BB0131 and flanking DNA (1.8 kb) was PCR amplified with engineered restriction sites using primers 3' BB0131 mutF1 (XmaI) and 3' BB0131 mutR1 (SacII) and TA cloned into pCR2.1 to generate pBB0131.4; ix) plasmids pBB0131.3B and pBB0131.4 were digested with XmaI and SacII and separated by gel electrophoresis; x) the 1.8 kb fragment from pBB0131.4 was gel extracted and cloned into the gel extracted fragment from pBB0131.3B to generate the final construct pBB0131.5. All plasmids were confirmed by restriction digest and DNA sequencing. In summary, 877 of the 1098 bp constituting the *recA* coding region were deleted and replaced with the streptomycin resistance gene transcribed in the opposite orientation and under the control of the constitutive *B. burgdorferi flgB* promoter. Plasmid pBB0131.5 was confirmed by PCR using primers flanking the antibiotic insertion site (BB0131 mut confirm F1 and 5' recA SalI) that resulted in a 1.7 kb fragment (Fig. 9, lane 3) compared to a 1.2 kb fragment generated with the wild-type genomic DNA as a template (Fig. 9, lane 2).

To generate the *recA* deletion mutant, competent B31-A cells were prepared and transformed with 15 µg of pBB0131.5 as previously described (Samuels et al. 1994). Briefly, transformed cells were resuspended in 10 ml of complete BSK-II and allowed to recover

Adaptation to Glucosamine Starvation in *Borrelia burgdorferi* is Mediated by *recA* 131

Primer Name Sequence (5' 3')

pKFSS1 R1 TGATGAACAGGGTCACGTCGTC

BB0131 mut confirm F1 CAAGGCTGATAGAGTAACTACCCA

5' BB0131 mutF1 (KpnI) GCTAGGGTACCTGCTTATATCGCCTCAAAGCTCG 5' BB0131 mutR2 (XbaI) GCTAGTCTAGACTTAGCAAAGAAGTAGAACTTGC

3' BB0131 mutF1 (XmaI) GCTAGCCCGGGGCTTTTTCTATTTGAACTCTTGCAAGC 3' BB0131 mutR1 (SacII) GCTAGCCGCGGTATGGCCATGTCGGCTTTATCTCC

BB0131 comp F1 (KpnI) GCTAGGGTACCCAAGGCTGATAGAGTAACTACCCA BB0131 comp R1 (XbaI) GCTAGTCTAGATGCAGGCGAGAGAATGGAAATTGG

5' recA (SalI) GTCGACATGTCAAAGTTAAAGGAAAAAAGAGAAAAAGC

PflaB F1 (SalI) GTCGACATATCATTCCTCCATGATAAAATTT

PflaB R1 (NotI) GCGGCCGCTGTCTGTCGCCTCTTGTGG

ErmC F1 GGTTCCATAATATGTTCTCCCTTTCTCAG

BbrecA F1 CAAGGCTGATAGAGTAACTACCCA

ErmC R1 CCCAACGCTCGAATTTAAAGACCC recA operon F1 CACGCTCTTTAAATAAGGGCTGAACC recA operon F3 AATCTCAAGAGCTTGCTCTCCGGT recA operon F4 CCAATGCCGAGAGCCTCATCTAAT recA operon F5 TGGCCCATACTTCTCTGATTCCTG recA operon F6 ACGTCCTTTAAATGCCTGCCAC recA operon F8 CAAGGCTTTCAACTTGACCATTCCA recA operon R1 CAGGCTCATGGTATTCATTGGGAG recA operon R1A TACTTGGGCCTTGGGAATCAAACC

recA operon R2 AAAGCTCGTGAACTTGGTGA

recA operon R2A ATCGGTCTTAGAGGTCGTGAGAGA recA operon R2B AGGATTTGCATATTGAAGAAGAGATGC

recA operon R3 TATGGCCATGTCGGCTTTATCTCC recA operon R4 GAAGATAGAGCACGTATTGATGAG recA operon R5 CCGAAGAGGCTCATTCTCTTGA

BB0131 F1 ACATCATCGCTTGAGCCCGATCTA BB0131 R1 ACCACTACCGGTGGGAATGCTTTA

Table 5. Oligonucleotide primers used in this study

oligo dT-anchor primer GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV

overnight at 33C. The transformation reaction was plated in solid BSK-II medium containing 100 g ml-1 streptomycin to select for the allelic exchange event. Streptomycin resistant colonies were screened by PCR using primers that flanked the insertion site. One clone (JA10) was chosen for further experimentation and PCR of the genomic DNA from this strain with the primers BB0131 mut confirm F1 and 5' recA SalI demonstrated the presence of the mutant *recA* allele (Fig. 9, lane 5). In addition, the insertion was confirmed by DNA sequencing of the PCR product.

Table 4. Strains and plasmids used in this study.

overnight at 33C. The transformation reaction was plated in solid BSK-II medium containing 100 g ml-1 streptomycin to select for the allelic exchange event. Streptomycin resistant colonies were screened by PCR using primers that flanked the insertion site. One clone (JA10) was chosen for further experimentation and PCR of the genomic DNA from this strain with the primers BB0131 mut confirm F1 and 5' recA SalI demonstrated the presence of the mutant *recA* allele (Fig. 9, lane 5). In addition, the insertion was confirmed by

Strain or Plasmid Genotype and Description Reference

B31-A High passage non-infectious wild-type (Elias et al.

 RR45 GlcNAc starvation adapted clone This study JA10 StrR; B31-A *recA* mutant This study

JR1 StrR KanR; JA10 complemented with precA.2 This study

pKFSS1 StrR; *B. burgdorferi* shuttle vector, cp9 based (Frank et al.

pBSV2 KanR; *B. burgdorferi* shuttle vector, cp9 based (Stewart et

pBB0450.1 ErmR; *ermC*::BB0450 (Rhodes et

pBB0131.5 StrR; *aadA*::BB0131 (*recA*) This study

pBB0131comp.2 This study

cp32 based This study

with 512 bp of upstream DNA This study

fused to constitutive *flaB* promoter This study

JA15 StrR KanR; JA10 complemented with

 DH5 *supE*44 F- *lac*U169 (80*lac*Z M15) *hsdR*17 *relA*<sup>1</sup> *endA*1 *gyrA*96 *thi*-1 *relA*1

pCE320 KanR ZeoR; *B. burgdorferi* shuttle vector, cp32

ErmC/pCE320 KanR ZeoR, ErmR; *B. burgdorferi* shuttle vector,

pBB0131comp.2 KanR; BB0131 complementation construct - *recA*

pRecA.3 KanR; BB0131 complementation construct –*recA*

based

Table 4. Strains and plasmids used in this study.

2000)

(Hanahan 1983)

2003)

al. 2001)

(Eggers et al. 2002)

al. 2009)

DNA sequencing of the PCR product.

*B . burgdorferi*

Strains

*E. coli*

Plasmids

Table 5. Oligonucleotide primers used in this study

Adaptation to Glucosamine Starvation in *Borrelia burgdorferi* is Mediated by *recA* 133

gel electrophoresis. The 360 bp product from pPflaB was gel extracted and ligated into the gel extracted fragment of pRecA.1 to generate pRecA.2. The *flaB* promoter-*recA* fusion was removed from pRecA.2 by digestion with KpnI and XbaI (sites in pCR2.1 flanking the TA insertion site), separated by gel electrophoresis, gel extracted and cloned into like sites in pCE320 (Eggers et al. 2002) (a *B. burgdorferi* shuttle vector with a circular plasmid 32 (cp32) origin for replication that confers kanamycin resistance) to generate pRecA.3. The complementation construct pRecA.3 was concentrated and transformed into JA10 as described above. Transformants were screened by PCR using primers BB0131 mut confirm F1 and 5' recA SalI, and one clone was designated JR1 and used for further experiments. PCR using 50 ng of JR1 genomic DNA resulted in amplification of both wild-type and

A homologous recombination assay similar to that described previously by Liveris *et al* (Liveris et al. 2008) was used to determine transformation and recombination frequencies for the wild-type, *recA* mutant and *recA* complemented mutant strains. To determine transformation frequency we generated plasmid ErmC/pCE320 in which the pCE320 shuttle vector was modified to confer erythromycin resistance. Specifically, primers ErmC F1 and ErmC R1 were used to amplify the erythromycin resistance gene and its corresponding promoter from plasmid pBB0450.1, which was then TA cloned into pCR2.1 to generate pCR2.1/ErmC. Plasmid pCR2.1/ErmC was digested with BamHI and NotI, separated by gel electrophoresis, and the fragment containing *ermC* was gel extracted and cloned into like sites in pCE320 to generate ErmC/pCE320. Plasmid ErmC/pCE320 was concentrated to >1 g l-1 and cells were electroporated with 15 g of plasmid as previously described (Samuels et al. 1994). After recovery overnight in BSK-II, transformants were selected on solid BSK-II containing 1 g ml-1 erythromycin and transformation frequency was calculated by dividing the number of transformant colonies obtained by the total number of cells plated (CFU) as determined by dilution and plating without antibiotic selection. The presence of ErmC/pCE320 was confirmed in at least ten erythromycin resistant colonies from each experiment by colony PCR with primers specific for *ermC*.

The ability of *recA* expressing and non-expressing strains to mediate allelic exchange was determined by calculating the recombination frequency following electroporation with plasmid pBB0450.1 (*rpoN::ermC* with flanking DNA) (Rhodes et al. 2009). Transformation and plating with pBB0450.1 was carried out as described above for ErmC/pCE320 and recombinants were selected on solid BSK-II containing 1 g ml-1 erythromycin. Recombination frequency was calculated by dividing the number of erythromycin resistant colonies by the total number of cells plated (CFU) as determined by dilution and plating without selection. Allelic exchange was confirmed in at least ten erythromycin resistant colonies from each

RNA was extracted from wild-type cells (B31-A) for use in RT-PCR and qRT-PCR experiments. Briefly, cells were harvested during mid to late log phase (1.0 to 5.0 107 cells ml-1) by centrifugation (10,000 *g*, 12 min, 4C). Pellets were resuspended in 500 l of cold BSK-II and transferred to a 2 ml microcentrifuge tube. One milliliter of Bacteria RNAProtect

experiment by colony PCR with primers flanking the insertion site in *rpoN*.

mutant *recA* alleles (Fig. 9; lane 7).

**4.6 RNA extraction**

**4.5 Homologous recombination assay**

Fig. 9. PCR confirmation of the *recA* mutant and complemented mutant strains. PCR confirmation of JA10 (*recA* deletion/insertion mutant), JA15 (JA10 complemented with pBB0131comp.2) and JR1 (JA10 complemented with pRecA.3) using primers BB0131 mut confirm F1 and 5' recA SalI which flank the insertion site. The larger PCR product is 1.7 kb (upper arrow) and contains the streptomycin resistance gene within *recA*. The smaller PCR product is 1.2 kb (lower arrow) and represents the wild-type *recA* gene. PCR reactions using the following templates were separated by electrophoresis: lane 1 – 1 kb ladder (Promega), lane 2 – B31-A genomic DNA, lane 3 – pBB0131.5 (*recA* mutation construct), lane 4 – pRecA.3 (complementation plasmid for JR1), lane 5 – JA10 genomic DNA (*recA* mutant), lane 6 – JA15 genomic DNA (JA10 complemented with pBB0131comp.2) and lane 7 – JR1 genomic DNA (JA10 complemented with pRecA.3).

#### **4.4 Complementation of the** *recA* **mutant**

Two approaches were used to complement the *recA* mutation in JA10. First, the *recA* gene and 512 bp of upstream DNA were PCR amplified using primers BB0131 comp F1 (KpnI) and BB0131 comp R1 (XbaI), and the 1.6 kb fragment was TA cloned into pCR2.1 to generate pBB0131comp.1. Plasmids pBB0131comp.1 and pBSV2 (Stewart et al. 2001) (*E. coli*-*B. burgdorferi* shuttle conferring kanamycin resistance), were digested with KpnI and XbaI and separated by gel electrophoresis. The 1.6 kb fragment from pBB0131comp.1 was gel extracted and ligated with the gel extracted fragment from pBSV2 to generate pBB0131comp.2. Fifteen micrograms of pBB0131comp.2 was transformed into competent JA10 cells and transformants were selected by plating on solid BSK-II medium containing 100 g ml-1 streptomycin and 340 g ml-1 kanamycin. Clones were screened by PCR using primers BB0131 mut confirm F1 and 5' recA SalI. One clone, designated JA15, was selected for further experimentation and the presence of both the mutant and wild-type *recA* alleles was confirmed by PCR using 50 ng of genomic DNA as template (Fig. 9; lane 6).

We also generated a complementation construct in which we fused the wild-type *recA* gene to the constitutive *B. burgdorferi flaB* promoter. The *flaB* promoter was PCR amplified from B31-A genomic DNA using primers PflaB F1 (SalI) and PflaB R1 (NotI), and cloned into pCR2.1 to generate pPflaB. The *recA* coding region was PCR amplified from B31-A genomic DNA using primers 5' recA (SalI) and BbrecA F1, and cloned into pCR2.1 to generate pRecA.1. Plasmids pPflaB and pRecA.1 were digested with SalI and NotI and separated by gel electrophoresis. The 360 bp product from pPflaB was gel extracted and ligated into the gel extracted fragment of pRecA.1 to generate pRecA.2. The *flaB* promoter-*recA* fusion was removed from pRecA.2 by digestion with KpnI and XbaI (sites in pCR2.1 flanking the TA insertion site), separated by gel electrophoresis, gel extracted and cloned into like sites in pCE320 (Eggers et al. 2002) (a *B. burgdorferi* shuttle vector with a circular plasmid 32 (cp32) origin for replication that confers kanamycin resistance) to generate pRecA.3. The complementation construct pRecA.3 was concentrated and transformed into JA10 as described above. Transformants were screened by PCR using primers BB0131 mut confirm F1 and 5' recA SalI, and one clone was designated JR1 and used for further experiments. PCR using 50 ng of JR1 genomic DNA resulted in amplification of both wild-type and mutant *recA* alleles (Fig. 9; lane 7).

#### **4.5 Homologous recombination assay**

132 Lyme Disease

Fig. 9. PCR confirmation of the *recA* mutant and complemented mutant strains. PCR confirmation of JA10 (*recA* deletion/insertion mutant), JA15 (JA10 complemented with pBB0131comp.2) and JR1 (JA10 complemented with pRecA.3) using primers BB0131 mut confirm F1 and 5' recA SalI which flank the insertion site. The larger PCR product is 1.7 kb (upper arrow) and contains the streptomycin resistance gene within *recA*. The smaller PCR product is 1.2 kb (lower arrow) and represents the wild-type *recA* gene. PCR reactions using the following templates were separated by electrophoresis: lane 1 – 1 kb ladder (Promega), lane 2 – B31-A genomic DNA, lane 3 – pBB0131.5 (*recA* mutation construct), lane 4 – pRecA.3 (complementation plasmid for JR1), lane 5 – JA10 genomic DNA (*recA* mutant), lane 6 – JA15 genomic DNA (JA10 complemented with pBB0131comp.2) and lane 7 – JR1

Two approaches were used to complement the *recA* mutation in JA10. First, the *recA* gene and 512 bp of upstream DNA were PCR amplified using primers BB0131 comp F1 (KpnI) and BB0131 comp R1 (XbaI), and the 1.6 kb fragment was TA cloned into pCR2.1 to generate pBB0131comp.1. Plasmids pBB0131comp.1 and pBSV2 (Stewart et al. 2001) (*E. coli*-*B. burgdorferi* shuttle conferring kanamycin resistance), were digested with KpnI and XbaI and separated by gel electrophoresis. The 1.6 kb fragment from pBB0131comp.1 was gel extracted and ligated with the gel extracted fragment from pBSV2 to generate pBB0131comp.2. Fifteen micrograms of pBB0131comp.2 was transformed into competent JA10 cells and transformants were selected by plating on solid BSK-II medium containing 100 g ml-1 streptomycin and 340 g ml-1 kanamycin. Clones were screened by PCR using primers BB0131 mut confirm F1 and 5' recA SalI. One clone, designated JA15, was selected for further experimentation and the presence of both the mutant and wild-type *recA* alleles

was confirmed by PCR using 50 ng of genomic DNA as template (Fig. 9; lane 6).

We also generated a complementation construct in which we fused the wild-type *recA* gene to the constitutive *B. burgdorferi flaB* promoter. The *flaB* promoter was PCR amplified from B31-A genomic DNA using primers PflaB F1 (SalI) and PflaB R1 (NotI), and cloned into pCR2.1 to generate pPflaB. The *recA* coding region was PCR amplified from B31-A genomic DNA using primers 5' recA (SalI) and BbrecA F1, and cloned into pCR2.1 to generate pRecA.1. Plasmids pPflaB and pRecA.1 were digested with SalI and NotI and separated by

genomic DNA (JA10 complemented with pRecA.3).

**4.4 Complementation of the** *recA* **mutant**

A homologous recombination assay similar to that described previously by Liveris *et al* (Liveris et al. 2008) was used to determine transformation and recombination frequencies for the wild-type, *recA* mutant and *recA* complemented mutant strains. To determine transformation frequency we generated plasmid ErmC/pCE320 in which the pCE320 shuttle vector was modified to confer erythromycin resistance. Specifically, primers ErmC F1 and ErmC R1 were used to amplify the erythromycin resistance gene and its corresponding promoter from plasmid pBB0450.1, which was then TA cloned into pCR2.1 to generate pCR2.1/ErmC. Plasmid pCR2.1/ErmC was digested with BamHI and NotI, separated by gel electrophoresis, and the fragment containing *ermC* was gel extracted and cloned into like sites in pCE320 to generate ErmC/pCE320. Plasmid ErmC/pCE320 was concentrated to >1 g l-1 and cells were electroporated with 15 g of plasmid as previously described (Samuels et al. 1994). After recovery overnight in BSK-II, transformants were selected on solid BSK-II containing 1 g ml-1 erythromycin and transformation frequency was calculated by dividing the number of transformant colonies obtained by the total number of cells plated (CFU) as determined by dilution and plating without antibiotic selection. The presence of ErmC/pCE320 was confirmed in at least ten erythromycin resistant colonies from each experiment by colony PCR with primers specific for *ermC*.

The ability of *recA* expressing and non-expressing strains to mediate allelic exchange was determined by calculating the recombination frequency following electroporation with plasmid pBB0450.1 (*rpoN::ermC* with flanking DNA) (Rhodes et al. 2009). Transformation and plating with pBB0450.1 was carried out as described above for ErmC/pCE320 and recombinants were selected on solid BSK-II containing 1 g ml-1 erythromycin. Recombination frequency was calculated by dividing the number of erythromycin resistant colonies by the total number of cells plated (CFU) as determined by dilution and plating without selection. Allelic exchange was confirmed in at least ten erythromycin resistant colonies from each experiment by colony PCR with primers flanking the insertion site in *rpoN*.

#### **4.6 RNA extraction**

RNA was extracted from wild-type cells (B31-A) for use in RT-PCR and qRT-PCR experiments. Briefly, cells were harvested during mid to late log phase (1.0 to 5.0 107 cells ml-1) by centrifugation (10,000 *g*, 12 min, 4C). Pellets were resuspended in 500 l of cold BSK-II and transferred to a 2 ml microcentrifuge tube. One milliliter of Bacteria RNAProtect

Adaptation to Glucosamine Starvation in *Borrelia burgdorferi* is Mediated by *recA* 135

min at 95C, ii) 35 cycles of 95C for 30 sec, 58C for 30 sec and 72C for 1 to 4 min (depending on the size of the expected product), and iii) final extension at 72C for 10 min. PCR products

The approximate *recA* transcriptional start sites were determined using the 2nd Generation 5'/3' RACE Kit (Roche Applied Science; Mannheim, Germany) according to the manufacturer's instructions. Briefly, two first-strand cDNA synthesis reactions were performed with 1 g of total RNA and antisense primers recA operon F3 and recA operon F5 to determine the transcritptional start sites immediately upstream of BB0131 (*recA*) and BB0133. The two cDNA reactions were purified using the High Pure PCR Product Purification Kit (Roche Applied Science). The purified cDNAs were divided into two reaction tubes and a homopolymeric A or G-tail was added to the 3' end using recombinant terminal deoxynucleotidyl transferase and dATP or dGTP. A PCR product was amplified from each tailed cDNA using the appropriate anchor primer (oligo dT-AP or oligo dC-AP) and a nested gene specific primer. Nested primer recA operon F4 was used to amplify tailed cDNA generated with recA operon F3, and nested primer recA operon F6 was used to amplify tailed cDNA generated with recA operon F5. The PCR products were subjected to electrophoresis and the bands were gel extracted using the QIAquick PCR Purification Kit (Qiagen). The sequence of the purified PCR products were determined using either recA operon F4 or recA operon F8 and aligned to the upstream sequence of *recA* or BB0133 to determine the transcriptional start sites. Promoter analysis was carried out by visual inspection and comparison of the region upstream of the approximate transcriptional start sites with previously described RpoD, RpoS and RpoN-

Nucleic acid sequencing was performed by the Rhode Island Genomics and Sequencing Center using a 3130xl Genetic Analyzer (Applied Biosystems; Forest City, CA). Sequencing reactions were prepared using the BigDye Terminator v3.0 Cycle Sequencing Kit. Sequences were analyzed using DNASTAR Lasergene software (DNASTAR, Inc.; Madison,

In this study we demonstrate that *B. burgdorferi* undergoes a genetic adaptation during GlcNAc starvation that allows cells to grow to a high cell density when re-introduced to the starvation condition. We also show that *recA* mutant cells do not respond to GlcNAc starvation the same as wild-type cells, suggesting a role for RecA in this process. Finally, we characterize the *recA* transcriptional unit and provide evidence that this gene is controlled

We thank P. Rosa and J. Radolf for providing strains and plasmids. This research is based in part upon work conducted using the Rhode Island Genomics and Sequencing Center, which

were separated on a 1% agarose gel and visualized using ethidium bromide.

**4.9 5' Rapid Amplification of cDNA Ends (RACE) and promoter analysis**

dependent promoter sequences in *B. burgdorferi*.

WI) and Vector NTI (Invitrogen, Corp.).

transcriptionally from two separate promoters.

**5. Conclusions**

**6. Acknowledgments**

**4.10 Nucleotide sequencing and computer analysis**

(Qiagen; Valencia, CA) was added, mixed by vortexing and incubated for 5 min at room temperature. Cells were collected by centrifugation at 5,000 *g* for 10 min, the supernatant was decanted and pellets were stored at -80C for up to 4 weeks prior to RNA extraction. RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions and treated with RQ1 RNase-free DNase (Promega, Corp.) and RNasin (Promega, Corp.) to remove any contaminating genomic DNA. RNA was purified using the RNeasy Mini kit and following the RNA Clean-up protocol supplied by the manufacturer. RNA concentration (OD260) and purity (OD260/280) were determined by UV spectroscopy using the Nanodrop ND-1000 (Nanodrop products of Thermo Fisher Scientific; Wilmington, DE) and RNA integrity was verified by visualizing the intensity of the 16S and 23S rRNA bands on a 1% agarose gel. RNA was concentrated to greater that 1 g l-1 by isopropanol precipitation and stored at -80C.

#### **4.7 Quantitative RT-PCR (qRT-PCR) to determine** *recA* **expression**

qRT-PCR was performed using the Mx3005P Multiplex Quantitative PCR System in conjunction with the Brilliant SYBR Green Single-Step qRT-PCR Master Mix kit (Stratagene; La Jolla, CA) according to the manufacturer's instructions. Briefly, a standard curve (101 to 107 copies per reaction) was prepared using a purified *recA* PCR product that was generated from B31-A genomic DNA using primers BB0131 F1 and BB0131 R1. Reactions (25 l) containing 10 ng of total RNA extracted from mid-log phase cells (~1.0 107 cells ml-1) were run under the following conditions: 1 cycle of 50C for 30 min and 95C for 15 min, followed by 40 cycles of 95C for 30 s and 58C for 30 s. Fluorescence was measured at the end of the 58C step every cycle. Triplicate samples for each experiment were averaged, and the average and standard deviations were calculated for three independent experiments. All qRT-PCR experiments included both no-reverse transcriptase (RT) and no-template controls. The *recA* copy number for each sample was calculated using the MxPro (Stratagene) data analysis software and the *recA* standard curve described above. Copy number in each sample was normalized based on the total RNA input (10 ng per reaction) and the fold difference in *recA* copy number was determined by comparing the expression in the *recA* mutant and complemented mutant strains to the expression in the wild-type strain.

#### **4.8 RT-PCR to determine the** *recA* **transcriptional unit**

RT-PCR was used to determine the organization of the *recA* transcriptional unit. Complementary DNA (cDNA) was generated from B31-A RNA using the SuperScript First Strand Synthesis for RT-PCR kit (Invitrogen, Corp.) according to the manufacturer's instructions. Briefly, 0.5 g of RNA was reverse transcribed with SuperScript II RT enzyme at 42C for one hour using gene specific primers recA operon F1 or F3. The RT enzyme was heat inactivated and RNA was degraded from the RNA-cDNA hybrid using RNaseH. To confirm there was no genomic DNA contamination a separate no RT reaction was run in conjunction with each RT reaction. For PCR, antisense primers used to generate the cDNA were used in various combinations with the sense primers recA operon R1, R1A, R2, R2A, R2B, R3, R4 or R5. Three reactions were prepared for each primer set and the following templates were added: i) 50 ng of B31-A genomic DNA, ii) 2 l of the appropriate cDNA reaction, and iii) 2 l of the appropriate no RT control reaction. Templates were amplified using TaKaRa ExTaq (Fisher Scientific; Pittsburgh, PA) and the following thermal cycle: i) initial denaturation for 10 min at 95C, ii) 35 cycles of 95C for 30 sec, 58C for 30 sec and 72C for 1 to 4 min (depending on the size of the expected product), and iii) final extension at 72C for 10 min. PCR products were separated on a 1% agarose gel and visualized using ethidium bromide.

#### **4.9 5' Rapid Amplification of cDNA Ends (RACE) and promoter analysis**

The approximate *recA* transcriptional start sites were determined using the 2nd Generation 5'/3' RACE Kit (Roche Applied Science; Mannheim, Germany) according to the manufacturer's instructions. Briefly, two first-strand cDNA synthesis reactions were performed with 1 g of total RNA and antisense primers recA operon F3 and recA operon F5 to determine the transcritptional start sites immediately upstream of BB0131 (*recA*) and BB0133. The two cDNA reactions were purified using the High Pure PCR Product Purification Kit (Roche Applied Science). The purified cDNAs were divided into two reaction tubes and a homopolymeric A or G-tail was added to the 3' end using recombinant terminal deoxynucleotidyl transferase and dATP or dGTP. A PCR product was amplified from each tailed cDNA using the appropriate anchor primer (oligo dT-AP or oligo dC-AP) and a nested gene specific primer. Nested primer recA operon F4 was used to amplify tailed cDNA generated with recA operon F3, and nested primer recA operon F6 was used to amplify tailed cDNA generated with recA operon F5. The PCR products were subjected to electrophoresis and the bands were gel extracted using the QIAquick PCR Purification Kit (Qiagen). The sequence of the purified PCR products were determined using either recA operon F4 or recA operon F8 and aligned to the upstream sequence of *recA* or BB0133 to determine the transcriptional start sites. Promoter analysis was carried out by visual inspection and comparison of the region upstream of the approximate transcriptional start sites with previously described RpoD, RpoS and RpoNdependent promoter sequences in *B. burgdorferi*.

#### **4.10 Nucleotide sequencing and computer analysis**

Nucleic acid sequencing was performed by the Rhode Island Genomics and Sequencing Center using a 3130xl Genetic Analyzer (Applied Biosystems; Forest City, CA). Sequencing reactions were prepared using the BigDye Terminator v3.0 Cycle Sequencing Kit. Sequences were analyzed using DNASTAR Lasergene software (DNASTAR, Inc.; Madison, WI) and Vector NTI (Invitrogen, Corp.).

#### **5. Conclusions**

134 Lyme Disease

(Qiagen; Valencia, CA) was added, mixed by vortexing and incubated for 5 min at room temperature. Cells were collected by centrifugation at 5,000 *g* for 10 min, the supernatant was decanted and pellets were stored at -80C for up to 4 weeks prior to RNA extraction. RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions and treated with RQ1 RNase-free DNase (Promega, Corp.) and RNasin (Promega, Corp.) to remove any contaminating genomic DNA. RNA was purified using the RNeasy Mini kit and following the RNA Clean-up protocol supplied by the manufacturer. RNA concentration (OD260) and purity (OD260/280) were determined by UV spectroscopy using the Nanodrop ND-1000 (Nanodrop products of Thermo Fisher Scientific; Wilmington, DE) and RNA integrity was verified by visualizing the intensity of the 16S and 23S rRNA bands on a 1% agarose gel. RNA was concentrated to greater that 1 g l-1 by isopropanol

qRT-PCR was performed using the Mx3005P Multiplex Quantitative PCR System in conjunction with the Brilliant SYBR Green Single-Step qRT-PCR Master Mix kit (Stratagene; La Jolla, CA) according to the manufacturer's instructions. Briefly, a standard curve (101 to 107 copies per reaction) was prepared using a purified *recA* PCR product that was generated from B31-A genomic DNA using primers BB0131 F1 and BB0131 R1. Reactions (25 l) containing 10 ng of total RNA extracted from mid-log phase cells (~1.0 107 cells ml-1) were run under the following conditions: 1 cycle of 50C for 30 min and 95C for 15 min, followed by 40 cycles of 95C for 30 s and 58C for 30 s. Fluorescence was measured at the end of the 58C step every cycle. Triplicate samples for each experiment were averaged, and the average and standard deviations were calculated for three independent experiments. All qRT-PCR experiments included both no-reverse transcriptase (RT) and no-template controls. The *recA* copy number for each sample was calculated using the MxPro (Stratagene) data analysis software and the *recA* standard curve described above. Copy number in each sample was normalized based on the total RNA input (10 ng per reaction) and the fold difference in *recA* copy number was determined by comparing the expression in the *recA*

mutant and complemented mutant strains to the expression in the wild-type strain.

RT-PCR was used to determine the organization of the *recA* transcriptional unit. Complementary DNA (cDNA) was generated from B31-A RNA using the SuperScript First Strand Synthesis for RT-PCR kit (Invitrogen, Corp.) according to the manufacturer's instructions. Briefly, 0.5 g of RNA was reverse transcribed with SuperScript II RT enzyme at 42C for one hour using gene specific primers recA operon F1 or F3. The RT enzyme was heat inactivated and RNA was degraded from the RNA-cDNA hybrid using RNaseH. To confirm there was no genomic DNA contamination a separate no RT reaction was run in conjunction with each RT reaction. For PCR, antisense primers used to generate the cDNA were used in various combinations with the sense primers recA operon R1, R1A, R2, R2A, R2B, R3, R4 or R5. Three reactions were prepared for each primer set and the following templates were added: i) 50 ng of B31-A genomic DNA, ii) 2 l of the appropriate cDNA reaction, and iii) 2 l of the appropriate no RT control reaction. Templates were amplified using TaKaRa ExTaq (Fisher Scientific; Pittsburgh, PA) and the following thermal cycle: i) initial denaturation for 10

**4.8 RT-PCR to determine the** *recA* **transcriptional unit**

**4.7 Quantitative RT-PCR (qRT-PCR) to determine** *recA* **expression**

precipitation and stored at -80C.

In this study we demonstrate that *B. burgdorferi* undergoes a genetic adaptation during GlcNAc starvation that allows cells to grow to a high cell density when re-introduced to the starvation condition. We also show that *recA* mutant cells do not respond to GlcNAc starvation the same as wild-type cells, suggesting a role for RecA in this process. Finally, we characterize the *recA* transcriptional unit and provide evidence that this gene is controlled transcriptionally from two separate promoters.

#### **6. Acknowledgments**

We thank P. Rosa and J. Radolf for providing strains and plasmids. This research is based in part upon work conducted using the Rhode Island Genomics and Sequencing Center, which

Adaptation to Glucosamine Starvation in *Borrelia burgdorferi* is Mediated by *recA* 137

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is supported in part by the National Science Foundation under EPSCoR Grant No. 0554548. This work was supported by NIH grant 5 R01AI03723010 awarded to DRN.

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**7. References**

**1. Introduction**

remarkably unique characteristics.

**7**

**Porins in the Genus** *Borrelia*

Sven Bergström3 and Roland Benz1,2

*1Jacobs University Bremen 2University of Wuerzburg 3University of Umea*

> *1,2Germany 3Sweden*

Iván Bárcena-Uribarri1, Marcus Thein2, Mari Bonde3,

**1.1 The cell envelope of** *Borrelia* **compared to other gram-negative bacteria**

response avoidance, as well as pathways for the acquisition of nutrients.

*Borreliae* belong to the spirochete phylum, an ancient evolutionary branch only remotely related to Gram-negative and Gram-positive bacteria, although their envelope is regarded to be Gram-negative. Spirochetes of the genus *Borrelia* exhibit a complex life style, characterized by the ability to shuttle between hematophagous arthropods and various vertebrates, which exposes them to a variety of niches that are different in nutritional composition as well as immunological pressure. Survival, transmission and ultimately pathogenesis during infection obviously require a large degree of adaptive biological capacity. Thus, the interactions between the host and *Borrelia* pathogens need to be optimized for interaction with different environments such as the tick midgut, and various mammalian tissues. The different surface-exposed proteins play an important role in this process by providing ligands for receptor-mediated adhesion, mechanisms of host immune

*Borreliae* are considered to be Gram-negative bacteria because they possess two membranes and a periplasmic peptidoglycan layer, which in *Borrelia* is anchored to the inner membrane [1]. In spite of this, if *Borrelia* are compared to typical Gram-negative bacteria such as *Escherichia coli*, their cell envelope differs in many aspects (Fig. 1). While flagella in typical Gram-negative bacteria are placed on the surface of the outer membrane, the flagella in *Borreliae* are found in the periplasmic space between inner and outer membranes. This peculiar characteristic is shared among the spirochetes. Another large difference between the common Gram-negative eubacteria and *Borreliae* spirochetes is the absence of lipopolysaccharides [2, 3]. Instead, the outer membrane in *Borreliae* is very rich in lipoproteins, which represent the primary interface between the bacterium and the host [4]. The borrelial outer membranes are considerably more fluid than those of other Gram negative bacteria. They also possess a lower density of membrane spanning proteins [5, 6]. Porins, the focus of this review, are also found in the outer membrane of spirochetes with

### **Porins in the Genus** *Borrelia*

Iván Bárcena-Uribarri1, Marcus Thein2, Mari Bonde3,

 Sven Bergström3 and Roland Benz1,2 *1Jacobs University Bremen 2University of Wuerzburg 3University of Umea 1,2Germany 3Sweden*

#### **1. Introduction**

138 Lyme Disease

Rhodes, R., W. Coy & D. Nelson (2009). "Chitobiose utilization in *Borrelia burgdorferi* is

Rhodes, R. G., J. A. Atoyan & D. R. Nelson (2010). "The chitobiose transporter, *chbC*, is required for chitin utilization in *Borrelia burgdorferi*." *BMC Microbiology* 10: 21. Rosenberg, S. M., S. Longerich, P. Gee & R. S. Harris (1994). "Adaptive mutation by deletions

Saier MH, J., Paulsen IT (2000). "Whole genome analyses of transporters in spirochetes:

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Studholme, D. J. & M. Buck (2000). "The biology of enhancer-dependent transcriptional

Tilly, K., A. F. Elias, J. Errett, E. Fischer, R. Iyer, I. Schwartz, J. L. Bono & P. Rosa (2001).

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Steere, A. C. (2001). "Lyme Disease." *The New England Journal of Medicine* 345(2): 115-125. Steere, A. C., J. Coburn & L. Glickstein (2004). "The emergence of Lyme disease." *Journal of*

*Borrelia burgdorferi* and *Treponema pallidum*." *Journal of Molecular Microbiology and*

agent *Borrelia burgdorferi* with coumarin-resistant *gyrB*." *Journal of Bacteriology*

mechanism of gene amplification induced under stress in *Escherichia coli*." *Public*

sufficient for autonomous replication and transformation of infectious *Borrelia*

regulation in bacteria: insights from genome sequences." *Federation of European*

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176(19): 6045-9.

#### **1.1 The cell envelope of** *Borrelia* **compared to other gram-negative bacteria**

*Borreliae* belong to the spirochete phylum, an ancient evolutionary branch only remotely related to Gram-negative and Gram-positive bacteria, although their envelope is regarded to be Gram-negative. Spirochetes of the genus *Borrelia* exhibit a complex life style, characterized by the ability to shuttle between hematophagous arthropods and various vertebrates, which exposes them to a variety of niches that are different in nutritional composition as well as immunological pressure. Survival, transmission and ultimately pathogenesis during infection obviously require a large degree of adaptive biological capacity. Thus, the interactions between the host and *Borrelia* pathogens need to be optimized for interaction with different environments such as the tick midgut, and various mammalian tissues. The different surface-exposed proteins play an important role in this process by providing ligands for receptor-mediated adhesion, mechanisms of host immune response avoidance, as well as pathways for the acquisition of nutrients.

*Borreliae* are considered to be Gram-negative bacteria because they possess two membranes and a periplasmic peptidoglycan layer, which in *Borrelia* is anchored to the inner membrane [1]. In spite of this, if *Borrelia* are compared to typical Gram-negative bacteria such as *Escherichia coli*, their cell envelope differs in many aspects (Fig. 1). While flagella in typical Gram-negative bacteria are placed on the surface of the outer membrane, the flagella in *Borreliae* are found in the periplasmic space between inner and outer membranes. This peculiar characteristic is shared among the spirochetes. Another large difference between the common Gram-negative eubacteria and *Borreliae* spirochetes is the absence of lipopolysaccharides [2, 3]. Instead, the outer membrane in *Borreliae* is very rich in lipoproteins, which represent the primary interface between the bacterium and the host [4]. The borrelial outer membranes are considerably more fluid than those of other Gram negative bacteria. They also possess a lower density of membrane spanning proteins [5, 6]. Porins, the focus of this review, are also found in the outer membrane of spirochetes with remarkably unique characteristics.

Porins in the Genus *Borrelia* 141

OspA expression is up-regulated in unfed ticks while OspC is expressed in large amounts in

Beside the Osps, there is a wide variety of lipoproteins with different functionalities present in *B. burgdorferi*, such as the multicopy lipoproteins Mlps [22], Lp6.6, a small lipoprotein of 6.6 kDa [23], the decorin-binding proteins DbpA/B [24], the glycosaminoglycan-binding protein Bgp [25], P35 comprising two lipoproteins [26], the Vmp-like-sequence Vls [27], the Bmp family [28], P22 [29], the OspE/F related proteins Erps [30], the Elps containing OspE/F-like leader peptides [30] and the complement regulator acquiring surface proteins

In contrast to Lyme disease species, the most abundant proteins in the relapsing fever species' outer membranes are the variable major proteins Vmp. These lipoproteins are expressed with different surface epitopes through antigenic variation [31-33]. The lipoproteins of relapsing fever agents are subdivided into two families: the variable large proteins (Vlps) and the variable small proteins (Vsps) [7, 34, 35]. Interestingly, the expression of the single Vsp species is correlated to the pattern and the state of the disease. Vsps undergo antigenic variation and at least three mechanisms are known for surface

Porins are a group of proteins located in the outer membrane of Gram negative bacteria that facilitate the transfer of substances in both directions between the surrounding environment and the periplasmic space. Porins are also found in mycobacteria, chloroplasts and

Porins form water-filled channels that allow a passive transport of molecules down their concentration gradients [40, 41]. This transport through the outer membrane does not require energy in contrast to the uphill substrate translocation via transporters in the inner membrane. Most of the porins described to date form β barrels composed of antiparallel β-sheets. Frequently, they are associated in oligomers that confer a higher stability to the whole complex (Fig. 2). In the outer membrane of Gram negative bacteria, channel-tunnel proteins like TolC in *Escherichia* and BesC in *Borrelia* are also found. They form pores in the outer membrane and are part of bigger protein complexes involved in drug-resistance known as

Porins can be classified in two groups depending on whether they are selective for a class of

General diffusion porins allow the transport of small molecules, ions and water through a membrane. The permeability of general diffusion pores for substrates is dependent only on their size and charge. Examples for this kind of porins are OmpF and OmpC of *E. coli* [40]. Substrate-specific porins represent pores or channels that are specialized for the transport of certain molecules. These molecules bind to the channel interior and are guided to the periplasm. Tsx is an example of a substrate specific porin in *E. coli* that is specialized in the nucleoside transport from the surrounding media to the periplasmic space (Fig. 2) [43, 44]. Other examples include porins with specificity for carbohydrates [45], phosphate [46] and

feeding ticks and in the mammalian host [21].

alteration of relapsing fever lipoproteins [36, 37].

**1.2 Porins in gram-negative bacteria**

mitochondria [38, 39].

efflux pumps (Fig.2).

molecules or not [42]:

antibiotics [47].

CRASPs [10].

Fig. 1. Comparison of the cell envelope of a typical Gram negative bacterium such as *Escherichia coli* and *Borrelia burgdorferi*. While *E. coli* has a high content of lipopolysaccharides in its outer membrane, *B. burgdorferi* has a high amount of lipoproteins. Periplasmic flagella and a lower amount of external membrane spanning proteins are also typical *Borrelia* features.

#### **1.1.1 Lipoproteins in the outer membrane of** *Borrelia*

The protein content of the borrelial outer membrane is represented by a huge quantity of lipoproteins. As an indication of their importance in *Borrelia*, lipoproteins are the most abundant proteins in these species [7]. These proteins are located on the surface of the bacterial cell and anchored in the outer leaflet of the outer membrane. Borrelial lipoproteins play a major role in the activation of the innate immune response of the host as well as in the adaptive responses and pathogenicity of *Borrelia* spirochetes. Thus, this class of proteins is responsible for the activation of host inflammatory response as tissue inflammation which is a unifying feature during the manifestation of Lyme disease. Lipoproteins dominate pathogenicity mechanisms exploited by *B. burgdorferi*, including antigenic variation [8, 9], evasion of complement killing [10-12] and adhesion mechanisms [13].

The most abundant and best described outer membrane lipoproteins in the Lyme disease species *B. burgdorferi* are the so-called outer surface proteins (Osps). The most prominent lipoprotein species is OspA. OspA tertiary structure contains a single-layer β-sheet connecting the N- and C-terminal globular domains [14]. OspA acts as an adhesin in the tick environment [15] and it could have a protective function for P66 against proteases [16]. OspC, another well-studied *B. burgdorferi* lipoprotein, is homologous to variable small proteins (Vsps) of relapsing fever *Borrelia* [17]. The tertiary structure of OspC is also known and differs completely from OspA/B. It basically consists of five parallel α-helices and two short β-strands [18, 19]. This lipoprotein seems to be required to infect mice [20]. Interestingly, the OspA/B and OspC expression is reciprocally regulated and undergoes a switch during spirochete transition from the tick vector to the mammalian host. Thus, the

Fig. 1. Comparison of the cell envelope of a typical Gram negative bacterium such as

**1.1.1 Lipoproteins in the outer membrane of** *Borrelia*

evasion of complement killing [10-12] and adhesion mechanisms [13].

features.

*Escherichia coli* and *Borrelia burgdorferi*. While *E. coli* has a high content of lipopolysaccharides in its outer membrane, *B. burgdorferi* has a high amount of lipoproteins. Periplasmic flagella and a lower amount of external membrane spanning proteins are also typical *Borrelia*

The protein content of the borrelial outer membrane is represented by a huge quantity of lipoproteins. As an indication of their importance in *Borrelia*, lipoproteins are the most abundant proteins in these species [7]. These proteins are located on the surface of the bacterial cell and anchored in the outer leaflet of the outer membrane. Borrelial lipoproteins play a major role in the activation of the innate immune response of the host as well as in the adaptive responses and pathogenicity of *Borrelia* spirochetes. Thus, this class of proteins is responsible for the activation of host inflammatory response as tissue inflammation which is a unifying feature during the manifestation of Lyme disease. Lipoproteins dominate pathogenicity mechanisms exploited by *B. burgdorferi*, including antigenic variation [8, 9],

The most abundant and best described outer membrane lipoproteins in the Lyme disease species *B. burgdorferi* are the so-called outer surface proteins (Osps). The most prominent lipoprotein species is OspA. OspA tertiary structure contains a single-layer β-sheet connecting the N- and C-terminal globular domains [14]. OspA acts as an adhesin in the tick environment [15] and it could have a protective function for P66 against proteases [16]. OspC, another well-studied *B. burgdorferi* lipoprotein, is homologous to variable small proteins (Vsps) of relapsing fever *Borrelia* [17]. The tertiary structure of OspC is also known and differs completely from OspA/B. It basically consists of five parallel α-helices and two short β-strands [18, 19]. This lipoprotein seems to be required to infect mice [20]. Interestingly, the OspA/B and OspC expression is reciprocally regulated and undergoes a switch during spirochete transition from the tick vector to the mammalian host. Thus, the OspA expression is up-regulated in unfed ticks while OspC is expressed in large amounts in feeding ticks and in the mammalian host [21].

Beside the Osps, there is a wide variety of lipoproteins with different functionalities present in *B. burgdorferi*, such as the multicopy lipoproteins Mlps [22], Lp6.6, a small lipoprotein of 6.6 kDa [23], the decorin-binding proteins DbpA/B [24], the glycosaminoglycan-binding protein Bgp [25], P35 comprising two lipoproteins [26], the Vmp-like-sequence Vls [27], the Bmp family [28], P22 [29], the OspE/F related proteins Erps [30], the Elps containing OspE/F-like leader peptides [30] and the complement regulator acquiring surface proteins CRASPs [10].

In contrast to Lyme disease species, the most abundant proteins in the relapsing fever species' outer membranes are the variable major proteins Vmp. These lipoproteins are expressed with different surface epitopes through antigenic variation [31-33]. The lipoproteins of relapsing fever agents are subdivided into two families: the variable large proteins (Vlps) and the variable small proteins (Vsps) [7, 34, 35]. Interestingly, the expression of the single Vsp species is correlated to the pattern and the state of the disease. Vsps undergo antigenic variation and at least three mechanisms are known for surface alteration of relapsing fever lipoproteins [36, 37].

#### **1.2 Porins in gram-negative bacteria**

Porins are a group of proteins located in the outer membrane of Gram negative bacteria that facilitate the transfer of substances in both directions between the surrounding environment and the periplasmic space. Porins are also found in mycobacteria, chloroplasts and mitochondria [38, 39].

Porins form water-filled channels that allow a passive transport of molecules down their concentration gradients [40, 41]. This transport through the outer membrane does not require energy in contrast to the uphill substrate translocation via transporters in the inner membrane.

Most of the porins described to date form β barrels composed of antiparallel β-sheets. Frequently, they are associated in oligomers that confer a higher stability to the whole complex (Fig. 2). In the outer membrane of Gram negative bacteria, channel-tunnel proteins like TolC in *Escherichia* and BesC in *Borrelia* are also found. They form pores in the outer membrane and are part of bigger protein complexes involved in drug-resistance known as efflux pumps (Fig.2).

Porins can be classified in two groups depending on whether they are selective for a class of molecules or not [42]:

General diffusion porins allow the transport of small molecules, ions and water through a membrane. The permeability of general diffusion pores for substrates is dependent only on their size and charge. Examples for this kind of porins are OmpF and OmpC of *E. coli* [40].

Substrate-specific porins represent pores or channels that are specialized for the transport of certain molecules. These molecules bind to the channel interior and are guided to the periplasm. Tsx is an example of a substrate specific porin in *E. coli* that is specialized in the nucleoside transport from the surrounding media to the periplasmic space (Fig. 2) [43, 44]. Other examples include porins with specificity for carbohydrates [45], phosphate [46] and antibiotics [47].

Porins in the Genus *Borrelia* 143

P66 exhibits a dual function. P66 is a well-studied pore-forming outer membrane protein present in Lyme disease and relapsing fever *Borreliae* [49-55]. Interestingly, P66 exhibits dual functions. It was shown to act as an adhesin which can bind to β(1)- and β(3)-chain integrins [51-53]. And strikingly, it acts as a porin in the outer membrane with the capability of forming pores with an extremely high single-channel conductance. This could be demonstrated by studies in planar lipid membranes [50, 54, 55]. In addition, P66 is also of special interest because of the presence of surface-exposed loops [49, 56] exhibiting a certain immunogenic potential [57], a property of interest in the search of vaccine candidates

P66 is present in both Lyme disease and relapsing fever agents. Comparison of the deduced P66 amino acid sequences from published genomes of the Lyme disease species *B. burgdorferi* B31, *B. afzelii* pKo, *B. garinii* PBi and the relapsing fever species *B. duttonii* Ly, *B. recurrentis* A1 and *B. hermsii* HS1 revealed 41% inter-species identity with partially highly conserved domains. The three LD species' P66 share the high sequence identity of 90%. P66 has according to its name a molecular weight of 66 kDa as can be seen after SDS-PAGE of purified P66.

P66 exhibits an unusually high single channel conductance. The pore-forming properties of P66 channels from the LD species *B. burgdorferi*, *B. afzelii* and *B. garinii* have been studied in detail by different groups [50, 54, 55]. P66 is able to form pores in planar lipid bilayers with the enormously high single-channel conductance of 11 nS in 1 M KCl [55]. Thereby, the conductance for P66 of the LD species *B. burgdorferi*, *B. afzelii*, *B. garinii* in 1 M KCl were all similarly high, in a range between 9 and 11 nS [55]. Measurements in different KCl concentrations from 0.1 to 3 M revealed that the single channel conductance was approximately a linear function of the electrolyte concentration which indicates a lack of

The P66 channels are nonselective for small anions and cations [50, 55]. This was demonstrated by single-channel experiments with salts containing cations and anions of different size such as LiCl and KCH3COO and by the analysis of the membrane potential using the Goldman-Hodgin-Katz equation [58]. P66 channels are permeable for both anions and cations, because the calculated values of the permeability ratio of cations over anions (Pc/Pa) in KCl were in the range between 0.8 and 1.0 for all measured P66 homologues [55].

P66 channels exhibit voltage-dependent closure. This was demonstrated in multi-channel experiments. *B. burgdorferi* P66 started to show voltage-dependent closure at potentials as high as + 70 and -60 mV reaching a maximal conductance decrease of approximately 20%. P66 homologues of different LD species all showed similar voltage-dependent behavior with slight species-specific differences of the potential for the start of conductance decrease. For example, for P66 of *B. afzelii* and *B. garinii*, the voltage-dependent closure started at potentials as high as +/- 40 mV and the conductance decreased by approximately 30% at

The pore diameter of P66 is small related to the huge single-channel conductance. The above mentioned outstanding high single-channel conductance of 11 nS (in 1 M KCl) is atypical and rare for Gram-negative bacterial porins. However, certain spirochetal porins exhibit also an extremely high single-channel conductance, such as the one of the major outer membrane

Values close to one indicate equal pore permeability for anions and cations.

applied potentials of +/-80 mV and higher [55].

**2.1 P66: A porin with an unusual high single channel conductance**

against European LD *Borreliae*.

binding sites for ions.

Fig. 2. Bacterial outer-membrane pore-forming proteins. Diffusion channels have usually a β-barrel tertiary structure with longer loops to the outside and short turns towards the periplasmic space. In *E. coli*, Tsx is a substrate specific porin for nucleosides while OmpC is a general diffusion porin. Usually porins form trimers like OmpC although other associations are also possible like in the case of Tsx. TolC is a channel-tunnel that together with an inner membrane transport system and adaptor proteins forms an efflux pump for export of drugs and other substances. These channel-tunnels are also formed by trimers but they only form a single pore.

#### **2. Porins in the genus** *Borrelia*

*Borrelia* species possess a small genome composed of a small linear chromosome complemented with a set of up to 21 linear and circular plasmids [48]. Many of their genes are related to bacterial virulence and the adaptation of bacteria to the different hosts and vectors. The absence of genes in *Borrelia* involved in the biosynthesis of amino acids, fatty acids or nucleotides is very remarkable. This metabolic deficiency makes *Borrelia* species strictly dependent on substances provided by their host. Therefore, the role of the *Borrelia* porins in the acquisition of nutrients is of extreme importance.

Up to now, five porins and a channel-tunnel as part of a multi-drug efflux system have been described in the outer membrane of different *Borreliae*. P66, P13, BBA01 (a P13 paralogue) and BBA74 have been described in *Borrelia burgdorferi*. Oms38 has been described in relapsing fever species, and possess a homologue in *Borrelia burgdorferi* designated DipA. Another outer membrane pore-forming protein is BesC. BesC is a channel-tunnel which is part of a bigger complex spanning the whole cell envelope which is involved in secretion of toxic compounds for *Borrelia*, like for example antibiotics.

Fig. 2. Bacterial outer-membrane pore-forming proteins. Diffusion channels have usually a β-barrel tertiary structure with longer loops to the outside and short turns towards the periplasmic space. In *E. coli*, Tsx is a substrate specific porin for nucleosides while OmpC is

associations are also possible like in the case of Tsx. TolC is a channel-tunnel that together with an inner membrane transport system and adaptor proteins forms an efflux pump for export of drugs and other substances. These channel-tunnels are also formed by trimers but

*Borrelia* species possess a small genome composed of a small linear chromosome complemented with a set of up to 21 linear and circular plasmids [48]. Many of their genes are related to bacterial virulence and the adaptation of bacteria to the different hosts and vectors. The absence of genes in *Borrelia* involved in the biosynthesis of amino acids, fatty acids or nucleotides is very remarkable. This metabolic deficiency makes *Borrelia* species strictly dependent on substances provided by their host. Therefore, the role of the *Borrelia*

Up to now, five porins and a channel-tunnel as part of a multi-drug efflux system have been described in the outer membrane of different *Borreliae*. P66, P13, BBA01 (a P13 paralogue) and BBA74 have been described in *Borrelia burgdorferi*. Oms38 has been described in relapsing fever species, and possess a homologue in *Borrelia burgdorferi* designated DipA. Another outer membrane pore-forming protein is BesC. BesC is a channel-tunnel which is part of a bigger complex spanning the whole cell envelope which is involved in secretion of

a general diffusion porin. Usually porins form trimers like OmpC although other

porins in the acquisition of nutrients is of extreme importance.

toxic compounds for *Borrelia*, like for example antibiotics.

they only form a single pore.

**2. Porins in the genus** *Borrelia*

#### **2.1 P66: A porin with an unusual high single channel conductance**

P66 exhibits a dual function. P66 is a well-studied pore-forming outer membrane protein present in Lyme disease and relapsing fever *Borreliae* [49-55]. Interestingly, P66 exhibits dual functions. It was shown to act as an adhesin which can bind to β(1)- and β(3)-chain integrins [51-53]. And strikingly, it acts as a porin in the outer membrane with the capability of forming pores with an extremely high single-channel conductance. This could be demonstrated by studies in planar lipid membranes [50, 54, 55]. In addition, P66 is also of special interest because of the presence of surface-exposed loops [49, 56] exhibiting a certain immunogenic potential [57], a property of interest in the search of vaccine candidates against European LD *Borreliae*.

P66 is present in both Lyme disease and relapsing fever agents. Comparison of the deduced P66 amino acid sequences from published genomes of the Lyme disease species *B. burgdorferi* B31, *B. afzelii* pKo, *B. garinii* PBi and the relapsing fever species *B. duttonii* Ly, *B. recurrentis* A1 and *B. hermsii* HS1 revealed 41% inter-species identity with partially highly conserved domains. The three LD species' P66 share the high sequence identity of 90%. P66 has according to its name a molecular weight of 66 kDa as can be seen after SDS-PAGE of purified P66.