Novel Algorithms Reveal Streptococcal Transcriptomes and Clues about Undefined Genes
[['Microarray technology is now commonly used to reveal genome-wide transcriptional changes in bacterial pathogens during interactions with the host .', 'Several factors , however , limit the power of such analyses , including inadequate statistical analysis and insufficient sample replication , both of which do not account for experimental variability , and often result in arbitrary thresholds for significance [1 , 2] .', 'In addition , unknown bacterial genes can confound the interpretation of expression profiles , restricting many microarray studies to the differential expression of well-characterized genes .', 'Several methods are available to organize gene expression profiles and to assist in extracting functional or regulatory gene information from microarray datasets .', 'Clustering algorithms group genes by similarities in expression patterns , based on the assumption that co-expressed genes share common function or regulation [3 , 4]; however , clustering solely by co-expression patterns may not reveal a considerable amount of information contained in array data .', 'These methods often: ( 1 ) produce unreliable data by missing known gene members of biological pathways; ( 2 ) fail to distinguish truly related gene clusters from coincidental groupings; and ( 3 ) identify clusters containing only unknown genes that may lack either common function or regulation , a considerable limitation for genomes containing a large percentage of undefined genes [1 , 2] .', 'Because no tools exist to interpret unknown gene clusters or to assess their significance and completeness , a significant portion of bacterial expression profiles are not interpretable using current clustering methods .', 'We introduce neighbor clustering as a new tool for analyzing bacterial microarray data that addresses some of these limitations by incorporating the physical position of genes on the bacterial chromosome into the analysis of expression data .', 'Information about gene function and regulation is stored intrinsically in the bacterial genome structure , as genes with common function or regulation tend to be physically proximate on the chromosome and often linked as operons [5 , 6] .', 'We incorporated these positional data into a series of neighbor clustering algorithms , named GenomeCrawler , that identifies groupings of potentially related genes from array data by combining two informative characteristics of bacterial genes that share common function or regulation [3–6]: ( 1 ) similar gene expression profiles ( i . e . , co-expression ) ; and ( 2 ) physical proximity of genes on the chromosome .', 'The algorithms also recalculate the statistical significance of each gene as a member of a particular cluster , as well as the significance of each resulting grouping as a whole , to ensure accuracy of cluster assignments .', 'This process ultimately identifies significant clusters of co-expressed gene neighbors that likely share common function or regulation .', 'We used this approach to analyze microarray expression data from group A streptococci ( Streptococcus pyogenes ) during adherence to human pharyngeal cells , the first overt infection step [7] .', 'The ability of all bacterial pathogens to infect the human host depends upon coordinated regulation of diverse gene sets that are required for survival in host environments .', 'Although recent microarray studies have highlighted the molecular responses of streptococci in relevant host conditions [8–10] , characterizing differentially expressed loci during pharyngeal cell adherence is critical for understanding the molecular basis for host colonization .', 'Studies from our laboratory [11 , 12] and others [13] have demonstrated that in vitro association with pharyngeal cells results in streptococcal phage induction and the increased expression of phage-encoded virulence factors .', 'Although the mechanisms mediating these responses are not known , the results of these studies indicate that streptococci sense and , on a transcriptional level , respond to various signals and cues in the pharyngeal cell environment .', 'We undertook the present study to understand and to assess more accurately the genome-wide transcriptional responses of streptococci during one of the earliest recognized stages of infection , namely adherence to human pharyngeal cells .', 'We compared data generated before and after neighbor clustering to show that this method provides a more comprehensive view of transcription by: ( 1 ) identifying more differentially expressed genes than even traditional , rigorous statistical analyses; ( 2 ) reconstructing intact biological pathways that statistical significance analysis could not reconstruct; and ( 3 ) providing preliminary insight and clues about the function or regulation of uncharacterized genes by associating their co-expression with physically proximate , functionally defined genes .'], ['We developed spotted oligonucleotide arrays of the S . pyogenes SF370 ( an M1 serotype ) genome [14] and compared the transcriptomes of streptococci that adhere to Detroit 562 human pharyngeal cells to non-adherent ( “associated” ) streptococci within the same experiment .', 'Adherence assays were performed as described [15] with modifications to minimize eukaryotic cell disruption .', 'We replicated experiments independently and used dye-swaps to incorporate biological and technical variation [16 , 17] .', 'Following filtering and normalization [18 , 19] , we analyzed data from four biological replicates [16] with robust summary statistics [20] , Bayesian statistics [21 , 22] , and permutation algorithms [19] to identify genes differentially expressed with significance during pharyngeal cell adherence .', 'This analysis identified 79 genes ( 4% of the genome ) exhibiting statistically significant fold changes in expression ( PF value < 0 . 05 ) during adherence from 1 , 769 open reading frames represented on the array ( Table 1 ) .', 'We refer to such genes as “differentially expressed . ”', 'We present the entire dataset from all experiments as Table S1 .', 'Genes demonstrating upregulation ( n = 45 ) and downregulation ( n = 34 ) included virulence factors , prophage-encoded transcripts , metabolic genes , and transcriptional regulators ( Table 2 ) .', 'Undefined or hypothetical genes comprised 27% of differentially expressed genes ( n = 21; 11 chromosomally encoded genes , ten phage-encoded genes ) .', 'We conducted TaqMan ( qRT-PCR ) analysis [23] of 11 differentially expressed genes to validate selected microarray hybridization results ( see Table S2 for genes and primer–probe sequences ) .', 'Five genes chosen for validation demonstrated statistically significant fold changes in expression by microarray analysis ( PF value < 0 . 05; two upregulated , three downregulated ) .', 'The remaining six genes ( four upregulated , two downregulated ) did not have significant PF values , but were statistically significant as members of particular neighbor clusters in subsequent analyses ( PE < 0 . 05 ) as detailed in later sections ) .', 'We averaged the data to generate a value for each gene , creating a set of 11 paired values from quantitative real-time ( qRT ) -PCR and microarray analyses ( Table S3 ) .', 'Results of standard linear regression analysis demonstrated a strong positive correlation ( r = 0 . 9 ) between data obtained using the different techniques ( see Figure S1 ) .', 'Streptococci elaborate several factors implicated in infection , including surface-exposed adhesins and secreted toxigenic proteins ( reviewed in [7 , 14 , 24] ) .', 'The initial statistical analysis identified four differentially expressed virulence genes ( Tables 1 and 2 ) .', 'Genes encoding streptolysin O ( slo or spy0167 ) and the SpeB protease ( spy2039 ) were downregulated , while genes encoding pyrogenic exotoxin H ( speH or spy1008 ) and a putative fibronectin-binding protein ( spy0130 ) were upregulated .', 'We verified the differential expression of spy2039 and spy0130 by qRT-PCR .', 'The downregulation of virulence loci during presumably inappropriate stages of infection was not surprising .', 'Streptolysin O is a cytotoxin that damages human tissue and increases host cell cytotoxicity [7 , 25] .', 'The resulting cellular damage , particularly to polymorphonuclear leukocytes [26] , decreases internalization and subsequent intracellular killing of streptococci [27] .', 'Based on its downregulation during adherence , we infer that slo was transcribed during pre-adherence associations , perhaps , as previously reported , to protect streptococci from phagocytic killing in vivo [27] .', 'However , once adhered , our data suggest that streptococci downregulate production of this cytotoxin , presumably to prevent further host tissue destruction that could interfere with adherence .', 'SpeB ( encoded by spy2039 ) is a multifunctional cysteine protease implicated in numerous infection strategies [28 , 29] .', 'Although few studies have examined gene expression patterns during adherence , SpeB production ( as detected by Western blot analysis ) decreases during co-culture with human peripheral blood mononuclear cells [30] and in a mouse infection model [31] .', 'When SpeB expression is limited , several streptococcal proteins necessary for adherence remain intact [24 , 32 , 33]; thus , decreased SpeB production ( as indicated here ) may promote pharyngeal cell attachment .', 'Furthermore , SpeB abolishes internalization ( following adherence ) of certain streptococcal strains by epithelial cells ( including Detroit 562 cells ) , a process mediated in part by the fibronectin-binding protein F [34 , 35] .', 'We observed significant upregulation of the gene spy0130 , encoding a protein recently found to be associated with the production of surface-exposed pili on strain SF370 [36] .', 'The protein shares 60% sequence similarity to protein F , suggesting that it may coordinate a similar internalization mechanism or may be involved directly in adherence ( discussed later in detail ) .', 'SpeB downregulation also coincides with increased expression of pyrogenic exotoxins [33 , 37] that reportedly increase streptococcal survival in vivo .', 'We observed that the exotoxin-encoding speH gene [38] was upregulated .', 'Taken together , our results agree with previous reports on SpeB production during host cell interactions , suggesting that decreased expression may promote streptococcal adherence ( by preventing proteolytic degradation of key virulence factors or adhesins ) , enhance internalization ( perhaps through a fibronectin-mediated pathway ) , and increase survival ( through increased pyrogenic exotoxin production , discussed below ) .', 'SF370 contains one inducible prophage ( 370 . 1 ) and three defective prophages ( 370 . 2 , 370 . 3 , and 370 . 4 ) that produce no infectious phage [39] .', 'We identified 11 differentially expressed phage 370 . 2 genes , suggesting that this defective phage is not transcriptionally silent ( Table 1 ) .', 'The speH gene ( spy1008 ) was induced , and the remaining genes , hypothetically involved in replication and regulation [39] , were downregulated .', 'The speH gene encodes a mitogenic exotoxin [38] reportedly induced during polymorphonuclear leukocyte phagocytosis [8] but not implicated previously in adherence .', 'Increased expression of speH during pharyngeal cell adherence suggests that the SpeH exotoxin is either necessary for adherence , or is a component of a downstream infection process .', 'Adherence-mediated upregulation of speH is likely not the result of phage induction , as the remaining phage 370 . 2 genes identified in our analysis were downregulated .', 'To determine if SpeH plays a direct role in the adherence process , we created a deletion mutant in strain SF370 ( SF370ΔspeH ) , which was confirmed by PCR ( unpublished data ) and RT-PCR ( Figure 1A ) and tested in vitro for adherence to human pharyngeal cells .', 'We observed no significant difference in adherence between the wild-type ( SF370 ) and mutant strains ( Figure 1B ) , indicating that SpeH is not involved directly in attachment to the pharyngeal cell .', 'The significant upregulation of the speH gene during adherence suggests that the gene product may function instead during a subsequent stage of infection .', 'We identified a number of genes encoding proteins involved in housekeeping processes ( such as carbohydrate and coenzyme metabolism ) that were differentially expressed , indicating a shift in metabolic processes due to host cell adherence ( Tables 1 and 2 ) .', 'For example , genes encoding proteins involved in folate biosynthesis [40] were upregulated , suggesting that certain cofactors that may be necessary during adherence were unavailable .', 'Also upregulated were genes encoding subunits of the F0F1 ATPase [41] ( discussed in more detail later ) , which may indicate an acid stress response to maintain cytoplasmic pH or a need to generate ATP in response to increased energy requirements .', 'We also identified the adherence-mediated upregulation of four transcriptional regulators ( Table 1 ) , suggestive of an adaptive response to host cell contact that is dynamic and complex .', 'For example , RopB ( encoded by spy2042 ) , a member of the Rgg family of response regulators , interacts with a number of regulatory networks throughout the streptococcal genome ( e . g . , mga , csrRS , sagA , and fasBCA ) , affecting the transcription of numerous proteins , virulence factors , and two-component regulatory systems [42 , 43] .', 'Although the delineation of genes influenced by RopB ( or any identified transcriptional regulator ) is beyond the scope of this study , our initial analysis did identify the upregulation of a two-component regulatory system , encoded by spy1236–1237 .', 'The functions of these particular loci are not yet known , and their adherence-mediated upregulation represents new targets in the study of regulators that function during host cell contact .', 'Our initial analysis revealed the differential expression of a wide range of functionally diverse genes and provided insight into the adaptive response of streptococci to host cell contact .', 'However , despite a rigorous statistical approach , this analysis , like many previous microarray studies , identified the differential expression of a large number of unknown genes ( n = 21 ) and a number of incomplete biological pathways ( e . g . , F0F1 ATPase [41] and folate biosynthesis [40] ) by failing to detect the differential expression of a number of known gene pathway members ( Table 1 ) .', 'To overcome these limitations and to extract more functional information from the array dataset ( including more complete biological pathways ) , we developed the neighbor clustering algorithms to combine the physical position of genes on the streptococcal chromosome with gene expression data .', 'Neighbor clustering was designed to identify expanded groupings of potentially related genes from our array data by incorporating two reliable predictors of genes that share common function or regulation , namely physical proximity and similar expression profiles [5 , 6] .', 'We implemented this approach by developing an algorithm with dynamic windowing ( GenomeCrawler ) that sequentially stepped through the microarray data and identified clusters of adjacent genes exhibiting similar fold changes in expression .', "Because the genome contains many possible clusters , we restricted the algorithm's search space to identify only spatially related clusters .", "GenomeCrawler applied a separate permutation algorithm , using the sum of each gene's t-statistics to calculate adjusted P values ( PK ) for each cluster , which corresponded to the probability of assembling a cluster by chance .", 'Significance was assigned to clusters with PK < 0 . 05 , and the resulting groupings are listed in Table 3 .', 'Because individual genes could be members of many different significant clusters , GenomeCrawler then applied a distinct permutation algorithm to calculate the probability ( PC ) that a gene was clustered coincidentally .', "Calculation of PC values relies on Bayes' Theorem , in which the probability of a gene's log2-fold change ( PF value ) is combined with the cluster probability itself ( PK value ) .", 'We stress that PC reflects the significance of a gene based on its cluster context rather than a recapitulation of PF .', 'This ensures a strong dependency between PF and PC , preventing a gene with a relatively low log2-fold change from being scored as significant simply because it is clustered with a gene with a highly significant PF value .', 'Finally , GenomeCrawler calculated the overall significance of differentially expressed genes ( PE values ) by integrating differential expression probabilities ( PF ) and cluster context probabilities ( PC ) .', 'We developed a plotting application ( GenomeSpyer ) that represents the chromosome as a linear molecule to visualize GenomeCrawler output , with genes displayed on the x-axis and their log2-fold change magnitudes on the y-axis .', 'Applications and all datasets are available for download at http://www . rockefeller . edu/vaf/streparray . php .', 'We visually inspected the resulting clusters and disqualified those that violated our neighbor cluster definition ( see Methods for details ) .', 'All output prior to cluster disqualifications is included for comparison ( see Table S4 ) .', 'Of the 309 qualifying clusters ( Table S5 ) , 197 ( 63 . 8% ) were composed entirely of known , functionally defined genes; however , 26 ( 13% ) of these were incorrectly assembled , as they contained known genes that are functionally unrelated .', 'Because we did not incorporate functional annotations of genes into the algorithms ( i . e . , to keep the analysis “blind” ) , we anticipated the possibility that some groupings could be assembled incorrectly despite the statistical framework for assigning clusters .', 'Of the remaining 283 ( 91 . 6% ) groupings , a number of differently sized clusters contained the same gene ( Table S5 ) .', 'We report such clusters first by highest significance ( lowest PK value ) , then by largest number of genes .', 'Thus , if clusters containing a particular gene were of equal significance , we report the cluster with the most gene members .', 'This method identified 47 significant clusters containing 173 differentially expressed genes ( listed in Table 3 and visualized in Figures 2 and S2–S4 ) , a considerably larger group than could have been compiled using only the initial 79 significant genes .', 'A total of 56 of the original 79 significant genes became components of significant clusters , whereas 23 remained unclustered .', 'We subdivided all clusters into three qualitative types based on the functional annotation of gene members .', 'We present examples of Type I and II clusters: Type I clusters ( n = 25 ) contained only functionally defined and functionally related genes ( as reported in published studies ) , such as biological pathways components ( Figures 2B and S2 ) ; Type II clusters ( n = 20 ) included both known and unknown genes ( Figures 2C and S3 ) .', 'Type III clusters ( n = 2 ) were composed entirely of unknown genes ( Figures 2D and S4 ) , and are not discussed in detail .', 'We measured the performance of our algorithm by examining whether it identified gene groupings known to be functionally related ( Type I clusters ) .', 'Only four ( 16% ) of 25 Type I clusters ( spy0080–0081 , spy1236–1237 , spy1707–1711 , spy2041–2042 ) could have been identified in entirety by significance analysis because all clustered genes exhibited significant differential expression ( PF value < 0 . 05 ) .', 'A total of 11 ( 52 . 4% ) of the remaining 21 clusters would not have been identified in their entirety without GenomeCrawler because we initially identified significant fold-changes in only a subset of genes necessary to encode particular pathways or loci; this is intuitively unreasonable if all genes are essential for functionality .', 'GenomeCrawler expanded these clusters to contain more genes that encode intact loci ( Table 3 ) .', 'For example , we initially identified ( Table', '1 ) the significant upregulation of three of the five known gene members of the folate biosynthetic pathway [40] ( spy1096–1100 ) , but GenomeCrawler identified a significant cluster containing all five genes ( Table 3 and Figure 2B ) .', 'We obtained a similar result for the eight-gene operon encoding the F0F1-type proton translocating ATPase [41] ( spy0754–0761 ) .', 'The initial significance analysis identified only four atp genes ( Table 1 ) , but neighbor clustering identified a significant cluster containing all eight genes necessary to encode a functional ATPase ( Table 3 ) .', 'Each of the 11 neighbor clusters that could have been only partially identified by our initial analysis alone gained gene members after application of the algorithms and became more complete sets of functionally related genes than initially identified ( Table 3 ) .', 'These clusters encompass various metabolic processes , including purine biosynthesis ( spy0025–0028 ) , lactose metabolism ( spy1916–1923 ) , fatty acid biosynthesis ( spy1743–1747 ) , lipoteichoic acid synthesis ( spy1308–1312 ) , and sugar phosphotransferase transport ( spy1058–1060 ) [14] , suggesting that specific changes occur in the streptococcal metabolic program as the bacteria adhere to human pharyngeal cells in vitro .', 'Notably , the remaining ten Type I clusters were composed entirely of genes that individually were not significant; however , after applying our algorithms , the combined contribution of each gene resulted in a significant cluster .', 'For example , the nine-gene operon that spans genes spy0738–0746 encodes streptolysin S , a potent cytolytic toxin that promotes internalization and host tissue dissemination [25 , 44] .', 'Though the differential expression of the individual genes was not significant following our initial statistical analysis , GenomeCrawler identified a significant downregulated cluster containing all nine genes ( Table 3 ) .', 'Adherence-induced downregulation of streptolysin S is consistent with its previously determined role in host cell internalization [25]; however , without neighbor clustering , expression of this operon was not evident immediately .', 'Although individual gene members of Type I clusters may not be statistically significant as a result of technical variability within experiments [17] , the genetic structure of certain Type I operons may provide an alternative explanation .', 'For example , the streptolysin operon encodes an internal terminator downstream of the sagA gene ( the first gene in the operon ) , which modulates the abundance of particular mRNA species ( e . g . , sagA mRNA versus the polycistronic message for all nine genes ) under different environmental conditions [45] .', 'If transcription is internally disrupted by such a terminator , the abundance of the sagA transcript may be much greater than the polycistronic message; such disproportionate transcript levels would affect log2-fold change values and impact the statistical significance of individual genes within these types of clusters .', 'Thus , in addition to helping resolve clusters that would not be easily recognized because of experimental technical variability , the neighbor clustering method may help to resolve operons with such internal terminators and regulators .', 'These results demonstrate that neighbor clustering effectively reconstructed a number of complete pathways and loci from processed array data .', 'Importantly , because functional gene data are not incorporated into its algorithms , GenomeCrawler is not biased toward identifying “expected” clusters .', 'Curating the dataset following its application may make the algorithms less user-friendly; however , the elimination of such bias is essential for this type of analysis .', 'Based on the Type I cluster results , we speculated that genes contained in Type II clusters might be related by function or regulation .', 'Type II groupings contain a combination of both known and unknown gene members and could provide preliminary clues about the function of unknown genes within a particular cluster by associating their expression with neighboring genes of known and defined function .', 'Alternatively , co-expression of genes results from common regulation , and Type II associations may suggest shared regulatory mechanisms for clustered genes .', 'We note , however , that despite the statistical framework with which groupings are assigned , experimental evidence is necessary to confirm functional or regulatory relatedness .', 'We do not suggest simply assigning either based on cluster membership; rather , cluster associations may provide some preliminary functional or regulatory clues for gene members .', 'A total of 18 ( 90% ) of 20 Type II clusters ( Table 3 and Figure S3 ) may not have been identified without neighbor clustering: eight ( 44 . 4% ) of 18 gained additional gene members; the remaining ten comprised genes that demonstrated significant differential expression only after applying GenomeCrawler .', 'Only two clusters ( spy0127–0130 and spy1701–1704 ) could have been identified without neighbor clustering; however , a number of these genes were initially annotated as hypothetical proteins , so a potential relationship between the gene members may not have been readily apparent .', 'The upregulated spy0127–0130 cluster is part of a larger genomic region known as FCT ( for fibronectin- and collagen-binding proteins and T antigen–encoding loci ) , which spans spy0123–0136 in the SF370 genome and encodes surface proteins and transcriptional regulators [46] .', 'A search of both the PFAM database [47] ( http://pfam . wustl . edu ) and sortase database ( http://www . doe-mbi . ucla . edu/Services/Sortase ) predicted that spy0129 encodes a sortase enzyme , which are transpeptidases that cleave protein substrates at conserved C-terminal motifs ( often LPXTG ) and then anchor these proteins to the bacterial cell wall [48 , 49] .', 'Recently , it was reported that the four genes spanning spy0127–0130 encode , and are responsible for , the formation of surface-localized , trypsin-resistant pili that induce protective immunity against a lethal dose of group A streptococci in a mouse model of infection [36] .', 'This same report provided the first experimental evidence supporting the sortase prediction , indicating that the gene product of spy0129 is responsible for the cell-wall sorting of the proteins encoded by both spy0128 ( annotated as a Cpa homolog [50] ) and spy0130 ( annotated as a protein F homolog [14] ) .', 'Furthermore , the spy0128-encoded protein is the structural backbone of the pili , and the gene product of spy0130 may be involved in stabilizing the structure [36] .', 'Together with the identification of this cluster by GenomeCrawler , these results prompted us to study this cluster and the contributions of the gene products to pharyngeal cell adherence .', 'We determined experimentally that cluster spy0127–0130 is an operon , verifying both related function and regulation of the gene members .', 'Reverse transcription of SF370 RNA , with primer combinations that spanned all four genes , produced cDNA fragments of sizes that could only result from a polycistronic mRNA template ( Figure 3 ) .', 'In silico sequence inspection identified a single putative promoter sequence upstream of spy0127 ( see Table S6 ) .', 'Although GenomeCrawler is not an operon-identifying algorithm , these results show that it could ( 1 ) identify this commonly regulated gene cluster and ( 2 ) define the cluster boundaries , excluding other proximate genes , such as an additional sortase-encoding gene , spy0135 .', 'We created a spy0129 deletion mutant in strain SF370 ( SF370Δspy0129 ) to determine if genes contained within the spy0127–0130 cluster were directly involved in adherence to pharyngeal cells .', 'We posited that a deletion in the spy0129 sortase gene may have the greatest overall effect on the production and processing of the gene products of this cluster , since both the spy0128 and spy0130 gene products do not localize to the cell-wall surface in the absence of the sortase enzyme [36] .', 'Allelic replacement created two putative deletion mutants; however , RT-PCR analysis ( Figure 4A ) revealed that only one such clone ( SF370Δspy0129 . 2 ) was a true knock-out for the spy0129 gene and useful for further study .', 'Because the gene cluster is also an operon , expression of the downstream gene spy0130 , encoding the protein F homolog/pilus protein , was also eliminated in this mutant ( Figure 4A ) .', "In vitro pharyngeal cell adherence assays revealed that the SF370Δspy0129 . 2 mutant was approximately 66% less adherent than the parental control strain , SF370 ( Figure 4B; p = 0 . 03 as determined by the Student's t-test ) .", 'These results suggest that either the spy0130 gene product is involved directly in adherence , or that due to the elimination of the sortase , the pili , which may function in their entirety as adhesins , were not assembled on the surface of the mutant .', 'Because the spy0129 gene product is not expected to be found on the streptococcal surface ( i . e . , it lacks a cell-wall anchoring motif ) , it is not likely to be involved directly in adherence .', 'We are working to produce an in-frame deletion of spy0128 and a spy0130 single knock-out mutant to delineate the contribution of each individual clustered gene product to adherence .', 'These results show that neighbor clustering is able to identify biologically relevant gene clusters .', 'This attribute may be particularly important for datasets in which the relationship between clustered genes is not obvious , and may facilitate the organization of larger datasets into more manageable packages .', 'Another cluster , spy1725–1719 , contained six genes that together ( though not individually ) exhibited significant downregulation .', 'The genes spy1724 , spy1722 , spy1721 , and spy1719 share transcriptional order and predicted function with homologs in the nusA-infB protein biosynthesis operon of Bacillus subtilis and Escherichia coli [51] .', 'We examined the spy1725 and spy1723 gene products ( annotated as hypothetical proteins [14] ) for similarities with known proteins that might indicate a role for these gene products in protein biosynthesis .', 'BlastP analysis aligned the spy1725 gene product , which has homologs in all sequenced streptococcal genomes , with the SP14 . 3 protein from S . pneumoniae [52] ( 80% sequence similarity; 67% identity ) .', 'Based on structural characterization , SP14 . 3 is a predicted RNA-binding protein .', 'The spy1723 gene product has similar domain structure to the YlxR protein of S . pneumoniae , an RNA-binding protein implicated in transcription termination [53] .', 'These results indicate that both genes likely encode RNA-binding proteins , in agreement with their functionally defined cluster members .', 'Although domain and homology searches yielded the functional predictions , their membership within a protein biosynthetic cluster provided the initial indication of common function or regulation .', 'Although neighbor clustering is not an operon-predicting method , we wanted to identify additional putative operons among the groupings since neighbor clusters by definition share certain operon characteristics ( tandemly arranged genes , separated by <300 bp , with similar expression patterns ) .', 'Although operon-modeling methods exist [54 , 55] , we inspected clusters in silico for upstream regulatory elements and identified 17 candidates , including clusters such as streptolysin S that have been previously confirmed as operons [56]; the spy0127–0130 grouping , which was confirmed as an operon in this study; and others that have yet to be verified ( Table S6 ) .', 'Experimental confirmation of each candidate is beyond the scope of this study , but Northern blot and RT-PCR analyses could provide such information .', 'We applied the statistical analysis and the GenomeCrawler algorithms to data from a recently published streptococcal microarray study that is relevant for comparison to our own data ( same streptococcal strain , similar array platform ) [57] .', 'In this study , the transciptomes of S . pyogenes strain SF370 and an isogenic mutant deficient for the Mga regulon were compared during exponential growth in culture broth .', 'The Mga
['Introduction', 'Results/Discussion', 'Methods', 'Supporting Information']
['Bacteria–host interactions are dynamic processes , and understanding transcriptional responses that directly or indirectly regulate the expression of genes involved in initial infection stages would illuminate the molecular events that result in host colonization .', 'We used oligonucleotide microarrays to monitor ( in vitro ) differential gene expression in group A streptococci during pharyngeal cell adherence , the first overt infection stage .', 'We present neighbor clustering , a new computational method for further analyzing bacterial microarray data that combines two informative characteristics of bacterial genes that share common function or regulation: ( 1 ) similar gene expression profiles ( i . e . , co-expression ) ; and ( 2 ) physical proximity of genes on the chromosome .', 'This method identifies statistically significant clusters of co-expressed gene neighbors that potentially share common function or regulation by coupling statistically analyzed gene expression profiles with the chromosomal position of genes .', 'We applied this method to our own data and to those of others , and we show that it identified a greater number of differentially expressed genes , facilitating the reconstruction of more multimeric proteins and complete metabolic pathways than would have been possible without its application .', 'We assessed the biological significance of two identified genes by assaying deletion mutants for adherence in vitro and show that neighbor clustering indeed provides biologically relevant data .', 'Neighbor clustering provides a more comprehensive view of the molecular responses of streptococci during pharyngeal cell adherence .']
['Microarray technology is commonly used to reveal genome-wide transcriptional changes in bacterial pathogens during interactions with the host .', 'Clustering algorithms , which group genes with similar expression patterns , facilitate microarray data organization and are based on assumptions that co-expressed genes share common function or regulation; however , clustering solely by co-expression may not reveal all of the information contained in bacterial array data .', 'We introduce neighbor clustering , a new tool for analyzing bacterial gene expression profiles , which distinguishes itself from other programs by incorporating details unique to the architecture of bacterial chromosomes into the analysis .', 'Neighbor clustering combines two informative characteristics of bacterial genes that share common function or regulation— ( 1 ) similar expression profiles and ( 2 ) physical proximity on the chromosome—and extracts statistically significant clusters of gene neighbors that are potentially related by function or regulation .', 'We present the analysis of microarray data from group A streptococci during adherence to human pharyngeal cells , the first overt infection step .', 'We show that neighbor clustering identifies more differentially expressed genes than rigorous statistical analyses alone , and can provide functional clues about unknown genes .', 'We extended the analysis to include a previously published streptococcal array study to demonstrate the applicability of the method .']
['genetics and genomics', 'eubacteria', 'microbiology', 'computational biology']