Ampicillin concentrations varied from 5 μg mL-1 to 4500 μg mL-1

Ampicillin concentrations varied from 5 μg mL-1 to 4500 μg mL-1. Test of XylS expression levels using a synthetic operon and luciferase assay XylS amounts could be measured more directly Selleck RG7204 via luciferase activity in all constructs based on

pFS7. Luciferase activity was measured using the Luciferase Assay System from Promega, according to the manufacturer’s protocol. The luminometer used was a GloMax 20/20 (Promega). Strains were grown as described above. RNA isolation, cDNA synthesis and qRT-PCR Transcript amounts were determined by two-step quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR). RNAqueous (Ambion) was used for total RNA isolation. Isolated RNA was treated with Turbo DNAse (Ambion) and reverse transcription was performed using a first-strand cDNA synthesis kit with random pd(N)6 primers (Amersham Biosciences). PCR was carried out in the presence of Power SYBR Green PCR Master Mix (Applied Biosystems) using a 7500 Real Time PCR system (Applied Biosystems).

During PCR samples were heated to 95°C for 10 min, followed by 40 cycles of amplification (95°C for 15 s; 60°C for 1 min). Results were analysed by 7500 system selleck products software v1.3 using the 2-∆∆CT method [39]. Primers were designed using Primer Express software (Applied Biosystems). For xylS primers 5′-TGTTATCATCTGCAAATAATACTCAAAGG-3′ and 5′-GCCCGGCGCAAAATAGT-3′ were used. 16S rRNA was used as endogenous control with the primer pair 5′-ATTGACGTTACCCGCAGAAGAA-3′ and 5′-GCTTGCACCCTCCGTATTACC-3′. Protein analysis by SDS-PAGE For SDS-PAGE analysis cells were grown in a volume of 25 mL. Cultures containing plasmid pET16b.xylS were induced with 0.5 mM IPTG or grown in the absence

of inducer. After centrifugation the pellets were washed in 0.9% NaCl. 100 mg pellet (wet weight) were resuspended in 0.5 mL lysis buffer (50 mM Tris–HCl, pH 8.0, 1 mM EDTA, pH 8.0, 20% sucrose), 1 mg lysozyme and 62.5 U mL-1 benzonase nuclease (Sigma) were added and samples were left with shaking at Molecular motor room temperature for 2 hours. After centrifugation (13.000 rpm, 8 min) the supernatant was used as soluble fraction, while the pellet was resuspended in 0.5 mL SDS-PAGE running buffer, giving the insoluble fraction. Protein gels were run under denaturing conditions using ClearPAGE 10% gels and ClearPAGE SDS-R Run buffer (C.B.S. Scientific) followed by staining with Coomassie Brilliant blue R-250 (Merck). References 1. Brautaset T, Lale R, Valla S: Positively regulated bacterial expression systems. Microb Biotechnol 2009, 2:15–30.PubMedCrossRef 2. Mergulhão FJM, Monteiro GA, Cabral JMS, Taipa MA: Design of bacterial vector systems for the production of recombinant proteins in Escherichia coli. Microbiol Biotechnol 2004, 14:1–14. 3. Ramos JL, Marques S, Timmis KN: Transcriptional control of the Pseudomonas TOL plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators.

Data analysis The genetic diversity was measured by the Hunter-Ga

Data analysis The genetic diversity was measured by the Hunter-Gaston Diversity Index (DI) on http://​www.​hpa-bioinformatics.​org.​uk/​cgi-bin/​DICI/​DICI.​pl. A high DI with a narrow confident interval PLX-4720 purchase (CI) indicates accurate measurement of a highly variable locus. These loci may be sufficiently variable to be used as an indicator to discriminate

between samples or as a starting point for assay development. The genetic distances between two isolates i and j were calculated as following: One marker difference is equivalent to 15%, 5/7 different is 70%. In our study, the criteria sets provided by either MLVA or MLST analysis consider two strains similar having at least 70% similarity, i.e. a DLV difference. The interest of the method is to quantify the difference. The minimum spanning trees by MLST using the 7 house keeping genes and by MLVA were constructed using BioNumerics ver. 5.0 with the categorical coefficient. Priority rules were fixed as following: maximum number of i) Single-locus variants (SLVs);

ii) SLVs and double-locus variants (DLVs); iii) Maximum neighbour minimum cluster size of two loci (DLV) and 2 ST, when the seven BIBW2992 ic50 housekeeping gene markers were used by MLST; iv) Maximum neighbour minimum cluster size of two loci (DLV) and 2 MT, when 17 markers were used and one locus (SLV) and 2 MT when 7 markers are used by MLVA. The Congruence among Distance Matrices MLST/MLVA was calculated in % of difference of the genetic distance between two isolates depending on the number of markers used using Bionumerics ver.5.0 as well. The Inter-Matrix Difference (IMD) was calculated using the formula below, where d(i,j) is the genetic distance between i and j, and n the number of isolates. learn more Marker numbers refer to Table 2. The lower the IMD value is the closest is the distance matrices given by the two techniques. Table 2 Genetic diversity of the 331 isolates of S. pneumoniae Marker name DI* 95% CI † Marker set by author This paper Koeck 2008 [[19]] Pichon 2010 [[26]] Elberse 2011 [[25]]       (A)   (B)

(C) ms15_507bp_45bp_7U 0.607 [0.588-0.626]       + ms17_167bp_45bp_3U 0.852 [0.847-0.857] + + +   ms19_663pb_60pb_10U 0.674 [0.658-0.691] + + +   ms25_426bp_45bp_4U 0.788 [0.779-0.797] + + + + ms26_492bp_51bp_6U 0.714 [0.703-0.726]         ms27_326bp_45bp_3U 0.561 [0.543-0.579] +       ms31_594bp_45bp_9U 0.695 [0.683-0.708]         ms32_280pb_45bp_2U 0.598 [0.585-0.611]       + ms33_407bp_45bp_2U 0.737 [0.725-0.748] + +   + ms34_239bp_45bp_1U 0.682 [0.670-0.695]     +   ms35_349bp_49bp_4U 0.572 [0.557-0.587]         ms36_274pb_45pb_2U 0.793 [0.786-0.801]     +   ms37_501bp_45bp_7U 0.855 [0.851-0.859] + + + + ms38_309bp_45bp_2U 0.557 [0.535-0.578]       + ms39_275bp_45bp_2U 0.812 [0.804-0.819] +   +   ms40_376bp_45bp_3U 0.789 [0.782-0.797]   +   + ms41_166pb_14pb_2U 0.567 [0.548-0.586]   +     All markers 0.989 [0.987-0.991]         Congruence (%)     47.2 59 65.