All these studies examined relatively short-term responses, with follow-up times no longer than 2 years. Moreover, the characteristics of the patients (e.g. the clinical and biological features of their HIV infection, their geographical origins, whether they were pretreated or naïve to cART, and their adherence to treatment), the definition of the virological response (e.g. 50 or 500 copies/mL) and follow-up times varied among the studies. Our study, which is probably the first to assess the impact of this deletion over a long follow-up period in a large number of treated patients, showed a significantly better response

after 5 years of treatment in Δ32 heterozygous patients. Previously, C59 wnt mw the longest follow-up time was 24 months in the study of Bogner et al. Etoposide purchase [11], in which a better virological response to cART was found in Δ32 heterozygotes among adherent Caucasian patients naïve to antiretroviral treatment. The discrepancy found between short-term and long-term virological responses to cART in our study might explain some of the differences among previous studies. The interpretation of such a moderate effect of the deletion on response to cART would be in favour of the absence of an effect among treated patients, or of limited effect only detectable after

extensive follow-up. In order to take into account differences existing at baseline or occurring during follow-up that might also influence response to cART, the multivariable analysis was adjusted for potential confounders. After this adjustment, we found that heterozygous patients Epothilone B (EPO906, Patupilone) still showed a better

long-term virological response, suggesting that there is an independent effect of the CCR5 Δ32 deletion on long-term virological response in the context of a multifactorial determination of response. The potential disadvantage of the wild-type profile might be counterbalanced by the beneficial effect of high adherence and initiation of cART at an optimum time. In view of the conflicting results obtained in previous studies, a meta-analysis including other observational cohorts would be useful to elucidate the long-term effect of this mutation. The authors would like to thank Rodolphe Thiebaut for his helpful suggestions concerning the statistical methodology. Scientific committee: Steering Committee: Principal Investigators: C. Leport, F. Raffi; Methodology: G. Chêne, R. Salamon; Social Sciences: J-P. Moatti, J. Pierret, B. Spire; Virology: F. Brun-Vézinet, H. Fleury, B. Masquelier; Pharmacology: G. Peytavin, R. Garraffo. Other members: D. Costagliola, P. Dellamonica, C. Katlama, L. Meyer, D. Salmon, A. Sobel. Events validation committee: L. Cuzin, M. Dupon, X. Duval, V. Le Moing, B. Marchou, T. May, P. Morlat, C. Rabaud, A. Waldner-Combernoux. Project co-ordination: F. Collin-Filleul.

This study highlights the need for detailed profiling of the

huge uncultured component of the rumen bacterial community in order to understand their role in the degradation of feed in the rumen. The authors acknowledge Prof. R.I. Mackie for his helpful suggestions and proofreading of the manuscript. “
“The genome sequence of a Sphingobium strain capable of tolerating high concentrations of Ni ions, and exhibiting natural kanamycin resistance, is presented. The presence of a transposon derived kanamycin resistance gene and several genes for efflux-mediated Pifithrin-�� molecular weight metal resistance may explain the observed characteristics of the new Sphingobium isolate. “
“Toxin–antitoxin (TA) systems are small genetic elements found on plasmids or chromosomes of countless

bacteria, archaea, and possibly also unicellular fungi. Under normal growth conditions, the activity of the toxin protein or its translation is counteracted by an antitoxin protein or noncoding RNA. Five types of TA systems have been proposed that differ markedly in their genetic architectures and modes of activity control. Subtle regulatory properties, frequently responsive to environmental cues, impact Belinostat purchase the behavior of TA systems. Typically, stress conditions result in the degradation or depletion of the antitoxin. Unleashed toxin proteins impede or alter cellular processes including translation, DNA replication, or ATP or cell wall synthesis. TA Non-specific serine/threonine protein kinase toxin activity can

then result in cell death or in the formation of drug-tolerant persister cells. The versatile properties of TA systems have also been exploited in biotechnology and may aid in combating infectious diseases. “
“Horizontal gene transfer plays an important role in bacterial evolution. DNA acquired by horizontal gene transfer has to be incorporated into existing regulatory networks. The histone-like nucleoid structuring protein H-NS acts as a silencer of horizontally acquired genes to avoid potential damage. However, specific regulators can overcome H-NS repression, resulting in the integration of newly acquired genes into existing regulatory networks. Here, we analyzed the influence of H-NS on the transcription of the Yersinia enterocolitica hreP gene and its regulators pypA, pypB, and pypC by establishing a dominant-negative H-NS version. Using transcriptional fusions and electrophoretic mobility shift assays, we show that H-NS silences hreP, pypA, pypB, and pypC by direct interactions. While the H-NS antagonist RovA activates pypC, it has no effect on pypA and pypB. Furthermore, H-NS affects biofilm formation in Y. enterocolitica. “
“In Pseudomonas putida, as in many other eubacteria, cyclopropane fatty acids (CFAs) accumulate in the membrane during the stationary phase of growth. Here, we show that cfaB gene expression in P. putida KT2440 is dependent on the RpoS sigma factor that recognizes the sequence 5′-CTACTCT-3′ between −8 and −14.

Following incubation, media was discarded and the formazan crystals were solubilized by adding 200 μL DMSO and the absorbance measured at A560 nm. The percentage toxicity was calculated as A phage library displaying random 7-residue peptides was

panned against (His)6-DevR protein. Five rounds of panning were PFT�� purchase performed (three rounds with (His)6-DevR immobilized on Ni2+ NTA magnetic agarose beads and two rounds with (His)6-DevR coated on a well in a polystyrene ELISA plate) to select DevR binders and to exclude bead and plastic binding phages. Selective enrichment of DevR binding phages was achieved using this approach as demonstrated by approximately fourfold more efficient binding to DevR of the phages derived from the fifth round of panning compared to the unpanned phage pool. Furthermore, the enriched phage did not bind to either BSA or plastic (Fig. 1a). A total of 194 phage clones from DevS~P and glycine elutions from the final round of panning were individually amplified and screened by ELISA to select DevR binding phages. Nineteen phage clones were selected for sequencing based on their binding selectivity to DevR (not shown). The sequence ‘TLHLHHL’ was repeated 15 times and a 7-mer peptide, DevRS1, bearing this sequence was synthesized and further characterized. In an ELISA performed with purified full-length N-terminal-tagged

glutathione-S-transferase [GST]-DevR (Bagchi et al., 2005) and its individual Seliciclib N- and C-terminal domains, DevRS1 sequence displaying phage clone

G43 bound relatively more efficiently to the DevR C-terminal domain (DevRC, containing 144–217 amino acids of DevR expressed with a N-terminal tag of GST) as compared to the N-terminal domain of DevR (DevRN, containing 1–144 amino acids of DevR expressed with a N-terminal GST tag) and poorly to GST alone or to BSA or plastic (Fig. 1b). The binding specificity of DevRS1 was confirmed by a competition ELISA wherein the peptide DevRS1 inhibited the binding of TLHLHHL-displaying phage (G43) to (His)6-DevR but not of nonspecific binder phage (Fig. 1c). The effect of DevRS1 peptide on gene expression and viability of M. tb was examined next. Exposure to DevRS1 peptide at 5 mM concentration resulted in ~ 55–60% inhibition of Rv3134c promoter activity (a DevR-regulated Interleukin-2 receptor promoter, Fig. 2a, black bars) with respect to DMSO control under both aerobic and hypoxic conditions. The observed inhibition of promoter activity in the aerobic set up is ascribed to the development of hypoxia in standing cultures (Chauhan & Tyagi, 2008a). The activity of the constitutively expressed sigA promoter was not affected under identical conditions (Fig. 2a), indicating the target specificity of the peptide. It is expected that inhibition of Rv3134c promoter activity would be associated with the inhibition of other regulon promoters as observed by Gupta et al.

To study the temperature stability of the ethanolic extract or of the fatty acid mixture, aliquots ERK signaling pathway inhibitor were either heated to 55, 75, or 100 °C for 30 min or frozen at −20 °C, and the residual hemolytic activities were measured

as described earlier. The effects of different pH values on the hemolytic activity were determined following the pH adjustment of the erythrocyte buffer solution to pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5. To study the effects of ionic strength on the hemolytic activity of the ethanolic extract, the ionic strength of the erythrocyte buffer was first adjusted to 100, 140, 180, 220, 260, 300, 340, 380, 420, 460, 500, and 540 mM NaCl. To study the interactions of the hemolytically active compounds in the ethanolic extract from W. sebi with lipid vesicles, various GSK1120212 ic50 small unilamellar vesicles (SUVs) at a final

concentration of 2 mg mL−1 were prepared as described by Rebolj et al. (2006). The dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin, and cholesterol that were used were from Avanti Polar Lipids and Merck. The permeabilization of SUVs loaded with fluorescent calcein (Sigma) was assayed as described by Rebolj et al. (2006). The membrane binding of hemolytically http://www.selleck.co.jp/products/wnt-c59-c59.html active compounds from the W. sebi ethanolic extract was estimated by measuring the residual hemolytic activity of unbound compounds after a 30-min incubation period of SUVs

with the extract at 25 °C (Sepčić et al., 2003). Here, 80 μL SUVs (2 mg mL−1) of various compositions were prepared in vesicle buffer and pipetted into multiwell plates, followed by the addition of 20 μL (TS = 0.54 mg mL−1) of the ethanolic extract. The residual hemolytic activity of the extract was then assessed after this incubation period by adding 100 μL erythrocyte suspension to each well (Sepčić et al., 2003). Vesicle permeabilization by the ethanolic extract was determined by combining 20 μL of the extract (TS = 0.54 mg mL−1) and 2 μL calcein-loaded SUVs in 1 mL of vesicle buffer (erythrocyte buffer supplemented with 1 mM EDTA), as described by Rebolj et al. (2006). Significant differences among experimental groups were compared with one-way anova, using least significant difference (LSD) post hoc tests (P < 0.05). All of the statistical tests were performed using spss for Windows, version 15.00 (SPSS Inc. 2006). The composition of the ethanolic extract from the W. sebi mycelia was determined by GC/MS analysis. Twenty-one compounds were identified in the extract, the majority of which were variously saturated and unsaturated fatty acids and sterols, as summarized in Table 1. The most intense chromatographic peaks were recorded at the retention times of 19.4, 20.

To study the temperature stability of the ethanolic extract or of the fatty acid mixture, aliquots ROCK inhibitor were either heated to 55, 75, or 100 °C for 30 min or frozen at −20 °C, and the residual hemolytic activities were measured

as described earlier. The effects of different pH values on the hemolytic activity were determined following the pH adjustment of the erythrocyte buffer solution to pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5. To study the effects of ionic strength on the hemolytic activity of the ethanolic extract, the ionic strength of the erythrocyte buffer was first adjusted to 100, 140, 180, 220, 260, 300, 340, 380, 420, 460, 500, and 540 mM NaCl. To study the interactions of the hemolytically active compounds in the ethanolic extract from W. sebi with lipid vesicles, various PI3K Inhibitor Library small unilamellar vesicles (SUVs) at a final

concentration of 2 mg mL−1 were prepared as described by Rebolj et al. (2006). The dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingomyelin, and cholesterol that were used were from Avanti Polar Lipids and Merck. The permeabilization of SUVs loaded with fluorescent calcein (Sigma) was assayed as described by Rebolj et al. (2006). The membrane binding of hemolytically Venetoclax mw active compounds from the W. sebi ethanolic extract was estimated by measuring the residual hemolytic activity of unbound compounds after a 30-min incubation period of SUVs

with the extract at 25 °C (Sepčić et al., 2003). Here, 80 μL SUVs (2 mg mL−1) of various compositions were prepared in vesicle buffer and pipetted into multiwell plates, followed by the addition of 20 μL (TS = 0.54 mg mL−1) of the ethanolic extract. The residual hemolytic activity of the extract was then assessed after this incubation period by adding 100 μL erythrocyte suspension to each well (Sepčić et al., 2003). Vesicle permeabilization by the ethanolic extract was determined by combining 20 μL of the extract (TS = 0.54 mg mL−1) and 2 μL calcein-loaded SUVs in 1 mL of vesicle buffer (erythrocyte buffer supplemented with 1 mM EDTA), as described by Rebolj et al. (2006). Significant differences among experimental groups were compared with one-way anova, using least significant difference (LSD) post hoc tests (P < 0.05). All of the statistical tests were performed using spss for Windows, version 15.00 (SPSS Inc. 2006). The composition of the ethanolic extract from the W. sebi mycelia was determined by GC/MS analysis. Twenty-one compounds were identified in the extract, the majority of which were variously saturated and unsaturated fatty acids and sterols, as summarized in Table 1. The most intense chromatographic peaks were recorded at the retention times of 19.4, 20.

A sample size of at least 50 mothers of children with JIA was calculated based on standard deviations with 95% confidence intervals as used in previous studies using the PSI to calculate maternal stress in other chronic childhood illnesses.[14] Correlations were sought between joint count, CHAQ, Physician and Parent Global VAS and PSI using Spearman’s r correlation coefficient. Level of significance was set at 0.05 and all tests were two-tailed t-tests were used to compare the maternal stress scores with those of mothers of children with other chronic childhood illnesses. PSI scores were expressed as means and 95% CI. Complete data was obtained

for 50 mothers and children. The children BMN 673 had a mean age of 6 years (SD ± 2.9 years) and 33 (66%) were female. Twenty-eight (56%) had oligoarticular arthritis, 10 (20%) had polyarticular arthritis, 10 (20%) had systemic onset arthritis and two (4%) had psoriatic arthritis. Twenty-five (50%) were on methotrexate and 10 (20%) were on a biologic therapy (five tozilizumab, four etanercept, one anakinra). Full demographic data for both mothers and children are shown in Table 1. The mean PSI scores for mothers with children with JIA are shown in Table 2 which also shows a comparison to normative and other chronic disease samples. The mean total stress score for mothers of children with U0126 molecular weight JIA was 235.4 (95% CI 218.5–252.3),

was greater than the mean total stress scores for mothers of normal children 222.8(95% CI 221.4–224.2) and children with other chronic disorders such as insulin-dependent diabetes 218.1 Phosphatidylinositol diacylglycerol-lyase (95% CI 204.7–231.6) and profound deafness 221.7 (95% CI 206.4–237.0). This reached statistical significance (P < 0.05). A similar trend was also seen in the parent domain. The level of maternal stress was higher in mothers of children with moderate to severe eczema and enteral feeding with mean total stress scores of 259.6 (95% CI 244.9–274.3) and 251.4 (95% CI 242.1–260.7), respectively.

The mean parent domain was greater, 128.7 (95% CI 120.4–136.9) than for mothers of normal children, 123.1 (95% CI 122.2–124.0) and children with deafness, 125.4 (95% CI 114.5–136.3) and diabetes, 117.2 (95% CI 109.7–124.7). This reached statistical significance only when looking at the normative group. There was only data available for the child domain in cystic fibrosis (CF). The mean PSI for the child domain in our study was 110.7 (95% CI 103.2–118.2), which was greater than the child domain reported in CF, PSI 109.4 (95% CI 104–114.5) but was not statistically significant. Seventeen (34%) mothers scored within the clinical range (PSI > 260) for total stress scores, 17 (34%) within the child domain (> 118) and seven (14%) in the parent domain (> 150). Figure 1 shows the proportion of mothers within each subtype of JIA who scored in the clinical range. The mean active joint count of the children with JIA was 1.1 (SD 1.5) with a range of 0–8 joints.

This should be reflected in an increase in MK-1775 order the number of peaks in the alpha topography from the undivided condition to the divided condition. For the blinking spotlight model

of attention (VanRullen et al., 2007), we derived three possible predictions for suppression of the to-be-ignored stimuli. In this theory, the attentional spotlight is thought to constantly move between all available stimuli. Therefore, the first prediction is that all unattended stimuli will be suppressed individually. That is, we assume that a similar mechanism exists for both suppression and excitation. For the current experimental paradigm, such a mechanism would result in two peaks of suppression for both the divided attention condition and the undivided attention condition. The second prediction is that there will be no suppression of to-be-ignored stimuli, as the blinking spotlight of attention might only selectively enhance target locations. This should obviously result in alpha topographies that do not possess www.selleckchem.com/products/abt-199.html distinctive occipito-parietal peaks. The third prediction is that, while the attentional focus switches rhythmically between all possible target locations, suppression will be allocated to distracter

locations in a static fashion. This would result in the same topographic distribution and increase in the number Silibinin of peaks in the divided attention condition as for the divided spotlight account, and indicate a static split of suppression. Participants were successful at performing the difficult attentional tasks.

With chance level at 33.3%, the mean percentages of correct responses were approximately 50% for the attentional task conditions involving the outer right stimulus, and approximately 45% for those involving the left outer stimulus (Fig. 3). These performance values are somewhat lower than in other studies of attention, but the experimental task was more difficult, owing to the randomly flickering stimuli that were necessary to estimate the brain’s impulse response to all four stimuli. For the C1 time-frame, the repeated measures anova revealed no significant main effects (F1,54 = 0.2; P = 0.657). Only for the inner left stimulus was there significant modulation of activity with attention (F1,13 = 4.78; P = 0.048). This indicates that there was no influence of attention on cortical processing in this very early time-frame, or that the locations of the four different stimuli were not optimal for obtaining C1 responses.

Data from Brazil (most of which were from Amazonia and a few from French Guiana) have identified P vivax relapse rates of 39.6% after primaquine regimens (total doses ranging from 2.2 to 4.9 mg/kg), half of which occurred

within 108 days of radical cure[10]; the study advocates that the primaquine total dose above which Navitoclax nmr relapses do not occur is 3.6 mg/kg. The failure rate of the 30 mg/day regimen in the present study (roughly 30%) is not so different from those observed by Pedro and colleagues[10] but higher than the other data found in the literature (efficacy of 95%).[4] However, all three relapsing patients were prescribed primaquine total doses of above 3.6 mg/kg, which seems contradictory to the findings in Brazil,[10] and would suggest that some strains from French Guiana need higher primaquine doses, closer to the Chesson type of P vivax. More data from records of travelers who acquired

P vivax in French Guiana would be required to discern whether the high risk of relapse observed after standard radical cure on a small sample of records reflects the current risk of relapse in this area. If Z-VAD-FMK molecular weight so, efficacy of potentially more effective alternative regimens should be comparatively assessed. The fact that two of the patients who relapsed had body weight >70 kg (100 and 105 kg) may have played a role as the initial regimen for them was 0.3 mg/kg daily whereas the second one was 0.5 mg/kg daily. On the basis of a trend of higher risk of relapse after standard radical cure in high body weight Evodiamine patients with P vivax infections, Baird and colleagues[6] advocated for a regimen of 0.5 mg/kg primaquine daily for 14 days for patients weighing more than 70 kg. This recommendation has since been partially integrated

by the CDC experts meeting: although the standard recommended course is still 30 mg/day over 14 days, it is now specified that for individuals weighing more than 70 kg, the treatment could be extended to provide a total dose of 6 mg/kg.[3] In our case series, radical cure of P ovale and P vivax infections used primaquine alone; however, as higher efficiency of primaquine was demonstrated when given concurrently with blood schizonticides,[4] an alternative could be to give a combinative treatment as first-line radical cure. Relapses of P vivax infections from French Guiana were frequently observed in our experience of radical cure with primaquine at 30 mg daily. More data are needed to estimate properly the relapse rate of P vivax infections from French Guiana after primaquine radical cure and further comparative studies would be required to test suitably the hypothesis that radical cure dosage could be adapted to body weight in order to reduce the risk of relapse in this population. The authors state they have no conflicts of interest to declare.

“
“Listeria monocytogenes is a food-borne pathogen that can survive

under a wide range of environmental and energy stress conditions. The general stress response controlled by σB largely contributes to stress resistance in L. monocytogenes. Moreover, the bacterial cell wall is the first defense GSK2118436 price against cellular stress and as such is the target of numerous antibiotics. We therefore hypothesize that σB contributes to monitoring the integrity of cell walls. We evaluated σB activity in wild type and ΔsigB mutant L. monocytogenes containing reporter fusions (σB-dependent opuCA promoter and a lacZ reporter gene) during the early exponential growth phase by measuring the specific activity of β-galactosidase after vancomycin (2 μg mL−1 final concentration) stress. σB activity is significantly induced only in the wild-type strain by addition of vancomycin. In

addition, we identified σB-dependent vancomycin-inducible proteins using LC-ESI-MS/MS analysis. Two independent proteomic analyses confirmed the minimum twofold upregulation of 18 vancomycin-inducible σB-dependent stress response proteins in the wild-type strain compared with the ΔsigB mutant. The functions buy Trametinib of these proteins are associated with cell wall biogenesis, intracellular transport, general stress response, cell metabolism and virulence. These results suggest that the σB protein may contribute to the monitoring of cell wall integrity. Listeria monocytogenes is a widely distributed intracellular pathogen that causes listeriosis, a serious illness from which children, pregnant

women and immunocompromised individuals are at risk. Infection is most often caused by the ingestion of contaminated foods, such as those most frequently associated with raw milk, soft cheeses, raw vegetables and refrigerated ready-to-eat products (Farber & Peterkin, 1991). Listeria monocytogenes has the ability to grow in very diverse environments; it can survive in a wide temperature range (−1 to 45 °C), a broad pH range (4.5–9.0) and in high salt concentrations (10% NaCl) (Cole et al., 1990; Sleator et al., 2001). These unique resistance properties are related to the general stress response, which is controlled by the PLEKHB2 alternative sigma factor σB (Wiedmann et al., 1998; O’Byrne & Karatzas, 2008). Listeria monocytogenesσB was found to play a role in the general stress response. σB-null mutants demonstrate increased sensitivity under salt, acid, cold, heat, ethanol and oxidative stress, as well as carbon starvation (Becker et al., 1998, 2000; Wiedmann et al., 1998). About 150 σB-dependent genes are known to be expressed under stress conditions, where they contribute to stress resistance in L. monocytogenes (Raengpradub et al., 2008).

, 2010; Shoji et al., 2011). Studies of Gram-negative bacteria have identified at least eight different protein secretion systems, including types I–VI, the two-partner

secretion system and the chaperone/usher system (Economou et al., 2006). PorSS is not related to the previously known bacterial protein secretion systems. PorK, PorL, PorM, PorN, PorW, PorT and Sov involved in the P. gingivalis PorSS share similarity in amino acid sequence with Flavobacterium johnsoniae gliding motility proteins, GldK, GldL, GldM, GldN, SprE, SprT and SprA, respectively (Braun et al., 2005; Rhodes et al., 2010; Sato et al., 2010). In F. johnsoniae, disruption of the sprT gene resulted in defects in translocation of the gliding motility protein SprB and secretion of chitinase, suggesting www.selleckchem.com/products/DAPT-GSI-IX.html that the PorSS is linked to gliding motility of bacteria in the Bacteroidetes phylum (Sato et al., 2010). Genes homologous find more to the PorSS-related genes are also found in genomes of other members of the Bacteroidetes phylum, including important periodontal pathogens such as P. intermedia and T. forsythia (Sato et al., 2010). In the

present study, proteomic analyses of particle-free culture supernatants and vesicle fractions from porK+ and porK strains with the genetic background of rgpA rgpB kgp and outer membrane fractions from wild-type and porK strains were performed to identify P. gingivalis proteins that were secreted into the extracellular milieu by the PorSS. Bacterial strains and plasmids used in this study are listed in Table 1. Porphyromonas gingivalis cells were grown anaerobically (10% CO2, 10% H2, 80% N2) in enriched brain heart infusion medium and on enriched trypticase soy agar

(Nakayama et al., 1995). For blood agar plates, defibrinated laked sheep blood was added to enriched trypticase soy agar at 5%. For selection and Idelalisib order maintenance of antibiotic-resistant P. gingivalis strains, antibiotics were added to the medium at the following concentrations: erythromycin (Em), 10 μg mL−1; tetracycline (Tc), 0.7 μg mL−1. Porphyromonas gingivalis deletion mutants were constructed as follows. DNA regions upstream and downstream of a gene were PCR-amplified from the chromosomal DNA of P. gingivalis ATCC 33277T using pairs of primers (PGN gene number-U-F plus PGN gene number-U-R and PGN gene number-D-F plus PGN gene number-D-R), respectively, where ‘U’ indicates upstream, ‘F’ indicates forward, ‘D’ indicates downstream and ‘R’ indicates reverse. Primers used in this study are listed in Supporting information, Table S1. Amplified DNAs upstream and downstream of each gene were double-digested with NotI plus BamHI and KpnI plus BamHI, respectively. Both digested products were ligated together with pBluescript II SK(−) which had been digested with NotI plus KpnI, resulting in pKD945 (for rgpA mutagenesis) and pKD947 (for rgpB mutagenesis). The 1.5-kb BamHI cepA (pKD1002) and 2.