Article Figures & Data
Figures
- Fig 1
Formation of aberrantly shaped cells in V. parahaemolyticus 1137 incubated at 4°C in MMS–0.5% NaCl for 12 h. (A) Cells observed under a light microscope. Bar, 5 μm. (B and C) Cells observed under a transmission electron microscope. Arrows indicate the bulging of aberrantly shaped cells. Bars, 0.2 μm.
- Fig 2
Effects of antibiotics on the formation of aberrantly shaped cells in V. parahaemolyticus 1137. Cultures were incubated at 4°C for 12 h in MMS with 0.5% NaCl, either without antibiotics (control) or with d-cycloserine (50 μg/ml) or cephalosporin C (10 μg/ml), and cell shapes were determined by microscopy. The cell shapes were classified into three groups: rod, coccoid, and other (nonrod, noncoccoid) aberrant shapes (15). Open bars, coccoid cells; hatched bars, aberrantly shaped cells; stippled bars, total of coccoid and aberrantly shaped cells. The values for the antibiotic-supplemented groups were compared with the corresponding values for the control group. Asterisks indicate significantly different values (P < 0.05). For each determination, a total of 200 bacteria from 20 randomly selected fields were examined.
- Fig 3
Effect of salinity on the formation of aberrantly shaped cells in V. parahaemolyticus 1137. Cultures were suspended in MMS with 0.5, 1.0, or 1.5% NaCl and were incubated at 4°C for 12 h. Coccoid cells (open bars) and aberrantly shaped cells (hatched bars) were counted under a light microscope. Their total was also determined (stippled bars). The values obtained at different salt concentrations were compared. Asterisks indicate significantly different values (P < 0.05). For each determination, a total of 200 bacteria from 20 randomly selected fields were examined.
- Fig 4
Expression of genes associated with cell wall synthesis in V. parahaemolyticus 1137. Strain 1137 was cultured in MMS–0.5% NaCl and was incubated at 4°C. The expression of target genes at 12 h relative to their expression at 0 h was determined by RT-qPCR.
- Fig 5
Formation of aberrantly shaped cells in the V. parahaemolyticus wild-type strain 1137, Δ2468 (dacB deletion mutant), Δ2468C (the complementary strain with dacB restored), and Δ2468V (dacB deletion mutant containing pSCB01). Cultures were suspended in MMS with 0.5% NaCl and were incubated at 4°C for 12 h, and aberrantly shaped cells were counted under a light microscope. The values for the mutant and complemented strains were compared to that for the wild type. Asterisks indicate significant differences (P < 0.05).
Tables
- Table 1
Bacterial strains and plasmids used in this study
Strain or plasmid Characteristics/sequence Source or reference Strains V. parahaemolyticus 1137 Wild type, serotype O3:K6, KP+; clinical isolate This study Δ2468 1137 ΔdacB This study Δ2468C 1137 ΔdacB containing pSCB01 dacB This study Δ2468V 1137 ΔdacB containing pSCB01 This study E. coli XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacIqZΔM15 Tn10 (Tetr)] Stratagene SM10λpir thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu λ pirR6K; Kmr 46 Plasmids pGEM-T Easy Cloning vector; Apr Promega pDS132 R6K ori mobRP4 sacB Cmr 47 pBR328 Vector derived from pBR325 and pMB1; Apr Cmr Tcr Roche pSCB01 Derived from pBR328 and pDS132; mobRP4 Apr Cmr Tcr This study pGEM-T dacB pGEM-T containing dacB This study pDS132ΔdacB pDS132 with ΔdacB This study pSCB01 dacB pSCB01 containing dacB This study - Table 2
Primers used in cloning experiments of this study
Primer Sequence (5′ to 3′) Amplicon length (bp) Primer 1 CTTCACTGACGGTGAAAAAG 562 Primer 2 GGCCATTTTTATCTAAACCATAAGACAATAGCGGAAAAATAAGA Primer 3 TCTTATTTTTCCGCTATTGTCTTATGGTTTAGATAAAAATGGCC 579 Primer 4 AGTGTACATGCCAGTGATTG Primer C CAGTCTTTAGACTTGAATACTC 2,025a Primer 1+ CTATACGGTGTTGGTTTCTG 3,338 Primer 4+ TGTGTCATCTATCGGCATAA Primer F TACGTTGGCCTCTACTCATT 489 Primer R CTTGGACACAGTTGTTGTTG Seq 1F TAACCCTAGAAAATCTCACT 677 Seq 1R TAATTCGTAGCGATTGTCTG Seq 2F GCATCAACCTATTCGTGTTA 897 Seq 2R ATTTACTTTACTGTGGTGAA ↵a Amplified by primers C and 4.
- Table 3
Selected genes associated with cell wall synthesis in V. parahaemolyticus
Locus Gene Product VP0454 Penicillin-binding protein 3 VP0535 lspA Lipoprotein signal peptidase VP0457 mraY Phospho-N-acetylmuramoyl-pentapeptide transferase VP0548 tagE Putative ToxR-activated protein TagE, subfamily M23B unassigned peptidases (Vibrio harveyi) VP0564 mepA Non-penicillin-sensitive murein endopeptidase, d-alanyl-d-alanine endopeptidase; functions in hydrolyzing cell wall peptidoglycan VP0722 rodA Penicillin-binding protein 2 VP1385 Hypothetical protein, cell wall endopeptidase, subfamily M23B VP1485 nlpC Lipoprotein NlpC, Spr peptidase (Vibrio parahaemolyticus), outer membrane protein VP2369 mltA Murein transglycosylase A, catalyzes the cleavage of the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues VP2463 ftsH Cell division protein FtsH, family M41 unassigned peptidases (Vibrio parahaemolyticus) VP2468 dacB d-Alanyl-d-alanine carboxypeptidase/endopeptidase, LMW PBP; removes terminal d-alanine from pentapeptide side chains VP2471 Hypothetical protein, subfamily M23B nonpeptidase homologs (Vibrio sp. strain EJY3) VP2497 mrcB Penicillin-binding protein 1B VP2658 murA UDP-N-acetylglucosamine 1-carboxyvinyltransferase; adds enolpyruvyl to UDP-N-acetylglucosamine as a component of cell wall formation VP2751 Penicillin-binding protein 1A VPA0517 Hypothetical protein, zoocin A (Vibrio sp.), a PBPa VPA1649 tagE Putative TagE protein, subfamily M23B unassigned peptidases (Vibrio alginolyticus); outer membrane protein related to metalloendopeptidases, cell envelope biogenesis ↵a See reference 48.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Primers used in RT-qPCR (Table S1); preliminary data on the determination of antibiotic concentrations to be used in this study (Fig. S1).
PDF, 126K
- Supplemental file 1 -
