Association of a D-alanyl-D-alanine carboxypeptidase gene with the formation of aberrantly shaped cells during the induction of viable but nonculturable Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus is a halophilic Gram-negative bacterium that causes human gastroenteritis. When the viable but nonculturable (VBNC) state of this bacterium was induced by incubation at 4°C in Morita minimal salt solution containing 0.5% NaCl, the rod-shaped cells became coccoid, and various aberrantly shaped intermediates were formed in the initial stage. This study examined the factors that influence the formation of these aberrantly shaped cells. The proportion of aberrantly shaped cells was not affected in a medium containing D-cycloserine (50 μg/ml) but was lower in a medium containing cephalosporin C (10 μg/ml) than in the control medium without antibiotics. The proportion of aberrantly shaped cells was higher in a culture medium that contained 0.5% NaCl than in culture media containing 1.0 or 1.5% NaCl. The expression of 15 of 17 selected genes associated with cell wall synthesis was enhanced, and the expression of VP2468 (dacB), which encodes D-alanyl-D-alanine carboxypeptidase, was enhanced the most. The proportion of aberrantly shaped cells was significantly lower in the dacB mutant strain than in the parent strain, but the proportion was restored in the presence of the complementary dacB gene. This study suggests that disturbance of the dynamics of cell wall synthesis by enhanced expression of the VP2468 gene is associated with the formation of aberrantly shaped cells in the initial stage of induction of VBNC V. parahaemolyticus cells under specific conditions.

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).