Vibrio cholerae is an aetiological agent of cholera that inhabits marine and estuarine environments. It can survive harsh environments by entering the viable but non-culturable (VBNC) state, but the related changes in gene expression have not been described. Here, we experimentally induced the VBNC state in V. cholerae O1, by incubation in artificial seawater at 4°C. Bacterial cells that were incubated for 70 days retained their membrane integrity and were pathogenic, colonizing the gut of iron-dextran-treated mice, even though they formed no colonies on tryptic soy agar (TSA) or TSA amended with pyruvate. We therefore used this stage of cells as the VBNC bacteria. We compared the global transcription pattern of the VBNC cells with that of stationary-phase cells grown in rich medium. A total of 100 genes were induced by more than fivefold in the VBNC state, and the modulated genes were mostly those responsible for cellular processes. Furthermore, real-time RT-PCR analysis verified the changes in the expression levels, showing that the VC0230 [iron(III) ABC transporter], VC1212 (polB), VC2132 (fliG) and VC2187 (flaC) mRNAs were increased in the non-culturable state. Thus, these genes may be suitable markers for the detection of VBNC V. cholerae. To our knowledge, this is the first report of a comprehensive transcriptome analysis of V. cholerae in the VBNC state. The significance of this gene expression profile compared with those of in vivo isolates and non-stressed bacteria (culturable in vitro) is its potential to provide information about the public health risk from dormant bacteria.
Erysipelothrix rhusiopathiae is a gram-positive rod capable of causing erysipelas in swine. To establish a method for specifically detecting E. rhusiopathiae for practical applications, such as for the inspection of slaughterhouses, the feasibility of using primers derived from the DNA sequence coding for 16S rRNA in a PCR-specific detection system was investigated. Oligonucleotide primers were designed to amplify a 407-bp DNA fragment by PCR. The amplification was specific to the Erysipelothrix DNA but not to that of other bacterial genera tested. This PCR-based method efficiently and specifically detected the Erysipelothrix DNA sequence in joint and spleen samples from mice within 6 h, and application of the 407-bp DNA segment from samples containing very low numbers of bacteria (<20 bacteria per spleen from mice) was possible. Although this PCR amplification is specific for the Erysipelothrix genus, which contains at least two species, E. rhusiopathiae and E. tonsillarum, it can be concluded that all Erysipelothrix strains detected by this PCR system in diseased pigs are E. rhusiopathiae because only E. rhusiopathiae is virulent for pigs. These results show that this PCR amplification system using the DNA sequence coding for 16S rRNA is very rapid and reliable and avoids cumbersome and lengthy cultivation steps, demonstrating that this system could be used for practical applications.
Transposon insertional mutants of Listeria monocytogenes were constructed to identify genes involved in osmotolerance, and one mutant that showed reduced growth under high osmotic pressure was obtained. The cloned gene from the transposon insertion site of the mutant, named rel, was 2,214 bp in length and had very high homology to relA of Bacillus subtilis, which encodes guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp) [collectively designated (p)ppGpp] synthetase during stringent response. The mutant showed a deficiency in (p)ppGpp accumulation. In the parental strain, the amount of intracellular (p)ppGpp was not increased after an osmotic upshift but was slightly decreased compared with the level before the upward shift. The reduced osmotolerance of the mutant was restored to a level almost equal to that of the parent strain when the chromosomal region that included rel of L. monocytogenes was introduced into the mutant. After exposure to methyl glucoside, the rel mutant accumulated (p)ppGpp at a higher level than the basal level and partially restored the ability to grow in NaCl-supplemented brain heart infusion broth. The mutant was found to grow in chemically defined minimal medium supplemented with glycine betaine or carnitine, so-called compatible solutes, and 4% NaCl. Our results suggest that the appropriate intracellular concentration of (p)ppGpp is essential for full osmotolerance in L. monocytogenes and that its mechanism is different from that for the accumulation of compatible solutes.Listeria monocytogenes is the causative agent of listeriosis and is transmitted mainly by food to humans (6). Its characteristics, such as ubiquitous distribution in the environment (33) and strong tolerances to NaCl and refrigeration temperatures (26), make it difficult to prevent food contamination with L. monocytogenes. Recent studies have shown that Listeria accumulates so-called compatible solutes, such as glycine betaine (betaine), carnitine, and proline, under hyperosmotic stress to counteract the outward flow of water (2, 12). Such accumulations have also been observed in a wide range of other organisms, including Bacillus subtilis (3) and Escherichia coli (4). In L. monocytogenes, the betL gene and gbu operon have been identified as betaine transporter-encoding genes (27, 13), and opuC has been identified as a carnitine transporter-encoding operon (7). Disruption of these genes reduces the osmotolerance of L. monocytogenes (7,13,27). Both betL (7) and opuC (27) have B -dependent promoters, and the sigB (the B -encoding gene) mutant strain also shows a reduced ability to accumulate betaine, resulting in lower osmotolerance than that in the parent strain (1). On the other hand, there are some reports about the influence of disruption of osmotic stress-related genes to virulence of L. monocytogenes; sigB (32) and proBA (proline transporter-encoding gene) (28) mutants showed no differences from the parental strain in rates of recovery from organs of mice. But strains with mutations in opuC (2...
The activity of prokaryotic and mammalian thymine glycol (Tg) glycosylases including Escherichia coli endonuclease III (Endo III) and endonuclease VIII (Endo VIII) and mouse Endo III homologue (mNth1) for formamidopyrimidine (Fapy) has been investigated using defined oligonucleotide substrates. 2,6-Diamino-4-hydroxy-5-N-methylformamidopyrimidine, a methylated Fapy derived from guanine, was site specifically incorporated in the oligonucleotide. The substrates contain- DNA damage caused by exogenous and endogenous agents is a major threat to genetic integrity of cells, and when unrepaired, it results in lethal and/or mutagenic events of cells (1). The deleterious effects of DNA damage have been also implicated in carcinogenesis and aging (2, 3). Among the diverse DNA lesions thus far identified, alterations of the base moiety by reactive oxygen species constitute one of the major classes of DNA damage. In cells, this class of damage is generally restored by the base excision repair (BER) 1 pathway, a highly conserved mechanism across species (4, 5). The BER process for oxidized bases is initiated by DNA N-glycosylases that remove damaged bases from DNA. In Escherichia coli, DNA N-glycosylases responsible for this reaction are basically classified into two subgroups depending on the substrate, albeit some overlapping specificities. Oxidized pyrimidine bases such as thymine glycol (Tg) and 5-hydroxycytosine, etc., are excised from DNA by endonuclease III (Endo III) and endonuclease VIII (Endo VIII) (5, 6) encoded by the nth and nei genes, respectively (7-9). In contrast, oxidized purine bases such as 7,8-dihydro-8-oxoguanine (8-oxoG) and formamidopyrimidine (Fapy) are removed by formamidopyrimidine DNA glycosylase (Fpg) (10, 11) encoded by the fpg/mutM gene (12). The nth nei double mutant of E. coli lacking Endo III and Endo VIII exhibits a mutator phenotype and a increased sensitivity to hydrogen peroxide and ionizing radiation (8, 9). The mutations observed for the double mutant are mostly C 3 T transitions probably arising from impaired excision of oxidized cytosine lesions (13).The fpg mutant of E. coli deficient in Fpg shows a mutator phenotype though the sensitivity to ionizing radiation or hydrogen peroxide remains unchanged (14, 15). The frequent mutations observed for the fpg mutant are G 3 T transversions due to impaired excision of 8-oxoG (15). Thus, the substrate specificity of the two subgroups of DNA N-glycosylases correlates fairly well with the phenotype of their mutants. Recently, functional homologues of Endo III (Ntg1 and Ntg2 proteins (also called Scr1 and Scr2)) and Fpg (yOgg1 protein) have been identified from Saccharomyces cerevisiae, and the substrate specificities of the expressed proteins have been studied. The yOgg1 protein recognizes 8-oxoG and Fapy, showing a substrate specificity similar to Fpg (16,17). Ntg1 and Ntg2 proteins recognize a variety of oxidized pyrimidines, such as Tg and 5-hydroxycytosine, that are also substrates for Endo III (18 -23). Despite such similar substrate speci...
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