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Induction of bacteriolysis of Vibrio vulnificus cells by 10 mM hydrogen peroxide (H(2)O(2)) was analyzed. All Vibrio species examined, except for Vibrio hollisae, were lysed by 10 mM H(2)O(2). Bacteriophage induction was not the cause of H(2)O(2)-induced bacteriolysis. Autolysis is also known to cause bacteriolysis. VvpS protein is a serine protease of V. vulnificus essential for autolysis. vvpS mutant underwent H(2)O(2)-induced bacteriolysis in the same manner as the wild type. Protease inhibitors including serine protease inhibitors did not inhibit H(2)O(2)-induced bacteriolysis, which means that bacteriolysis is not due to autolysis. Unexpectedly, H(2)O(2)-induced bacteriolysis was accelerated by adding 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) and phenylmethylsulfonyl fluoride which are serine protease inhibitors. The hydroxyl radical was generated by H(2)O(2)-AEBSF interaction. It was considered that H(2)O(2)-induced bacteriolysis was caused by the hydroxyl radical which was generated by Fenton reaction, and possibly mediated by AEBSF. Deferoxamine, an agent chelating ferric ion and Fenton reaction inhibitor, suppressed both H(2)O(2)-induced bacteriolysis and its acceleration by AEBSF. This suggests that both phenomena were Fenton reaction dependent, and hydroxyl radical generated by Fenton reaction caused bacteriolysis of V. vulnificus though the reason for high susceptibility of Vibrio species to hydroxyl radical is not known.

Vibrio vulnificus is a bacterium known to cause fatal necrotizing soft tissue infection in humans. Here, a remarkable therapeutic effect of hyperbaric oxygen (HBO) on V. vulnificus infection provoked by its injection into mouse footpads is described. HBO was shown to be bactericidal to this bacterium in vitro as well as in the infected tissue. The bactericidal activity of HBO was shown to be due to reactive oxygen species (ROS), the efficacy of HBO against V. vulnificus infection being accounted for by the high sensitivity of this bacterium to ROS. Besides being somewhat weak in ROS-inactivating enzyme activities, this bacterium is also unusually sensitive to ultraviolet light and other DNA-damaging agents. It seems likely that the sensitivity of V. vulnificus to HBO is mainly due to its poor ability to repair oxidative damage to DNA. These findings encourage clinical application of HBO against potentially fatal V. vulnificus infection in humans.

Legionella pneumophila grows in amoebae and has achieved the ability to grow at various temperatures, although the mechanisms controlling this ability remain poorly understood. The Icm/Dot type IVB secretion system is composed of more than 25 proteins and is known to be essential for intracellular growth. The role of the icmN gene in intracellular multiplication and the effects of culture temperatures on it are not precisely understood. We conducted our investigation using an icmN mutant made by gene replacement mutagenesis. Intracellular growth of the mutant was impaired both in mammalian macrophages and amoeba at 37 °C. In particular, intracellular growth in amoebae was completely impaired at 25 °C. It was found that genes from icmN to icmC formed an operon, i.e., icmN, -M, -L, -E, -G, -C,, and the promoter activity of the icmN operon was stronger at 25 than at 37 °C. It was suggested that icmM and its downstream genes had a secondary promoter that enables icmN mutant grow in amoebae at lower temperatures and macrophages at 37 °C. These results show that the icmN promoter has a low temperature inducible nature, and gene products of the icmN operon require high expression for bacterial proliferation at low temperatures within amoeba.

Streptococcus mutans, a major etiological agent of dental caries, is resistant to bacitracin. Microarray analysis revealed that mbrA and mbrB, encoding a putative ATP-binding cassette transporter, are prominently induced in the presence of bacitracin. On the basis of the latest report that MbrC, a putative response regulator in a two-component signaling system, binds the promoter region of mbrA and thus regulates its transcription, we cut into the mechanism by generating a mutant MbrC (D54N-MbrC) that substituted asparagine for aspartate at position 54, the predicted phosphorylation site. MbrC, but not the mutant D54N-MbrC, showed affinity for a DNA probe that contained the hypothetical mbrA promoter sequence. Furthermore, we introduced a point mutation (D54N-MbrC) into UA159; this mutant strain exhibited neither mbrA induction nor resistance in the presence of bacitracin. These data suggest that the aspartate residue at position 54 of MbrC is a promising candidate for phosphorylation in a bacitracin-sensing system and indispensable for S. mutans bacitracin resistance.

This study examined the antimicrobial/antifungal ability of a tissue conditioner containing a photocatalyst for Escherichia coli, Streptococcus mutans, Staphylococcus aureus and Candida albicans. The photocatalyst was mixed with tissue conditioners powders at concentrations of 0, 10, 15, and 20 wt%. Tissue conditioners powders containing a photocatalyst were mixed with liquid to make test specimens. Test specimens inoculated by each microorganism were irradiated by ultraviolet light for 0-, 2- and 4 hours. The antimicrobial/antifungal effects were evaluated by the CFU technique. The CFU values of each microorganism for tissue conditioners containing a photocatalyst showed significant decrease following UV-irradiation. The improvement in antimicrobial/antifungal effects was concomitant with the increase of the mixing ratio and the irradiation time. Therefore, the results indicated that tissue conditioners containing a photocatalyst might have photocatalytic ability.

Legionella (Fluoribacter) dumoffii is a resident of various aquatic environments and occasionally causes pneumonia in humans. We found that L. dumoffii strain TEX-KL carries a 66-kb circular plasmid. As predicted by the presence of tra genes similar to those of other transferable plasmids, we showed that pLD-TEX-KL was actually capable of transferring itself to a plasmid-cured derivative of the original strain. Unexpectedly, this plasmid-free derivative turned out to be partially defective in terms of growth at temperatures 30 degrees C or lower. Subsequent works revealed that the growth defect was attributable to the loss of the plasmid gene traA(Ti) homologous to the traA gene of Ti plasmid from Agrobacterium tumefaciens, and that the growth was restored by the introduction of the mobA/repB gene of plasmid pMMB207. Since the existence of a DNA nickase domain is the only feature common to the traA(Ti) and mobA/repB gene products, we hypothesized that this growth defect at low temperature is related to insufficient DNA transactions, which can somehow be alleviated by the nickase activity of those plasmid-encoded proteins. It was also noted that the above features of growth defect at low temperatures were seen in L. dumoffii cells parasitizing the amebic host Acanthamoeba culbertsoni.

Unlike its phage T4 counterpart (also known as endonuclease V), Micrococcus luteus UV endonuclease (pyrimidine dimer DNA glycosylase/apurinic-apyrimidinic endonuclease) has suffered from lack of genetic evidence to implicate it in the promotion of UV survival of the cell, i.e., mutants with its deficiency are no more UV-sensitive than the wild type. On the assumption that the contribution of UV endonuclease is obscured by the presence of a homolog of Escherichia coli UvrABC endonuclease, which has recently been identified in this bacterium, survival studies were carried out in its absence. With 254-nm UV irradiation, which generates not only pyrimidine dimers but also 6-4 photoproducts as lethal lesions, a double mutant defective in both UV endonuclease and the Uvr homolog was shown to be more sensitive than a single mutant defective only in the latter, with a dose reduction factor of approximately 2 at the survival level of 37%. Furthermore, molecular photosensitization, which produces only pyrimidine dimers, revealed an even greater difference in sensitivity, the dose reduction factor being about 3.4. These results indicate that the contribution to cell survival of UV endonuclease, an enzyme specific for pyrimidine dimers, is manifest if the backup by the Uvr homolog is absent.

Restriction fragments of Micrococcus luteus DNA containing the gene affected by a mutation in the UV-sensitive mutant DB7 were cloned both from the wild type and from the mutant in an Escherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to UV, mitomycin C, and 4-nitroquinoline 1-oxide by a one-step transformation. Determination of the nucleotide sequences revealed a potential open reading frame coding for a protein of 992 (tentative) amino acid residues, within which the DB7 mutation was identified as a CG-to-TA transition causing a translation termination. The putative product of the open reading frame shares an extensive amino acid sequence homology with the E. coli UvrA protein comprising 940 residues. The homology extends over the greater part of both polypeptides except for two extra sequences of 31 and 24 amino acid residues located at the amino-terminal and in the interior, respectively, of the M. luteus protein. In the homologous region, 56.7% and 16.7% of the 933 pairs of the aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. These results indicate that the gene defined by the mutation in DB7 represents a homolog of the E. coli uvrA gene. Hence, it has to be concluded that DB7, known for its deficiency in UV endonuclease (pyrimidine dimer DNA glycosylase/apurinicapyrimidinic endonuclease) activity, is a double mutant which is also defective in an enzyme complex similar to the E. coli UvrABC excinuclease.

Like recF and recQ mutants studied earlier, two other classes of Escherichia coli mutants defective in the RecF conjugal recombination pathway, recJ and recO, were found to be partially resistant to thymineless death. In contrast, a recN mutant, also belonging to the pathway, was indistinguishable from the wild type with respect to thymineless death.

SYNOPSIS The regeneration kinetics of Chlamydomonas reinhardtii mutants TS-6 and TS-79, whose flagella were mechanically amputated, indicated that the flagellar precursor in cytoplasm was used for regeneration when cycloheximide was present. The TS-6 cells rendered nonflagellate by regression at 35 C did not regenerate in the presence of cycloheximide, indicating that the precursor was inactivated by the high temperature. Neither mutant was able to use the absorbed flagellar components for regeneration in the presence of cycloheximide.

Restriction fragments of Micrococcus luteus DNA that contained the gene defined by the mutation of an excision repair-deficient mutant, UVsN1, were cloned from both the parental and mutant strains with the Escherichia coli host-vector system. The wild-type fragment was able to reverse the multiple sensitivity of the mutant to ultraviolet, mitomycin C, and 4-nitroquinoline-1-oxide by one-step transformation. Determination of the nucleotide sequences revealed an open reading frame potentially coding for a protein of 709 amino acid residues, within which the mutation was identified as a CG----TA transition causing a change from serine to phenylalanine. The putative product of the open reading frame showed an extensive amino acid sequence homology to the E. coli UvrB protein comprising 673 residues; the homologous region extended over the greater parts of both polypeptides, in which 55% and 17% of the 659 pairs of aligned amino acids were accounted for by conserved residues and conservative substitutions, respectively. This indicates that the gene defined by the UVsN1 mutation represents a homolog of the E. coli uvrB gene, implying the presence in M. luteus of an enzyme complex homologous to the E. coli UvrABC excinuclease.