Filament formation of Salmonella Paratyphi A accompanied by FtsZ assembly impairment and low level ppGpp.

Previously, we reported that Salmonella enterica serovar Paratyphi A strain S602 grew into multinuclear, nonseptate, and nonlethal filaments on agar plates containing nitrogenous salts. Strain S602 was more sensitive to osmotic and oxidative stress than the reference strain 3P243 of nonfilamentous Salmonella Paratyphi A. Strain S602 had an amber mutation (C154T) in rpoS. The revertant of this mutant, SR603, was repressed to form filaments under conditions with abundant nitrogenous salts. However, 3PR244, an rpoS mutant of 3P243 (C154T), did not form filaments, which implies that the rpoS mutation is not the sole cause of filamentation in strain S602. Next, we examined whether the level of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) in S602 strain is involved in filament formation. The intracellular ppGpp level in filamentous cells was lower than that in nonfilamentous cells. Furthermore, cells belonging to strain RE606, a derivative of S602 where the intracellular concentration of ppGpp was increased by overexpression of the relA gene, exhibited normal Z-ring formation and cell division. In the S602 strain, the decrease in the ppGpp level induced by the presence of nitrogenous salt and the rpoS mutation led to the inhibition of Z-ring formation and the subsequent filamentation of cells.

Elevated guanosine 5'-diphosphate 3'-diphosphate level inhibits bacterial growth and interferes with FtsZ assembly.

FtsZ, a protein essential for prokaryotic cell division, forms a ring structure known as the Z-ring at the division site. FtsZ has a GTP binding site and is assembled into linear structures in a GTP-dependent manner in vitro. We assessed whether guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a global regulator of gene expression in starved bacteria, affects cell division in Salmonella Paratyphi A. Elevation of intracellular ppGpp levels by using the relA expression vector induced repression of bacterial growth and incorrect FtsZ assembly. We found that FtsZ forms helical structures in the presence of ppGpp by using the GTP binding site; however, ppGpp levels required to form helical structures were at least 20-fold higher than the required GTP levels in vitro. Furthermore, once formed, helical structures did not change to the straight form even after GTP addition. Our data indicate that elevation of the ppGpp level leads to inhibition of bacterial growth and interferes with FtsZ assembly.

Hydrogen peroxide causes Vibrio vulnificus bacteriolysis accelerated by sulfonyl fluoride compounds.

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.

Supervised machine learning-based classification of oral malodor based on the microbiota in saliva samples.

OBJECTIVE: This study presents an effective method of classifying oral malodor from oral microbiota in saliva by using a support vector machine (SVM), an artificial neural network (ANN), and a decision tree. This approach uses concentrations of methyl mercaptan in mouth air as an indicator of oral malodor, and peak areas of terminal restriction fragment (T-RF) length polymorphisms (T-RFLPs) of the 16S rRNA gene as data for supervised machine-learning methods, without identifying specific species producing oral malodorous compounds. METHODS: 16S rRNA genes were amplified from saliva samples from 309 subjects, and T-RFLP analysis was carried out with the DNA fragments. T-RFLP analysis provides information on microbiota consisting of fragment lengths and peak areas corresponding to bacterial strains. The peak area is equivalent to the frequency of a specific fragment when one molecule is selected from terminal fragments. Another frequency is obtained by dividing the number of species-containing samples by the total number of samples. An SVM, an ANN, and a decision tree were trained based on these two frequencies in 308 samples and classified the presence or absence of methyl mercaptan in mouth air from the remaining subject. RESULTS: The proportion that trained SVM expressed as entropy achieved the highest classification accuracy, with a sensitivity of 51.1% and specificity of 95.0%. The ANN and decision tree provided lower classification accuracies, and only classification by the ANN was improved by weighting with entropy from the frequency of appearance in samples, which increased the accuracy to 81.9% with a sensitivity of 60.2% and a specificity of 90.5%. The decision tree showed low classification accuracy under all conditions. CONCLUSIONS: Using T-RF proportions and frequencies, models to classify the presence of methyl mercaptan, a volatile sulfur-containing compound that causes oral malodor, were developed. SVM classifiers successfully classified the presence of methyl mercaptan with high specificity, and this classification is expected to be useful for screening saliva for oral malodor before visits to specialist clinics. Classification by a SVM and an ANN does not require the identification of the oral microbiota species responsible for the malodor, and the ANN also does not require the proportions of T-RFs.

Effect of hyperbaric oxygen on Vibrio vulnificus and murine infection caused by it.

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.

Importance of the icmN gene in the growth of Legionella pneumophila in amoebic cells at low temperature.

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.

Antimicrobial and antifungal effects of tissue conditioners containing a photocatalyst.

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.

Characterization of MbrC involved in bacitracin resistance in Streptococcus mutans.

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 (D(54) N-MbrC) that substituted asparagine for aspartate at position 54, the predicted phosphorylation site. MbrC, but not the mutant D(54) N-MbrC, showed affinity for a DNA probe that contained the hypothetical mbrA promoter sequence. Furthermore, we introduced a point mutation (D(54) N-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.

Glucose metabolism in Legionella pneumophila: dependence on the Entner-Doudoroff pathway and connection with intracellular bacterial growth.

Glucose metabolism in Legionella pneumophila was studied by focusing on the Entner-Doudoroff (ED) pathway with a combined genetic and biochemical approach. The bacterium utilized exogenous glucose for synthesis of acid-insoluble cell components but manifested no discernible increase in the growth rate. Assays with permeabilized cell preparations revealed the activities of three enzymes involved in the pathway, i.e., glucokinase, phosphogluconate dehydratase, and 2-dehydro-3-deoxy-phosphogluconate aldolase, presumed to be encoded by the glk, edd, and eda genes, respectively. Gene-disrupted mutants for the three genes and the ywtG gene encoding a putative sugar transporter were devoid of the ability to metabolize exogenous glucose, indicating that the pathway is almost exclusively responsible for glucose metabolism and that the ywtG gene product is the glucose transporter. It was also established that these four genes formed part of an operon in which the gene order was edd-glk-eda-ywtG, as predicted by genomic information. Intriguingly, while the mutants exhibited no appreciable change in growth characteristics in vitro, they were defective in multiplication within eukaryotic cells, strongly indicating that the ED pathway must be functional for the intracellular growth of the bacterium to occur. Curiously, while the deficient glucose metabolism of the ywtG mutant was successfully complemented by the ywtG(+) gene supplied in trans via plasmid, its defect in intracellular growth was not. However, the latter defect was also manifested in wild-type cells when a plasmid carrying the mutant ywtG gene was introduced. This phenomenon, resembling so-called dominant negativity, awaits further investigation.

Conjugative plasmid pLD-TEX-KL promotes growth of host bacterium Legionella dumoffii at low temperatures.

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.

The ecological proportion of indigenous bacterial populations in saliva is correlated with oral health status.

To obtain deeper insights into the etiology of oral disease, an understanding of the composition of the surrounding bacterial environments that lead to health or disease is required, which is attracting increasing attention. In this study, the bacterial compositions in the saliva of 200 subjects aged 15-40 years were depicted as peak patterns by terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes. The subjects were classified into three clusters by partitioning around medoids clustering based on their T-RFLP profiles, and the clinical oral health parameters of the clusters were compared. The clustering of the T-RFLP profiles in this study was mainly based on differences in the abundance distribution of the dominant terminal restriction fragments (TRFs) detected in most of the subjects. Predicted from the sizes of the TRFs, the characteristically more predominant members of each were Prevotella and Veillonella species in cluster I; Streptococcus species in cluster II and Neisseria, Haemophilus or Aggregatibacter species and Porphyromonas species in cluster III. The parameters associated with periodontal disease were significantly different among the clusters. Clusters I and II had a higher percentage of sites of periodontal pockets greater than 4 mm than cluster III, and cluster I contained sites exhibiting bleeding on probing more often than cluster II or III; no significant differences were observed in other parameters. These results suggest that the abundance distribution of commensal bacteria in saliva is correlated with periodontal health, and might be involved in the susceptibility of an individual to periodontal disease.

Development and application of a T-RFLP data analysis method using correlation coefficient matrices.

Environmental microbiology studies commonly use terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes, for example, to analyze changes in community structure in relation to changing physicochemical and biological conditions over space and time. Although T-RFLP is most useful for comparing samples from different environments, a large number of samples makes effective analysis difficult using the Web-based tools that are currently available. To resolve this dilemma, we used a new approach for calculating data from multiple T-RFLP samples by estimating terminal fragment combinations, then applying a correlation analysis using two different fluorescent dyes generated simultaneously from all samples. This calculation was based on the expectation that the proportions of two terminal fragments from one full-length polymerase chain reaction fragment would be nearly the same in each analysis. Using this program, the oral microflora in 73 human saliva samples were analyzed, and 24 bacterial groups, with peak areas of at least 0.5% and correlation coefficients of 0.55 or greater, were identified from the T-RFs within 40 s.

Concerted action of lactate oxidase and pyruvate oxidase in aerobic growth of Streptococcus pneumoniae: role of lactate as an energy source.

Streptococcus pneumoniae was shown to possess lactate oxidase in addition to well-documented pyruvate oxidase. The activities of both H(2)O(2)-forming oxidases in wild-type cultures were detectable even in the early exponential phase of growth and attained the highest levels in the early stationary phase. For each of these oxidases, a defective mutant was constructed and compared to the parent regarding the dynamics of pyruvate and lactate in aerobic cultures. The results obtained indicated that the energy-yielding metabolism in the wild type could be best described by the following scheme. (i) As long as glucose is available, approximately one-fourth of the pyruvate formed is converted to acetate by the sequential action of pyruvate oxidase and acetate kinase with acquisition of additional ATP; (ii) the rest of the pyruvate is reduced by lactate dehydrogenase to form lactate, with partial achievement of redox balance; (iii) the lactate is oxidized by lactate oxidase back to pyruvate, which is converted to acetate as described above; and (iv) the sequential reactions mentioned above continue to occur as long as lactate is present. As predicted by this model, exogenously added lactate was shown to increase the final growth yield in the presence of both oxidases.

Identification and characterization of novel isoforms of human DP-1: DP-1{alpha} regulates the transcriptional activity of E2F1 as well as cell cycle progression in a dominant-negative manner.

The cell cycle-regulating transcription factors DP-1 and E2F form a heterodimeric complex and play a central role in cell cycle progression. Two different DP subunits (DP-1 and DP-2) exist in humans. In this study, we identified two novel DP-1 isoforms (DP-1alpha and DP-1beta) and characterized their structure and function. DP-1alpha is composed of 278 amino acids and lacks a portion of the C-terminal heterodimerization domain, whereas DP-1beta is composed of 357 amino acids with a frameshift that causes truncation of the C-terminal domain. Yeast two-hybrid and immunoprecipitation assays demonstrated that DP-1alpha binding to E2F1 was significantly reduced as compared with that of wild-type DP-1 or DP-1beta. Immunofluorescence analysis revealed that the subcellular localization of both DP-1 isoforms changed from the cytoplasm to the nucleus in HEK 293 cells cotransfected with E2F1 and wild-type DP-1 or DP-1beta. However, such a translocation for DP-1alpha was barely observed. Reverse transcription-PCR results showed that the three DP-1 isoforms are expressed ubiquitously at equal levels in several normal human tissues. We also demonstrated the expression of these isoforms at the protein level by Western blotting. Interestingly, we observed a significant decrease in transcriptional activity, a marked delay of cell cycle progression, and an inhibition of cell proliferation in DP-1alpha-transfected HEK 293 cells. Together, the results of the present study suggest that DP-1alpha is a novel isoform of DP-1 that acts as a dominant-negative regulator of cell cycle progression.