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  • 1
    Publication Date: 2017-01-19
    Description: Although nitric oxide (NO) is an important signaling molecule in bacteria and higher organisms, excessive intracellular NO is highly reactive and dangerous. Therefore, living cells need strict regulation systems for cellular NO homeostasis. Recently, we discovered that Streptomyces coelicolor A3(2) retains the nitrogen oxide cycle (NO 3 – -〉NO 2 – -〉NO-〉NO 3 – ) and nitrite removal system. The nitrogen oxide cycle regulates cellular NO levels, thereby controlling secondary metabolism initiation (red-pigmented antibiotic, RED production) and morphological differentiation. Nitrite induces gene expression in neighboring cells, suggesting another role for this cycle as a producer of transmittable intercellular communication molecules. Here, we demonstrated that ammonium-producing nitrite reductase (NirBD) is involved in regulating NO homeostasis in S. coelicolor A3(2). NirBD was constitutively produced in culture independently of GlnR, a known transcriptional factor. NirBD cleared the accumulated nitrite from the medium. Nir deletion mutants showed increased NO-dependent gene expression at later culture stages, whereas the wild-type M145 showed decreased expression, suggesting that high NO concentration was maintained in the mutant. Moreover, the nir deletion mutant produced more RED than that produced by the wild-type M145. These results suggest that NO 2 – removal by NirBD is important to regulate NO homeostasis and to complete NO signaling in S. coelicolor .
    Keywords: Physiology & Biochemistry
    Print ISSN: 0378-1097
    Electronic ISSN: 1574-6968
    Topics: Biology
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  • 2
    Publication Date: 2017-01-13
    Description: Earlier, vitamin C was demonstrated to sterilize Mycobacterium tuberculosis culture via Fenton's reaction at high concentration. It alters the regulatory pathways associated with stress response and dormancy. Since (p)ppGpp is considered to be the master regulator of stress response and is responsible for bacterial survival under stress, we tested the effect of vitamin C on the formation of (p)ppGpp. In vivo estimation of (p)ppGpp showed a decrease in (p)ppGpp levels in vitamin C-treated M. smegmatis cells in comparison to the untreated cells. Furthermore, in vitro (p)ppGpp synthesis using Rel MSM enzyme was conducted in order to confirm the specificity of the inhibition in the presence of variable concentrations of vitamin C. We observed that vitamin C at high concentration can inhibit the synthesis of (p)ppGpp. We illustrated binding of vitamin C to Rel MSM by isothermal titration calorimetry. Enzyme kinetics was followed where K 0.5 was found to be increased with the concomitant reduction of V max value suggesting mixed inhibition. Both long-term survival and biofilm formation were inhibited by vitamin C. The experiments suggest that vitamin C has the potential to be developed as the inhibitor of (p)ppGpp synthesis and stress response, at least in the concentration range used here.
    Keywords: Physiology & Biochemistry
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  • 3
    Publication Date: 2016-12-23
    Description: The culturability of Escherichia coli , Ralstonia eutropha and Bacillus subtilis after incubation in phosphate-buffered saline at either 5°C or 30°C was determined. The culturability of B. subtilis showed little dependence on temperature. The culturability of E. coli rapidly decreased at 30°C but remained almost constant at 5°C. In contrast, the culturability of R. eutropha decreased by three orders of magnitude at 5°C within 24 h but only moderately decreased (one order of magnitude) at 30°C. Remarkably, prolonged incubation of R. eutropha at 30°C resulted in a full recovery of colony forming units in contrast to only a partial recovery at 5°C. Ralstonia eutropha cells at 30°C remained culturable for 3 weeks while culturability at 5°C constantly decreased. The effect of temperature was significantly stronger in a polyhydroxybutyrate-negative mutant. Our data show that accumulated polyhydroxybutyrate has a cold-protective function and can prevent R. eutropha entering the viable but not culturable state.
    Keywords: Physiology & Biochemistry
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  • 4
    Publication Date: 2016-12-16
    Description: Pseudomonas plecoglossicida is a facultative fish pathogen. Recent studies showed that P. plecoglossicida infection in fish was associated with temperature. The aim of this study was to compare the secretomes of P. plecoglossicida cultured in vitro at representative temperatures for pathogenic (20°C) and less pathogenic (30°C) phenotypes. Thirteen proteins in the culture supernatants of P. plecoglossicida showed significant difference in abundance at 20 vs. 30°C. Four proteins were strongly increased at 20°C, including two hemolysin co-regulated proteins (Hcp) that are part of the bacterial type VI secretion system (T6SS), flagellin and an unknown protein. Immunoblot analysis verified the induced secretion of Hcps at 20°C. Furthermore, the upregulation of Hcps at 20°C was confirmed at transcriptional level by RT-qPCR analysis, which also demonstrated the induction of expression of other T6SS-related genes at 20°C. Taken together, we demonstrate the presence of two functionally active T6SS proteins in fish pathogenic P. plecoglossicida strains, as evidenced by the secretion of the T6SS substrate Hcp, the production of which were found to be controlled by temperature. Our findings also support efforts to develop vaccines targeting secreted virulence factors as prophylactic strategies for diseases in fish caused by P. plecoglossicida .
    Keywords: Physiology & Biochemistry
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  • 5
    Publication Date: 2016-10-22
    Description: The hydrocarbonoclastic bacterium Acinetobacter venetianus RAG-1 has attracted substantial attention due to its powerful oil-degrading capabilities and its potential to play an important ecological role in the cleanup of alkanes. In this study, we compare the transcriptome of the strain RAG-1 grown in dodecane, the corresponding alkanol (dodecanol), and sodium acetate for the characterization of genes involved in dodecane uptake and utilization. Comparison of the transcriptional responses of RAG-1 grown on dodecane led to the identification of 1074 genes that were differentially expressed relative to sodium acetate. Of these, 622 genes were upregulated when grown in dodecane. The highly upregulated genes were involved in alkane catabolism, along with stress response. Our data suggest AlkMb to be primarily involved in dodecane oxidation. Transcriptional response of RAG-1 grown on dodecane relative to dodecanol also led to the identification of permease, outer membrane protein and thin fimbriae coding genes potentially involved in dodecane uptake. This study provides the first model for key genes involved in alkane uptake and metabolism in A. venetianus RAG-1.
    Keywords: Physiology & Biochemistry
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  • 6
    Publication Date: 2016-10-22
    Description: Dissimilatory sulfate reduction is the central microbial metabolism in global sulfur cycling. Understanding the importance of sulfate reduction to Earth's biogeochemical S cycle requires aggregating single-cell processes with geochemical signals. For sulfate reduction, these signals include the ratio of stable sulfur isotopes preserved in minerals, as well as the hydrogen isotope ratios and structures of microbial membrane lipids preserved in organic matter. In this study, we cultivated the model sulfate reducer, Desulfovibrio vulgaris DSM 644 T , to investigate how these parameters were perturbed by changes in expression of the protein DsrC. DsrC is critical to the final metabolic step in sulfate reduction to sulfide. S and H isotopic fractionation imposed by the wild type was compared to three mutants. Discrimination against 34 S in sulfate, as calculated from the residual reactant, did not discernibly differ among all strains. However, a closed-system sulfur isotope distillation model, based on accumulated sulfide, produced inconsistent results in one mutant strain IPFG09. Lipids produced by IPFG09 were also slightly enriched in 2 H. These results suggest that DsrC alone does not have a major impact on sulfate-S, though may influence sulfide-S and lipid-H isotopic compositions. While intriguing, a mechanistic explanation requires further study under continuous culture conditions.
    Keywords: Physiology & Biochemistry
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  • 7
    Publication Date: 2016-10-20
    Description: Corynebacterium glutamicum is able to metabolize different nitrogen and carbon sources. In standard minimal media, ammonium and urea typically serve as nitrogen source and glucose or sucrose as carbon and energy source; however, amino acids might also play a role as nitrogen, carbon and energy source. In this study, the function of the putative glutaminase GlsK was investigated. A glsK deletion strain showed impaired growth with L-glutamine as carbon and energy source, while growth was improved upon glsK overexpression. GlsK possesses a carboxy-terminal domain that seems to be restricted to Corynebacterium species . A truncated GlsK lacking this extension led to faster growth, indicating a regulatory function of this domain. In fact, GlsK activity is regulated in a pH-dependent manner depending on the carboxy-terminal extension, and is positively influenced by cAMP. Furthermore, the C-terminal extension seems to be important for oligomerization of GlsK.
    Keywords: Physiology & Biochemistry
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  • 8
    Publication Date: 2016-10-08
    Description: In a non-targeted analysis of thiol-containing compounds in the hyperthermophilic methanogen Methanocaldococcus jannaschii , we discovered three unexpected metabolites: 3-mercaptopropionic acid (MPA), 2-hydroxy-4-mercaptobutyric acid (HMBA) and 4-mercapto-2-oxobutyric acid (MOB). HMBA and MOB have never been reported as natural products, while MPA is highly prevalent in aquatic environments as a result of biotic and abiotic processing of sulfur-containing compounds. This report provides evidence that HMBA and MOB are part of a biosynthetic pathway to generate MPA in M. jannaschii . We show that HMBA can be biosynthesized from malate semialdehyde and hydrogen sulfide, likely using a mechanism similar to that proposed for coenzyme M, coenzyme B and homocysteine biosynthesis in methanogens, where an aldehyde is converted to a thiol. The L-sulfolactate dehydrogenase, derived from the MJ1425 gene, is shown to catalyze the NAD-dependent oxidation of HMBA to MOB. Finally, we demonstrate that HMBA can be used as a biosynthetic precursor to MPA in M. jannaschii cell extracts. This proposed pathway may contribute to the wide occurrence of MPA in marine environments and indicates that MPA must serve some important function in M. jannaschii .
    Keywords: Physiology & Biochemistry
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  • 9
    Publication Date: 2016-10-08
    Description: Upon transition of Mycobacterium smegmatis into the dormant state, accumulation of a dark brown fluorescent pigment was observed. This pigment gave bright red fluorescence in both cells and the culture medium. Based on 1 H-NMR, MALDI and UV spectra, the fluorescent compounds, extracted from the culture medium as well as from the dormant cells, were concluded to be a mixture of free coproporphyrin III and uroporphyrin III and their corresponding methyl esters. A possible significance of porphyrin pigment accumulation in the dormant cells is discussed.
    Keywords: Physiology & Biochemistry
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  • 10
    Publication Date: 2016-10-08
    Description: Secreted proteins, those involved in cell wall biogenesis, are likely to play a role in communication in the symbiotic interaction between the fungal endophyte Epichloë festucae with perennial ryegrass ( Lolium perenne ), particularly given the close association between fungal hyphae and the plant cell wall. Our hypothesis was that secreted proteins are likely to be responsible for establishing and maintaining a normal symbiotic relationship. We analyzed an endophyte EST database for genes with predicted signal peptide sequences. Here, we report the identification and characterization of rhgA ; a gene involved in the regulation of hyphal growth in planta . In planta analysis of rhgA mutants showed that disruption of rhgA resulted in extensive unregulated hyphal growth. This phenotype was fully complemented by insertion of the rhgA gene and suggests that rhgA is important for maintaining normal hyphal growth during symbiosis.
    Keywords: Physiology & Biochemistry
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