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  • American Society for Microbiology  (5)
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  • American Society for Microbiology  (5)
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  • Biology  (5)
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  • 1
    Online Resource
    Online Resource
    Organic Hydroperoxide Induces Prodigiosin Biosynthesis in Serratia sp. ATCC 39006 in an OhrR-Dependent Manner
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 5 ( 2022-03-08)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 5 ( 2022-03-08)
    Abstract: The biosynthesis of prodigiosin in the model prodigiosin-producing strain, Serratia sp. ATCC 39006, is significantly influenced by environmental and cellular signals. However, a comprehensive regulatory mechanism for this process has not been well established. In the present study, we demonstrate that organic hydroperoxide activates prodigiosin biosynthesis in an OhrR-dependent manner. Specifically, the MarR-family transcriptional repressor OhrR (Ser39006_RS05455) binds to its operator located far upstream of the promoter region of the prodigiosin biosynthesis operon (319 to 286 nucleotides [nt] upstream of the transcription start site) and negatively regulates the expression of prodigiosin biosynthesis genes. Organic hydroperoxide disassociates the binding between OhrR and its operator, thereby promoting the prodigiosin production. Moreover, OhrR modulates the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide by regulating the transcription of its own gene and the downstream cotranscribed ohr gene. These results demonstrate that OhrR is a pleiotropic repressor that modulates the prodigiosin production and the resistance of Serratia sp. ATCC 39006 to organic hydroperoxide stress. IMPORTANCE Bacteria naturally encounter various environmental and cellular stresses. Organic hydroperoxides generated from the oxidation of polyunsaturated fatty acids are widely distributed and usually cause lethal oxidative stress by damaging cellular components. OhrR is known as a regulator that modulates the resistance of bacteria to organic hydroperoxide stress. In the current study, organic hydroperoxide disassociates OhrR from the promoter of prodigiosin biosynthesis gene cluster, thus promoting transcription of pigA to - O genes. In this model, organic hydroperoxide acts as an inducer of prodigiosin synthesis in Serratia sp. ATCC 39006. These results improve our understanding of the regulatory network of prodigiosin synthesis and serve as an example for identifying the cross talk between the stress responses and the regulation of secondary metabolism.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.02041-21
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
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    Online Resource
  • 2
    Online Resource
    Online Resource
    Proteorhodopsin Overproduction Enhances the Long-Term Viability of Escherichia coli
    American Society for Microbiology ; 2019
    In:  Applied and Environmental Microbiology Vol. 86, No. 1 ( 2019-12-13)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 86, No. 1 ( 2019-12-13)
    Abstract: Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli . Quantitative mass spectrometry shows that E. coli , exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ∼148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans. IMPORTANCE Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis . We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.02087-19
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
    Location Call Number Limitation Availability
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    Online Resource
  • 3
    Online Resource
    Online Resource
    Transcriptional Regulator PhlH Modulates 2,4-Diacetylphloroglucinol Biosynthesis in Response to the Biosynthetic Intermediate and End Product
    American Society for Microbiology ; 2017
    In:  Applied and Environmental Microbiology Vol. 83, No. 21 ( 2017-11)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 83, No. 21 ( 2017-11)
    Abstract: Certain strains of biocontrol bacterium Pseudomonas fluorescens produce the secondary metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) to antagonize soilborne phytopathogens in the rhizosphere. The gene cluster responsible for the biosynthesis of 2,4-DAPG is named phlACBDEFGH and it is still unclear how the pathway-specific regulator phlH within this gene cluster regulates the metabolism of 2,4-DAPG. Here, we found that PhlH in Pseudomonas fluorescens strain 2P24 represses the expression of the phlG gene encoding the 2,4-DAPG hydrolase by binding to a sequence motif overlapping with the −35 site recognized by σ 70 factors. Through biochemical screening of PhlH ligands we identified the end product 2,4-DAPG and its biosynthetic intermediate monoacetylphloroglucinol (MAPG), which can act as signaling molecules to modulate the binding of PhlH to the target sequence and activate the expression of phlG . Comparison of 2,4-DAPG production between the Δ phlH , Δ phlG , and Δ phlHG mutants confirmed that phlH and phlG impose negative feedback regulation over 2,4-DAPG biosynthesis. It was further demonstrated that the 2,4-DAPG degradation catalyzed by PhlG plays an insignificant role in 2,4-DAPG tolerance but contributes to bacterial growth advantages under carbon/nitrogen starvation conditions. Taken together, our data suggest that by monitoring and down-tuning in situ levels of 2,4-DAPG, the phlHG genes could dynamically modulate the metabolic loads attributed to 2,4-DAPG production and potentially contribute to rhizosphere adaptation. IMPORTANCE 2,4-DAPG, which is synthesized by biocontrol pseudomonad bacteria, is a broad-spectrum antibiotic against bacteria, fungi, oomycetes, and nematodes and plays an important role in suppressing soilborne plant pathogens. Although most of the genes in the 2,4-DAPG biosynthetic gene cluster ( phl ) have been characterized, it is still not clear how the pathway-specific regulator phlH is involved in 2,4-DAPG metabolism. This work revealed the role of PhlH in modulating 2,4-DAPG levels by controlling the expression of 2,4-DAPG hydrolase PhlG in response to 2,4-DAPG and MAPG. Since 2,4-DAPG biosynthesis imposes a metabolic burden on biocontrol pseudomonads, it is expected that the fine regulation of phlG by PhlH offers a way to dynamically modulate the metabolic loads attributed to 2,4-DAPG production.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.01419-17
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2017
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
    Location Call Number Limitation Availability
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    Online Resource
  • 4
    Online Resource
    Online Resource
    Cys 2 His 2 Zinc Finger Transcription Factor BcabaR1 Positively Regulates Abscisic Acid Production in Botrytis cinerea
    American Society for Microbiology ; 2018
    In:  Applied and Environmental Microbiology Vol. 84, No. 17 ( 2018-09)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 84, No. 17 ( 2018-09)
    Abstract: Abscisic acid (ABA) is one of the five classical phytohormones involved in increasing the tolerance of plants for various kinds of stresses caused by abiotic or biotic factors, and it also plays important roles in regulating the activation of innate immune cells and glucose homeostasis in mammals. For these reasons, as a “stress hormone,” ABA has recently received attention as a candidate drug for agriculture and biomedical applications, prompting significant development of ABA synthesis. Some plant-pathogenic fungi can synthesize natural ABA. The fungus Botrytis cinerea has been used for biotechnological production of ABA. Identification of the transcription factors (TFs) involved in regulation of ABA biosynthesis in B. cinerea would provide new clues to understand how ABA is synthesized and regulated. In this study, we defined a novel Cys 2 His 2 TF, BcabaR1, that regulates the transcriptional levels of ABA synthase genes ( bcaba1 , bcaba2 , bcaba3 , and bcaba4 ) in an ABA-overproducing mutant, B. cinerea TBC-A. Electrophoretic mobility shift assays revealed that recombinant BcabaR1 can bind specifically to both a 14-nucleotide sequence motif and a 39-nucleotide sequence motif in the promoter region of bcaba1 to - 4 genes in vitro . A decreased transcriptional level of the bcabaR1 gene in B. cinerea led to significantly decreased ABA production and downregulated transcription of bcaba1 to - 4 . When bcabaR1 was overexpressed in B. cinerea , ABA production was significantly increased, with upregulated transcription of bcaba1 to - 4 . Thus, in this study, we found that BcabaR1 acts as a positive regulator of ABA biosynthesis in B. cinerea . IMPORTANCE Abscisic acid (ABA) could make a potentially important contribution to theoretical research and applications in agriculture and medicine. Botrytis cinerea is a plant-pathogenic fungus that was found to produce ABA. There has been a view that ABA is related to the interaction between pathogenic fungi and plants. Identification of regulatory genes involved in ABA biosynthesis may facilitate an understanding of the underlying molecular mechanisms of ABA biosynthesis and the pathogenesis of B. cinerea . Here, we present a positive regulator, BcabaR1, of ABA biosynthesis in B. cinerea that can affect the transcriptional level of the ABA biosynthesis gene cluster, bcaba1 to - 4 , by directly binding to the conserved sequence elements in the promoter of the bcaba1 to - 4 genes. This TF was found to be specifically involved in regulation of ABA biosynthesis. This work provides new clues for finding other ABA biosynthesis genes and improving ABA yield in B. cinerea .
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/AEM.00920-18
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2018
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
    Location Call Number Limitation Availability
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    Online Resource
  • 5
    Online Resource
    Online Resource
    Engineering Novel ( R )-Selective Transaminase for Efficient Symmetric Synthesis of d -Alanine
    American Society for Microbiology ; 2022
    In:  Applied and Environmental Microbiology Vol. 88, No. 9 ( 2022-05-10)
    Details
    In: Applied and Environmental Microbiology, American Society for Microbiology, Vol. 88, No. 9 ( 2022-05-10)
    Abstract: d -Alanine belongs to nonessential amino acids that have diverse applications in the fields of food and health care. ( R )-transaminase [( R )-TA]-catalyzed asymmetric amination of pyruvate is a feasible alternative for the synthesis of d -alanine, but low catalytic efficiency and thermostability limit enzymatic utilization. In this work, several potential ( R )-TAs were discovered using NCBI database mining synchronously with enzymatic structure-function analysis, among which Capronia epimyces TA (CeTA) showed the highest activity for amination of pyruvate using ( R )-α-methylbenzylamine as the donor. Furthermore, enzymatic residues surrounding a large catalysis pocket were subjected to saturation and combinatorial mutagenesis, and positive mutant F113T showed dramatic improvement in activity and thermostability. Molecular modeling indicated that the substitution of phenylalanine with threonine afforded alleviation of steric hindrance in the pocket and induced formation of additional hydrogen bonds with neighboring residues. Finally, using recombinant cells containing F113T as a biocatalyst, the conversion yield of amination of 100 mM pyruvate to d -alanine achieved up to 95.2%, which seemed to be the highest level in the literature regarding synthesis of d -alanine using TAs. The inherent characteristics rendered CeTA F113T a promising platform for efficient preparation of d -alanine operating with high productivity. IMPORTANCE d -Alanine is an important compound with many valuable applications. Its asymmetric synthesis employing ( R )-ω-TA is considered an attractive choice. According to the stereoselectivity, ω-TAs have either ( R )- or ( S )-enantiopreference. There has been a variety of literature regarding screening, characterizing, and molecular modification of ( S )-ω-TAs; in contrast, the research about ( R )-ω-TA has lagged behind. In this work, we identify several ( R )-ω-TAs and succeeded in creating mutant F113T, which showed not only better efficiency toward pyruvate but also higher thermostability compared with the original enzyme. The obtained original enzymes and positive mutants displayed important application value for pushing symmetric synthesis of d -alanine to a higher level.
    Type of Medium: Online Resource
    ISSN: 0099-2240 , 1098-5336
    URL: Article
    DOI: 10.1128/aem.00062-22
    RVK:
    WA 15000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2022
    detail.hit.zdb_id: 223011-2
    detail.hit.zdb_id: 1478346-0
    SSG: 12
    Location Call Number Limitation Availability
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    Online Resource
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