The role of two SARP family transcriptional regulators in regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1629-1639 ◽  
Author(s):  
Renata Novakova ◽  
Alena Rehakova ◽  
Peter Kutas ◽  
Lubomira Feckova ◽  
Jan Kormanec
Keyword(s):  
Gene Cluster ◽  
Wild Type Strain ◽  
Negative Regulator ◽  
Wild Type ◽  
Late Exponential Phase ◽  
Streptomyces Aureofaciens ◽  
Upstream Part ◽  
Complex Regulation

Two regulators, Aur1P and Aur1R, have been previously found to control expression of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239 in a cascade mechanism. Here, we describe the characterization of two additional regulatory genes, aur1PR2 and aur1PR3, encoding homologues of the SARP family of transcriptional activators that were identified in the upstream part of the aur1 cluster. Expression of both genes is directed by a single promoter, aur1PR2p and aur1Pr3p, respectively, induced in late exponential phase. Disruption of aur1PR2 in S. aureofaciens CCM 3239 had no effect on auricin production. However, the disruption of aur1PR3 dramatically reduced auricin compared with its parental wild-type strain. Transcription from the aur1Ap promoter, directing expression of the first biosynthetic gene in the auricin gene cluster, was similarly decreased in the S. aureofaciens CCM 3239 aur1PR3 mutant. Transcription from the aur1PR3p promoter increased in the S. aureofaciens CCM 3239 aur1R mutant strain, and the TetR family negative regulator Aur1R was shown to specifically bind the aur1PR3p promoter. These results indicate a complex regulation of the auricin cluster by the additional SARP family transcriptional activator Aur1PR3.

scholarly journals Characterization of the Second LysR-Type Regulator in the Biphenyl-Catabolic Gene Cluster of Pseudomonas pseudoalcaligenes KF707

2003 ◽  
Vol 185 (12) ◽  
pp. 3575-3582 ◽  
Author(s):  
Takahito Watanabe ◽  
Hidehiko Fujihara ◽  
Kensuke Furukawa
Keyword(s):  
Gene Cluster ◽  
Wild Type Strain ◽  
Dienoic Acid ◽  
Sole Source ◽  
Wild Type ◽  
Transcription Level ◽  
Pseudomonas Pseudoalcaligenes ◽  
High Level ◽  
Lysr Family

ABSTRACT Pseudomonas pseudoalcaligenes KF707 possesses a biphenyl-catabolic (bph) gene cluster consisting of bphR1A1A2-(orf3)-bphA3A4BCX0X1X2X3D. The bphR1 (formerly orf0) gene product, which belongs to the GntR family, is a positive regulator for itself and bphX0X1X2X3D. Further analysis in this study revealed that a second regulator belonging to the LysR family (designated bphR2) is involved in the regulation of the bph genes in KF707. The bphR2 gene was not located near the bph gene cluster, and its product (BphR2) exhibited a high level of similarity to NahR (the naphthalene- and salicylate-catabolic regulator belonging to the LysR family) in plasmid NAH7 of Pseudomonas putida. A strain containing a disrupted bphR2 gene failed to grow on biphenyl as a sole source of carbon, and the BphD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) activity was significantly reduced compared to that of wild-type strain KF707. Furthermore, the same strain exhibited extremely low transcription of bphR1, bphA1, bphC, bphX0, and bphD. However, when the bphR2 gene was provided in trans to the bphR2-disrupted strain, the transcription level of these genes was restored. These results indicate that bphR2 regulates the bph genes positively as a second regulator together with BphR1.

scholarly journals Role of the Type VI Secretion System in the Pathogenicity of Pseudomonas syringae pv. actinidiae, the Causative Agent of Kiwifruit Bacterial Canker

2021 ◽  
Vol 12 ◽  
Author(s):  
Nana Wang ◽  
Ning Han ◽  
Runze Tian ◽  
Jiliang Chen ◽  
Xiaoning Gao ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Pseudomonas Syringae ◽  
Biofilm Formation ◽  
Wild Type Strain ◽  
Deletion Mutants ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Bacterial Competition ◽  
Type Vi Secretion

The type VI secretion system (T6SS), a macromolecular machine, plays an important role in the pathogenicity of many Gram-negative bacteria. However, the role of T6SS in the pathogenicity of Pseudomonas syringae pv. actinidiae (Psa), the pathogen of kiwifruit bacterial canker, is yet to be studied. Here, we found a T6SS gene cluster consisting of 13 core genes (A-J) in the genome of Psa M228 based on a genome-wide analysis. To determine whether the T6SS gene cluster affects the pathogenicity of Psa M228, T6SS and its 13 core gene deletion mutants were constructed and their pathogenicity was determined. The deletion mutants showed different degrees of reduction in pathogenicity compared with the wild-type strain M228; in tssM and tssJ mutants, pathogenicity was significantly reduced by 78.7 and 71.3%, respectively. The pathogenicity results were also confirmed by electron microscopy. To further confirm that the reduction in pathogenicity is related to the function of T6SS, we selected the T6SS gene cluster, comprising tssM and tssJ, for further analyses. Western blot results revealed that tssM and tssJ were necessary for hemolytic co-regulatory protein secretion, indicating that they encode a functional T6SS. Further, we explored the mechanism by which T6SS affects the pathogenicity of Psa M228. The ability of bacterial competition, biofilm formation, hydrogen peroxide tolerance, and proteolytic activity were all weakened in the deletion mutants M228ΔT6SS, M228ΔtssM, and M228ΔtssJ. All these properties of the two gene complementation mutants were restored to the same levels as those of the wild-type strain, M228. Quantitative real-time results showed that during the interaction between the deletion mutant M228ΔT6SS and the host, expression levels of T3SS transcriptional regulatory gene hrpR, structural genes hrpZ, hrcC, hopP1, and effector genes hopH1 and hopM1 were down-regulated at different levels. Taken together, our data provide evidence for the first time that the T6SS plays an important role in the pathogenicity of Psa, probably via effects on bacterial competition, biofilm formation, and environmental adaptability. Moreover, a complicated relationship exists between T6SS and T3SS.

scholarly journals H-NS Is a Negative Regulator of the Two Hemolysin/Cytotoxin Gene Clusters in Vibrio anguillarum

2013 ◽  
Vol 81 (10) ◽  
pp. 3566-3576 ◽  
Author(s):  
Xiangyu Mou ◽  
Edward J. Spinard ◽  
Maureen V. Driscoll ◽  
Wenjing Zhao ◽  
David R. Nelson
Keyword(s):  
Gene Cluster ◽  
Hemolytic Activity ◽  
Type Strain ◽  
Wild Type Strain ◽  
Vibrio Anguillarum ◽  
Gene Clusters ◽  
Negative Regulator ◽  
Wild Type ◽  
Promoter Regions ◽  
Content Type

ABSTRACTHemolysins produced byVibrio anguillarumhave been implicated in the development of hemorrhagic septicemia during vibriosis, a fatal fish disease. Previously, two hemolysin gene clusters responsible for the hemolysis and cytotoxicity ofV. anguillarumwere identified: thevah1-plpgene cluster and thertxACHBDEgene cluster. In this study, we identified thehnsgene, which encodes the H-NS protein and acts as a negative regulator of both gene clusters. TheV. anguillarumH-NS protein shares strong homology with other bacterial H-NS proteins. Anhnsmutant exhibited increased hemolytic activity and cytotoxicity compared to the wild-type strain. Complementation of thehnsmutation restored hemolytic activity and cytotoxicity levels to nearly wild-type levels. Furthermore, expression ofrtxA,rtxH,rtxB,vah1, andplpincreased in thehnsmutant and decreased in thehns-complemented mutant strain compared to expression in the wild-type strain. Additionally, experiments using DNase I showed that purified recombinant H-NS protected multiple sites in the promoter regions of both gene clusters. Thehnsmutant also exhibited significantly attenuated virulence against rainbow trout. Complementation of thehnsmutation restored virulence to wild-type levels, suggesting that H-NS regulates many genes that affect fitness and virulence. Previously, we showed that HlyU is a positive regulator of expression for both gene clusters. In this study, we demonstrate that upregulation byhlyUishnsdependent, suggesting that H-NS acts to repress or silence both gene clusters and HlyU acts to relieve that repression or silencing.

scholarly journals The role of the TetR-family transcriptional regulator Aur1R in negative regulation of the auricin gene cluster in Streptomyces aureofaciens CCM 3239

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2374-2383 ◽  
Author(s):  
Renata Novakova ◽  
Peter Kutas ◽  
Lubomira Feckova ◽  
Jan Kormanec
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Transcriptional Repressors ◽  
Biosynthetic Gene ◽  
Response Regulators ◽  
Streptomyces Aureofaciens ◽  
Two Component Signal Transduction

Two regulatory genes, aur1P and aur1R, have been previously identified upstream of the aur1 polyketide gene cluster involved in biosynthesis of the angucycline-like antibiotic auricin in Streptomyces aureofaciens CCM 3239. The aur1P gene encodes a protein similar to the response regulators of bacterial two-component signal transduction systems and has been shown to specifically activate expression of the auricin biosynthetic genes. The aur1R gene encodes a protein homologous to transcriptional repressors of the TetR family. Here we describe the characterization of the aur1R gene. Expression of the gene is directed by a single promoter, aur1Rp, which is induced just before stationary phase. Disruption of aur1R in S. aureofaciens CCM 3239 had no effect on growth and differentiation. However, the disrupted strain produced more auricin than its parental wild-type S. aureofaciens CCM 3239 strain. Transcription from the aur1Ap and aur1Pp promoters, directing expression of the first biosynthetic gene in the auricin gene cluster and the pathway-specific transcriptional activator, respectively, was increased in the S. aureofaciens CCM 3239 aur1R mutant strain. However, Aur1R was shown to bind specifically only to the aur1Pp promoter in vitro. This binding was abolished by the addition of auricin and/or its intermediates. The results indicate that the Aur1R regulator specifically represses expression of the aur1P gene, which encodes a pathway-specific activator of the auricin biosynthetic gene cluster in S. aureofaciens CCM 3239, and that this repression is relieved by auricin or its intermediates.

scholarly journals ClpC acts as a negative regulator of competence in Streptococcus thermophilus

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1676-1684 ◽  
Author(s):  
Truls Johan Biørnstad ◽  
Leiv Sigve Håvarstein
Keyword(s):  
Sigma Factor ◽  
Wild Type Strain ◽  
Natural Transformation ◽  
Streptococcus Thermophilus ◽  
Negative Regulator ◽  
Wild Type ◽  
Environmental Stimulus ◽  
Mutant Strains ◽  
Low Levels

The alternative sigma factor ComX is a key regulator of natural transformation in members of the genus Streptococcus. ComX controls expression of the late competence genes, which are essential for DNA binding, uptake and recombination. In Streptococcus pneumoniae, it has been demonstrated that ComX is degraded by ClpEP at the end of the competence period. In the present study we show that a different Clp protease complex, ClpCP, contributes to ComX degradation in Streptococcus thermophilus. Mutant strains lacking the ClpC chaperone displayed significantly increased transformability compared with the wild-type strain under conditions where ComX was expressed at relatively low levels. At higher expression levels, ClpCP appears to become saturated and unable to prevent the accumulation of ComX. Together, our results suggest that the role of ClpC is to mediate degradation of ComX when the sigma factor is produced in low amounts, i.e. when the environmental stimulus promoting competence development is weak. This would prevent S. thermophilus from developing the competent state at an inappropriate time and/or place.

scholarly journals Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

2021 ◽  
Author(s):  
Chang-Hun Shin ◽  
Hang Soo Cho ◽  
Hyung-Jin Won ◽  
Ho Jeong Kwon ◽  
Chan-Wha Kim ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Mutant Selection ◽  
Enhanced Production ◽  
Industrial Strains ◽  
Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

scholarly journals A Novel CO-Responsive Transcriptional Regulator and Enhanced H2Production by an Engineered Thermococcus onnurineus NA1 Strain

2014 ◽  
Vol 81 (5) ◽  
pp. 1708-1714 ◽  
Author(s):  
Min-Sik Kim ◽  
Ae Ran Choi ◽  
Seong Hyuk Lee ◽  
Hae-Chang Jung ◽  
Seung Seob Bae ◽  
...  
Keyword(s):  
Production Rate ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory System ◽  
Transcriptional Regulator ◽  
Bioreactor Culture ◽  
Wild Type ◽  
Content Type ◽  
Transcriptional Regulatory

ABSTRACTGenome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism inThermococcus onnurineusNA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes ofThermococcusspecies and “CandidatusKorarchaeum cryptofilum” OPF8. In-frame deletion of eithercorQorcorRcaused a severe impairment in CO-dependent growth and H2production. WhencorQandcorRdeletion mutants were complemented by introducing thecorQRgenes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integratedcorQR(ΔCorR/corQR↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR↑strain exhibited a much higher H2production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2production rate (191.9 mmol liter−1h−1) and the specific H2production rate (249.6 mmol g−1h−1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that thecorQRgenes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2production.

Characterization of Enterococcus faecium mutants resistant to mundticin KS, a class IIa bacteriocin

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2901-2908 ◽  
Author(s):  
Youko Sakayori ◽  
Mizuho Muramatsu ◽  
Satoshi Hanada ◽  
Yoichi Kamagata ◽  
Shinichi Kawamoto ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Antimicrobial Agents ◽  
Wild Type Strain ◽  
Unsaturated Fatty Acids ◽  
Class I ◽  
Wild Type ◽  
Food Preservatives ◽  
In The Wild ◽  
Resistant Mutants

The emergence and spread of mutants resistant to bacteriocins would threaten the safety of using bacteriocins as food preservatives. To determine the physiological characteristics of resistant mutants, mutants of Enterococcus faecium resistant to mundticin KS, a class IIa bacteriocin, were isolated. Two types of mutant were found that had different sensitivities to other antimicrobial agents such as nisin (class I) and kanamycin. Both mutants were resistant to mundticin KS even in the absence of Mg2+ ions. The composition of unsaturated fatty acids in the resistant mutants was significantly increased in the presence of mundticin KS. The composition of the phospholipids in the two resistant mutants also differed from those in the wild-type strain. The putative zwitterionic amino-containing phospholipid in both mutants significantly increased, whereas amounts of phosphatidylglycerol and cardiolipin decreased. These changes in membrane structure may influence resistance of enterococci to class IIa and class I bacteriocins.

Characterization of the Role of the FluG Protein in Asexual Development of Aspergillus nidulans

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1027-1036 ◽  
Author(s):  
Cletus A D'Souza ◽  
Bee Na Lee ◽  
Thomas H Adams
Keyword(s):  
Aspergillus Nidulans ◽  
Negative Influence ◽  
Asexual Development ◽  
Wild Type ◽  
Significant Similarity ◽  
Developmental Defects ◽  
Asexual Sporulation ◽  
Type Strains

Abstract We showed previously that a ΔfluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (∼400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of ΔfluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from ΔflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

scholarly journals Accumulation of Inorganic Polyphosphate in phoU Mutants of Escherichia coli and Synechocystis sp. Strain PCC6803

2002 ◽  
Vol 68 (8) ◽  
pp. 4107-4110 ◽  
Author(s):  
Tomohiro Morohoshi ◽  
Tatsuya Maruo ◽  
Yoko Shirai ◽  
Junichi Kato ◽  
Tsukasa Ikeda ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Biological Process ◽  
Negative Regulator ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Insertional Inactivation ◽  
Synechocystis Sp ◽  
Polyp Accumulation

ABSTRACT The biological process for phosphate (Pi) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the Pi regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more Pi from the medium than the wild-type strain removed.

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