scholarly journals The Pathway-Specific Regulator ClaR of Streptomyces clavuligerus Has a Global Effect on the Expression of Genes for Secondary Metabolism and Differentiation

2015 ◽  
Vol 81 (19) ◽  
pp. 6637-6648 ◽  
Author(s):  
Yolanda Martínez-Burgo ◽  
Rubén Álvarez-Álvarez ◽  
Antonio Rodríguez-García ◽  
Paloma Liras
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Aerial Mycelium ◽  
Gene Clusters ◽  
Streptomyces Clavuligerus ◽  
Plasmid Copy Number ◽  
Wild Type ◽  
Plasmid Copy ◽  
Content Type ◽  
Expression Of Genes

ABSTRACTStreptomyces clavuligerusclaR::aphis aclaR-defective mutant, but in addition to itsclaRdefect it also carries fewer copies of the resident linear plasmids pSCL2 and pSCL4 (on the order of 4 × 105-fold lower than the wild-type strain), as shown by qPCR. To determine the function of ClaR without potential interference due to plasmid copy number, a new strain,S. clavuligerusΔclaR::aac, withclaRdeleted and carrying the wild-type level of plasmids, was constructed. Transcriptomic analyses were performed inS. clavuligerusΔclaR::aacandS. clavuligerusATCC 27064 as the control strain. The new ΔclaRmutant did not produce clavulanic acid (CA) and showed a partial expression of genes for the early steps of the CA biosynthesis pathway and a very poor expression (1 to 8%) of the genes for the late steps of the CA pathway. Genes for cephamycin C biosynthesis were weakly upregulated (1.7-fold at 22.5 h of culture) in the ΔclaRmutant, but genes for holomycin biosynthesis were expressed at levels from 3- to 572-fold higher than in the wild-type strain, supporting the observed overproduction of holomycin byS. clavuligerusΔclaR::aac. Interestingly, three secondary metabolites produced by gene clusters SMCp20, SMCp22, and SMCp24, encoding still-cryptic compounds, had partially or totally downregulated their genes in the mutant, suggesting a regulatory role for ClaR wider than previously reported. In addition, theamfRgene was downregulated, and consequently, the mutant did not produce aerial mycelium. Expression levels of about 100 genes in the genome were partially up- or downregulated in the ΔclaRmutant, many of them related to the upregulation of the sigma factor-encodingrpoEgene.

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 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.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

scholarly journals The Riemerella anatipestiferAS87_01735Gene Encodes Nicotinamidase PncA, an Important Virulence Factor

2016 ◽  
Vol 82 (19) ◽  
pp. 5815-5823 ◽  
Author(s):  
Xiaolan Wang ◽  
Beibei Liu ◽  
Yafeng Dou ◽  
Hongjie Fan ◽  
Shaohui Wang ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Vaccine Candidate ◽  
Wild Type ◽  
Salvage Pathway ◽  
Effective Protection ◽  
Domestic Ducks ◽  
Content Type ◽  
Key Enzyme ◽  
Important Virulence Factor

ABSTRACTRiemerella anatipestiferis a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of theAS87_01735gene significantly decreased the bacterial virulence ofR. anatipestiferstrain Yb2 (mutant RA625). TheAS87_01735gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD+salvage pathway. In this study, theAS87_01735gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated thatR. anatipestiferPncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncAin this study) showed a similar growth rate but decreased NAD+quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncAimmunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that theR. anatipestiferAS87_01735gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncAmutant can be used as a novel live vaccine candidate.IMPORTANCERiemerella anatipestiferis reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. ThepncAgene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD+salvage pathway. In this study, we identified and characterized thepncA-homologous geneAS87_01735inR. anatipestiferstrain Yb2.R. anatipestiferPncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of thepncAmutant Yb2ΔpncAled to a decrease in the NAD+content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2ΔpncAimmunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence ofR. anatipestiferand that the Yb2ΔpncAmutant can be used as a novel live vaccine candidate.

scholarly journals Influence of Chemotaxis and Swimming Patterns on the Virulence of the Coral PathogenVibrio coralliilyticus

2018 ◽  
Vol 200 (15) ◽  
Author(s):  
Blake Ushijima ◽  
Claudia C. Häse
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Bacterial Colonization ◽  
Valuable Insight ◽  
Wild Type ◽  
Coral Mucus ◽  
Content Type ◽  
Swimming Pattern ◽  
Vibrio Coralliilyticus ◽  
Insight Into

ABSTRACTChemotaxis, the directed movement toward or away from a chemical signal, can be essential to bacterial pathogens for locating hosts or avoiding hostile environments. The coral pathogenVibrio coralliilyticuschemotaxes toward coral mucus; however, chemotaxis has not been experimentally demonstrated to be important for virulence. To further examine this, in-frame mutations were constructed in genes predicted to be important forV. coralliilyticuschemotaxis. MostVibriogenomes contain multiple homologs of various chemotaxis-related genes, and two paralogs of each forcheB,cheR, andcheAwere identified. Based on single mutant analyses, the paralogscheB2,cheR2, andcheA1were essential for chemotaxis in laboratory assays. As predicted, the ΔcheA1and ΔcheR2strains had a smooth-swimming pattern, while the ΔcheB2strain displayed a zigzag pattern when observed under light microscopy. However, these mutants, unlike the parent strain, were unable to chemotax toward the known attractants coral mucus, dimethylsulfoniopropionate, andN-acetyl-d-glucosamine. The ΔcheB2strain and an aflagellate ΔfliG1strain were avirulent to coral, while the ΔcheA1and ΔcheR2strains were hypervirulent (90 to 100% infection within 14 h on average) compared to the wild-type strain (66% infection within 36 h on average). Additionally, the ΔcheA1and ΔcheR2strains appeared to better colonize coral fragments than the wild-type strain. These results suggest that although chemotaxis may be involved with infection (the ΔcheB2strain was avirulent), a smooth-swimming phenotype is important for bacterial colonization and infection. This study provides valuable insight into understandingV. coralliilyticuspathogenesis and how this pathogen may be transmitted between hosts.IMPORTANCECorals are responsible for creating the immense structures that are essential to reef ecosystems; unfortunately, pathogens like the bacteriumVibrio coralliilyticuscan cause fatal infections of reef-building coral species. However, compared to related human pathogens, the mechanisms by whichV. coralliilyticusinitiates infections and locates new coral hosts are poorly understood. This study investigated the effects of chemotaxis, the directional swimming in response to chemical signals, and bacterial swimming patterns on infection of the coralMontipora capitata. Infection experiments with different mutant strains suggested that a smooth-swimming pattern resulted in hypervirulence. These results demonstrate that the role of chemotaxis in coral infection may not be as straightforward as previously hypothesized and provide valuable insight intoV. coralliilyticuspathogenesis.

scholarly journals The Ser/Thr Kinase PrkC Participates in Cell Wall Homeostasis and Antimicrobial Resistance inClostridium difficile

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Elodie Cuenot ◽  
Transito Garcia-Garcia ◽  
Thibaut Douche ◽  
Olivier Gorgette ◽  
Pascal Courtin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
Hamster Model ◽  
Content Type ◽  
Physiological Processes ◽  
Signal Transduction Networks ◽  
Mean Size

ABSTRACTClostridium difficileis the leading cause of antibiotic-associated diarrhea in adults. During infection,C. difficilemust detect the host environment and induce an appropriate survival strategy. Signal transduction networks involving serine/threonine kinases (STKs) play key roles in adaptation, as they regulate numerous physiological processes. PrkC ofC. difficileis an STK with two PASTA domains. We showed that PrkC is membrane associated and is found at the septum. We observed that deletion ofprkCaffects cell morphology with an increase in mean size, cell length heterogeneity, and presence of abnormal septa. A ΔprkCmutant was able to sporulate and germinate but was less motile and formed more biofilm than the wild-type strain. Moreover, a ΔprkCmutant was more sensitive to antimicrobial compounds that target the cell envelope, such as the secondary bile salt deoxycholate, cephalosporins, cationic antimicrobial peptides, and lysozyme. This increased susceptibility was not associated with differences in peptidoglycan or polysaccharide II composition. However, the ΔprkCmutant had less peptidoglycan and released more polysaccharide II into the supernatant. A proteomic analysis showed that the majority ofC. difficileproteins associated with the cell wall were less abundant in the ΔprkCmutant than the wild-type strain. Finally, in a hamster model of infection, the ΔprkCmutant had a colonization delay that did not significantly affect overall virulence.

scholarly journals Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Jan Kampf ◽  
Jan Gerwig ◽  
Kerstin Kruse ◽  
Robert Cleverley ◽  
Miriam Dormeyer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Selective Pressure ◽  
Wild Type ◽  
Master Regulator ◽  
Form Complex ◽  
Content Type

ABSTRACT Biofilm formation by Bacillus subtilis requires the expression of genes encoding enzymes for extracellular polysaccharide synthesis and for an amyloid-like protein. The master regulator SinR represses all the corresponding genes, and repression of these key biofilm genes is lifted when SinR interacts with its cognate antagonist proteins. The YmdB phosphodiesterase is a recently discovered factor that is involved in the control of SinR activity: cells lacking YmdB exhibit hyperactive SinR and are unable to relieve the repression of the biofilm genes. In this study, we have examined the dynamics of gene expression patterns in wild-type and ymdB mutant cells by microfluidic analysis coupled to time-lapse microscopy. Our results confirm the bistable expression pattern for motility and biofilm genes in the wild-type strain and the loss of biofilm gene expression in the mutant. Moreover, we demonstrated dynamic behavior in subpopulations of the wild-type strain that is characterized by switches in sets of the expressed genes. In order to gain further insights into the role of YmdB, we isolated a set of spontaneous suppressor mutants derived from ymdB mutants that had regained the ability to form complex colonies and biofilms. Interestingly, all of the mutations affected SinR. In some mutants, large genomic regions encompassing sinR were deleted, whereas others had alleles encoding SinR variants. Functional and biochemical studies with these SinR variants revealed how these proteins allowed biofilm gene expression in the ymdB mutant strains. IMPORTANCE Many bacteria are able to choose between two mutually exclusive lifestyles: biofilm formation and motility. In the model bacterium Bacillus subtilis, this choice is made by each individual cell rather than at the population level. The transcriptional repressor SinR is the master regulator in this decision-making process. The regulation of SinR activity involves complex control of its own expression and of its interaction with antagonist proteins. We show that the YmdB phosphodiesterase is required to allow the expression of SinR-repressed genes in a subpopulation of cells and that such subpopulations can switch between different SinR activity states. Suppressor analyses revealed that ymdB mutants readily acquire mutations affecting SinR, thus restoring biofilm formation. These findings suggest that B. subtilis cells experience selective pressure to form the extracellular matrix that is characteristic of biofilms and that YmdB is required for the homeostasis of SinR and/or its antagonists.

scholarly journals Glycerol Utilization Gene Cluster in Streptomyces clavuligerus

2009 ◽  
Vol 75 (9) ◽  
pp. 2991-2995 ◽  
Author(s):  
Sonia Baños ◽  
Rosario Pérez-Redondo ◽  
Bert Koekman ◽  
Paloma Liras
Keyword(s):  
Clavulanic Acid ◽  
Gene Cluster ◽  
Acid Production ◽  
Type Strain ◽  
Wild Type Strain ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Glycerol Utilization ◽  
In The Wild

ABSTRACT The Streptomyces clavuligerus ATCC 27064 glycerol cluster gylR-glpF1K1D1 is induced by glycerol but is not affected by glucose. S. clavuligerus growth and clavulanic acid production are stimulated by glycerol, but this does not occur in a glpK1-deleted mutant. Amplification of glpK1D1 results in transformants yielding larger amounts of clavulanic acid in the wild-type strain and in overproducer S. clavuligerus Gap15-7-30 or S. clavuligerus ΔrelA strains.

scholarly journals In VivoEfficacy of Human Simulated Regimens of Carbapenems and Comparator Agents against NDM-1-Producing Enterobacteriaceae

2013 ◽  
Vol 58 (3) ◽  
pp. 1671-1677 ◽  
Author(s):  
Dora E. Wiskirchen ◽  
Patrice Nordmann ◽  
Jared L. Crandon ◽  
David P. Nicolau
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Similar Efficacy ◽  
Infection Model ◽  
High Dose ◽  
Wild Type ◽  
Prolonged Infusion ◽  
Content Type

ABSTRACTDoripenem and ertapenem have demonstrated efficacy against several NDM-1-producing isolatesin vivo, despite having high MICs. In this study, we sought to further characterize the efficacy profiles of humanized regimens of standard (500 mg given every 8 h) and high-dose, prolonged infusion of doripenem (2 g given every 8 h, 4-h infusion) and 1 g of ertapenem given intravenously every 24 h and the comparator regimens of ceftazidime at 2 g given every 8 h (2-h infusion), levofloxacin at 500 mg every 24 h, and aztreonam at 2 g every 6 h (1-h infusion) against a wider range of isolates in a murine thigh infection model. An isogenic wild-type strain and NDM-1-producingKlebsiella pneumoniaeand eight clinical NDM-1-producing members of the familyEnterobacteriaceaewere tested in immunocompetent- and neutropenic-mouse models. The wild-type strain was susceptible to all of the agents, while the isogenic NDM-1-producing strain was resistant to ceftazidime, doripenem, and ertapenem. Clinical NDM-1-producing strains were resistant to nearly all five of the agents (two were susceptible to levofloxacin). In immunocompetent mice, all of the agents produced ≥1-log10CFU reductions of the isogenic wild-type and NDM-1-producing strains after 24 h. Minimal efficacy of ceftazidime, aztreonam, and levofloxacin against the clinical NDM-1-producing strains was observed. However, despitein vitroresistance, ≥1-log10CFU reductions of six of eight clinical strains were achieved with high-dose, prolonged infusion of doripenem and ertapenem. Slight enhancements of doripenem activity over the standard doses were obtained with high-dose, prolonged infusion for three of the four isolates tested. Similar efficacy observations were noted in neutropenic mice. These data suggest that carbapenems are a viable treatment option for infections caused by NDM-1-producingEnterobacteriaceae.

scholarly journals Sml1 Inhibits the DNA Repair Activity of Rev1 in Saccharomyces cerevisiae during Oxidative Stress

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Rui Yao ◽  
Pei Zhou ◽  
Chengjin Wu ◽  
Liming Liu ◽  
Jing Wu
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Survival Rate ◽  
Type Strain ◽  
Mutation Frequency ◽  
Wild Type Strain ◽  
Yeast Cells ◽  
Wild Type ◽  
Content Type

ABSTRACT In Saccharomyces cerevisiae, Y family DNA polymerase Rev1 is involved in the repair of DNA damage by translesion DNA synthesis (TLS). In the current study, to elucidate the role of Rev1 in oxidative stress-induced DNA damage in S. cerevisiae, REV1 was deleted and overexpressed; transcriptome analysis of these mutants along with the wild-type strain was performed to screen potential genes that could be associated with REV1 during response to DNA damage. When the yeast cells were treated with 2 mM H2O2, the deletion of REV1 resulted in a 1.5- and 2.8-fold decrease in the survival rate and mutation frequency, respectively, whereas overexpression of REV1 increased the survival rate and mutation frequency by 1.1- and 2.9-fold, respectively, compared to the survival rate and mutation frequency of the wild-type strain. Transcriptome and phenotypic analyses identified that Sml1 aggravated oxidative stress in the yeast cells by inhibiting the activity of Rev1. This inhibition was due to the physical interaction between the BRCA1 C terminus (BRCT) domain of Rev1 and amino acid residues 36 to 70 of Sml1; the cell survival rate and mutation frequency increased by 1.8- and 3.1-fold, respectively, when this interaction was blocked. We also found that Sml1 inhibited Rev1 phosphorylation under oxidative stress and that deletion of SML1 increased the phosphorylation of Rev1 by 46%, whereas overexpression of SML1 reduced phosphorylation of Rev1. Overall, these findings demonstrate that Sml1 could be a novel regulator that mediates Rev1 dephosphorylation to inhibit its activity during oxidative stress. IMPORTANCE Rev1 was critical for cell growth in S. cerevisiae, and the deletion of REV1 caused a severe growth defect in cells exposed to oxidative stress (2 mM H2O2). Furthermore, we found that Sml1 physically interacted with Rev1 and inhibited Rev1 phosphorylation, thereby inhibiting Rev1 DNA antioxidant activity. These findings indicate that Sml1 could be a novel regulator for Rev1 in response to DNA damage by oxidative stress.

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