scholarly journals Transcriptional activation of the Azotobacter vinelandii polyhydroxybutyrate biosynthetic genes phbBAC by PhbR and RpoS

Microbiology ◽  
2011 ◽  
Vol 157 (11) ◽  
pp. 3014-3023 ◽  
Author(s):  
Alberto Hernandez-Eligio ◽  
Mildred Castellanos ◽  
Soledad Moreno ◽  
Guadalupe Espín
Keyword(s):  
Stationary Phase ◽  
Transcriptional Activation ◽  
Azotobacter Vinelandii ◽  
Transcriptional Regulator ◽  
Gene Fusions ◽  
Wild Type ◽  
Rt Pcr ◽  
Regulation Of Expression ◽  
Expression Studies ◽  
Mutant Strains

We previously showed that in Azotobacter vinelandii, accumulation of polyhydroxybutyrate (PHB) occurs mainly during the stationary phase, and that a mutation in phbR, encoding a transcriptional regulator of the AraC family, reduces PHB accumulation. In this study, we characterized the roles of PhbR and RpoS, a central regulator during stationary phase in bacteria, in the regulation of expression of the PHB biosynthetic operon phbBAC and phbR. We showed that inactivation of rpoS reduced PHB accumulation, similar to the phbR mutation, and inactivation of both rpoS and phbR resulted in an inability to produce PHB. We carried out expression studies with the wild-type, and the rpoS, phbR and double rpoS-phbR mutant strains, using quantitative RT-PCR, as well as phbB : : gusA and phbR : : gusA gene fusions. These studies showed that both PhbR and RpoS act as activators of phbB and phbR, and revealed a role for PhbR as an autoactivator. We also demonstrated that PhbR binds specifically to two almost identical 18 bp sites, TGTCACCAA-N4-CACTA and TGTCACCAA-N4-CAGTA, present in the phbB promoter region. The activation of phbB and phbR transcription by RpoS reported here is in agreement with the observation that accumulation of PHB in A. vinelandii occurs mainly during the stationary phase.

Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae

2015 ◽  
Vol 25 (2-3) ◽  
pp. 120-128 ◽  
Author(s):  
Irfan Manzoor ◽  
Sulman Shafeeq ◽  
Muhammad Afzal ◽  
Oscar P. Kuipers
Keyword(s):  
Streptococcus Pneumoniae ◽  
Transcriptional Activation ◽  
Promoter Region ◽  
Transcriptional Regulator ◽  
Transcriptional Activator ◽  
Regulatory Site ◽  
Rt Pcr ◽  
The Impact

In this study, we explore the impact of fucose on the transcriptome of <i>S. pneumoniae</i> D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (<i>fcs</i> operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the <i>fcs</i> operon, as a transcriptional activator of the <i>fcs</i> operon. We also predict a 19-bp putative FcsR regulatory site (5′-ATTTGAACATTATTCAAGT-3′) in the promoter region of the <i>fcs</i> operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the <i>fcs</i> operon.

scholarly journals Investigation of the physiological relationship between the cyanide-insensitive oxidase and cyanide production in Pseudomonas aeruginosa

Microbiology ◽  
2006 ◽  
Vol 152 (5) ◽  
pp. 1407-1415 ◽  
Author(s):  
James E. A. Zlosnik ◽  
Gholam Reza Tavankar ◽  
Jacob G. Bundy ◽  
Dimitris Mossialos ◽  
Ronan O'Toole ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Stationary Phase ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Toxic Agent ◽  
Stationary Phase Culture ◽  
Prolonged Incubation ◽  
Mutant Strains ◽  
Electron Sink ◽  
Culture Supernatants

Pseudomonas aeruginosa is an opportunistic pathogen which demonstrates considerable respiratory versatility, possessing up to five terminal oxidases. One oxidase, the cyanide-insensitive oxidase (CIO), has been previously shown to be resistant to the potent respiratory inhibitor cyanide, a toxin that is synthesized by this bacterium. This study investigated the physiological relationship between hydrogen cyanide production and the CIO. It was found that cyanide is produced in P. aeruginosa at similar levels irrespective of its complement of CIO, indicating that the CIO is not an obligatory electron sink for cyanide synthesis. However, MICs for cyanide and growth in its presence demonstrated that the CIO provides P. aeruginosa with protection against the effects of exogenous cyanide. Nevertheless, the presence of cyanide did not affect the viability of cio mutant strains compared to the wild-type during prolonged incubation in stationary phase. The detection of the fermentation end products acetate and succinate in stationary-phase culture supernatants suggests that P. aeruginosa, irrespective of its CIO complement, may in part rely upon fermentation for energy generation in stationary phase. Furthermore, the decrease in cyanide levels during incubation in sealed flasks suggested that active breakdown of HCN by the culture was taking place. To investigate the possibility that the CIO may play a role in pathogenicity, wild-type and cio mutant strains were tested in the paralytic killing model of Caenorhabditis elegans, a model in which cyanide is the principal toxic agent leading to nematode death. The CIO mutant had delayed killing kinetics, demonstrating that the CIO is required for full pathogenicity of P. aeruginosa in this animal model.

scholarly journals Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Brett M. Barney ◽  
Mary H. Plunkett ◽  
Velmurugan Natarajan ◽  
Florence Mus ◽  
Carolann M. Knutson ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Stationary Phase ◽  
Gene Transcription ◽  
Biological Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Content Type ◽  
Ammonium Production ◽  
Biological Nitrogen

ABSTRACT Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene nifL result in the deregulation of the nif transcriptional activator NifA in the nitrogen-fixing bacterium Azotobacter vinelandii, triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the rnf1 gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium. IMPORTANCE The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (nifL-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in A. vinelandii as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.

scholarly journals Bypass of an epigenetic S-phase transcriptional module by convergent nutrient stress signals

2019 ◽  
Author(s):  
Marie Delaby ◽  
Gaël Panis ◽  
Coralie Fumeaux ◽  
Laurence Degeorges ◽  
Patrick H. Viollier
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Caulobacter Crescentus ◽  
Transcriptional Regulator ◽  
S Phase ◽  
Wild Type ◽  
Intracellular Growth ◽  
Obligate Intracellular ◽  
Stress Signals ◽  
Alpha Proteobacteria

AbstractThe signals feeding into bacterial S-phase transcription are poorly understood. Cellular cycling in the alpha-proteobacterium Caulobacter crescentus is driven by a complex circuit of at least three transcriptional modules that direct sequential promoter firing during the G1, early and late S cell cycle phases. In alpha-proteobacteria, the transcriptional regulator GcrA and the CcrM methyltransferase epigenetically activate promoters of cell division and polarity genes that fire in S-phase. By evolving Caulobacter crescentus cells to cycle and differentiate in the absence of the GcrA/CcrM module, we discovered that phosphate deprivation and (p)ppGpp alarmone stress signals converge on S-phase transcriptional activation. The cell cycle oscillations of the CtrA protein, the transcriptional regulator that implements G1 and late S-phase transcription, are essential in our evolved mutants, but not in wild-type cells, showing that the periodicity in CtrA abundance alone can sustain cellular cycling without GcrA/CcrM. While similar nutritional sensing occurs in other alpha-proteobacteria, GcrA and CcrM are not encoded in the reduced genomes of obligate intracellular relatives. We thus propose that the nutritional stress response induced during intracellular growth obviated the need for an S-phase transcriptional regulator.

Transformation of Azotobacter vinelandii strains unable to fix nitrogen with Rhizobium spp. DNA

1978 ◽  
Vol 24 (3) ◽  
pp. 209-214 ◽  
Author(s):  
William J. Page
Keyword(s):  
Transcriptional Control ◽  
Deoxyribonucleic Acid ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Spontaneous Reversion ◽  
Mutant Strains ◽  
Rhizobium Spp ◽  
Rhizobium Sp

The phenotypes of Azotobacter vinelandii ATCC 12837 strains defective in nitrogen fixation (Nif−) were characterized by intrageneric transformation with known Nif− strains of A. vinelandii OP. These former mutant strains were used as recipients for intergeneric transformation by deoxyribonucleic acid (DNA) prepared from Rhizobium spp. to determine if the rhizobia would transform the Azotobacter Nif− phenotypes to Nif+. The frequency of Nif+ transformants using Rhizobium DNA was always less than the frequency using Azotobacter wild-type DNA but was greater than the spontaneous reversion frequency. The Azotobacter Nif+ recombinants also were stable. DNA from all of the Rhizobium spp. transformed to Nif+Azotobacter mutants defective in the nitrogenase component I (molybdoferredoxin); however, some recombinants had a lower nitrogenase activity and a delayed nitrogenase depression time. Mutants defective in the pleiotrophic transcriptional control of both nitrogenase components were transformed to Nif+ by the asymbiotic nitrogen fixing Rhizobium sp. 32H1 and 41A1, but not the symbiotic nitrogen-fixing species. The significance of these results and the possible future applications of this system are discussed.

scholarly journals σB Activation under Environmental and Energy Stress Conditions in Listeria monocytogenes

2006 ◽  
Vol 72 (8) ◽  
pp. 5197-5203 ◽  
Author(s):  
Soraya Chaturongakul ◽  
Kathryn J. Boor
Keyword(s):  
Listeria Monocytogenes ◽  
Cumene Hydroperoxide ◽  
Stress Conditions ◽  
Wild Type ◽  
Regulation Of Expression ◽  
Transcript Levels ◽  
Energy Stress ◽  
Expression Studies ◽  
Genes Encoding ◽  
Gene Expression Studies

ABSTRACT To measure σB activation in Listeria monocytogenes under environmental or energy stress conditions, quantitative reverse transcriptase PCR (TaqMan) was used to determine the levels of transcripts for the σB-dependent opuCA and clpC genes in strains having null mutations in genes encoding regulator of sigma B proteins (rsbT and rsbV) and sigma B (sigB) and in the L. monocytogenes wild-type 10403S strain under different stress conditions. The ΔsigB, ΔrsbT, and ΔrsbV strains previously exhibited increased hemolytic activities compared to the hemolytic activity of the wild-type strain; therefore, transcript levels for hly were also determined. RsbT, RsbV, and σB were all required for opuCA expression during growth under carbon-limiting conditions or following exposure to pH 4.5, salt, ethanol, or the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Expression of clpC was RsbT, RsbV, and σB dependent in the presence of CCCP but not under the other conditions. hly expression was not RsbT, RsbV, or σB dependent in the presence of either CCCP or salt. opuCA transcript levels did not increase in the presence of rapidly lethal stresses (i.e., pH 2.5 or 13 mM cumene hydroperoxide) despite the enhanced survival of the wild type compared with the survival of the mutant strains under these conditions. These findings highlight the importance of complementing phenotypic characterizations with gene expression studies to identify direct and indirect effects of null mutations in regulatory genes, such as sigB. Overall, our data show that while σB activation occurs through a single pathway under both environmental and energy stress conditions, regulation of expression of some stress response and virulence genes in the σB regulon (e.g., clpC) appears to require networks involving multiple transcriptional regulators.

scholarly journals GalR Acts as a Transcriptional Activator of galKT in the Presence of Galactose in Streptococcus pneumoniae

2015 ◽  
Vol 25 (6) ◽  
pp. 363-371 ◽  
Author(s):  
Muhammad Afzal ◽  
Sulman Shafeeq ◽  
Irfan Manzoor ◽  
Oscar P. Kuipers
Keyword(s):  
Streptococcus Pneumoniae ◽  
Promoter Region ◽  
Regulatory Mechanism ◽  
Transcriptional Regulator ◽  
Transcriptional Activator ◽  
Regulatory Site ◽  
Wild Type ◽  
Rt Pcr ◽  
Leloir Pathway

We explored the regulatory mechanism of Leloir pathway genes in <i>Streptococcus pneumoniae</i> D39. Here, we demonstrate that the expression of <i>galKT</i> is galactose dependent. By microarray analysis and quantitative RT-PCR, we further show the role of the transcriptional regulator GalR, present upstream of <i>galKT</i>, as a transcriptional activator of <i>galKT</i> in the presence of galactose. Moreover, we predict a 19-bp regulatory site (5′-GATAGTTTAGTAAAATTTT-3′) for the transcriptional regulator GalR in the promoter region of <i>galK</i>, which is also highly conserved in other streptococci. Growth comparison of D39 &#x0394;<i>galK</i> with the D39 wild type grown in the presence of galactose shows that <i>galK</i> is required for the proper growth of <i>S. pneumoniae</i> on galactose.

σ B-dependent expression patterns of compatible solute transporter genes opuCA and lmo1421 and the conjugated bile salt hydrolase gene bsh in Listeria monocytogenes

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3247-3256 ◽  
Author(s):  
David Sue ◽  
Kathryn J. Boor ◽  
Martin Wiedmann
Keyword(s):  
Listeria Monocytogenes ◽  
Stationary Phase ◽  
Bile Salt ◽  
Expression Patterns ◽  
Exponential Phase ◽  
Compatible Solute ◽  
Bile Salt Hydrolase ◽  
Wild Type ◽  
Rt Pcr ◽  
Solute Transporter

Listeria monocytogenes is a food-borne pathogen that can persist and grow under a wide variety of environmental conditions including low pH and high osmolarity. The alternative sigma factor σ B contributes to L. monocytogenes survival under extreme conditions. The purpose of this study was to identify and confirm specific σ B-dependent genes in L. monocytogenes and to characterize their expression patterns under various stress conditions. opuCA, lmo1421 and bsh were identified as putative σ B-dependent genes based on the presence of a predicted σ B-dependent promoter sequence upstream of each gene. opuCA and lmo1421 encode known and putative compatible solute transporter proteins, respectively, and bsh encodes a conjugated bile salt hydrolase (BSH). Reporter fusions and semi-quantitative RT-PCR techniques were used to confirm σ B-dependent regulation of these stress-response genes and to determine their expression patterns in response to environmental stresses. RT-PCR demonstrated that opuCA, lmo1421 and bsh transcript levels are reduced in stationary-phase L. monocytogenes ΔsigB cells relative to levels present in wild-type cells. Furthermore, BSH activity is abolished in a L. monocytogenes ΔsigB strain. RT-PCR confirmed growth-phase-dependent expression of opuCA, with highest levels of expression in stationary-phase cells. The L. monocytogenes wild-type strain exhibited two- and threefold induction of opuCA expression and seven- and fivefold induction of lmo1421 expression following 10 and 15 min exposure to 0·5 M KCl, respectively, as determined by RT-PCR, suggesting rapid induction of σ B activity in exponential-phase L. monocytogenes upon exposure to salt stress. Single-copy chromosomal opuCA–gus reporter fusions also showed significant induction of opuCA expression following exposure of exponential-phase cells to increased salt concentrations (0·5 M NaCl or 0·5 M KCl). In conjunction with recent findings that indicate a role for opuCA and bsh in L. monocytogenes virulence, the data presented here provide further evidence of specific σ B-mediated contributions to both environmental stress resistance and intra-host survival in L. monocytogenes.

scholarly journals Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 605-616 ◽  
Author(s):  
Claudia S. López ◽  
Alejandro F. Alice ◽  
Horacio Heras ◽  
Emilio A. Rivas ◽  
Carmen Sánchez-Rivas
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Growth Curves ◽  
Membrane Lipid ◽  
Wild Type ◽  
Lipid Packing ◽  
Anionic Phospholipids ◽  
Large Unilamellar Vesicles ◽  
Mutant Strains ◽  
Lag Period

The importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure of Bacillus subtilis cultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (ywiE, ywnE and ywjE) were obtained. Only the ywnE mutation resulted in a complete deficiency in cardiolipin and thus corresponds to a true clsA gene. The osmotolerance of a clsA mutant was impaired: although at NaCl concentrations lower than 1·2 M the growth curves were similar to those of its wild-type control, at 1·5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of the clsA mutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, the clsA mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type and clsA mutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, the clsA mutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type and clsA mutant strains increased when they were grown in LBN. In a conditional IPTG-controlled pgsA mutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of the clsA and pgsA genes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth of B. subtilis in media containing elevated NaCl concentrations.

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