Transcriptional regulation of a hybrid cluster (prismane) protein

2005 ◽  
Vol 33 (1) ◽  
pp. 195-197 ◽  
Author(s):  
N.A. Filenko ◽  
D.F. Browning ◽  
J.A. Cole
Keyword(s):  
Physiological Function ◽  
Response Regulator ◽  
Anaerobic Growth ◽  
Hybrid Cluster ◽  
E Coli ◽  
Mutant Strains ◽  
Translation Initiation Codon ◽  
Nitric Oxide Reduction ◽  
Nitrate And Nitrite

HCP (hybrid-cluster protein) contains two Fe/S clusters, one of which is a hybrid [4Fe-2S-2O] cluster. Despite intensive study, its physiological function has not been reported. The Escherichia coli hcp gene is located in a two-gene operon with hcr, which encodes an NADH-dependent HCP reductase. E. coli HCP is detected after anaerobic growth with nitrate or nitrite: possible roles for it in hydroxylamine or nitric oxide reduction have been proposed. To study the regulation and role of HCP, an hcp::lacZ fusion was constructed and transformed into fnr, arcA and norR mutant strains of E. coli. Transcription from the hcp promoter was induced during anaerobic growth. Only the fnr mutant was defective in hcp expression. Nitrate- and nitrite-induced transcription from the hcp promoter was activated by the response regulator proteins NarL and NarP. Gel retardation assays were used to show that FNR (fumarate-nitrate regulation) and NarL form a complex with the hcp promoter. Transcription of the hcp-hcr operon initiates at a thymine nucleotide located 31 bp upstream of the translation-initiation codon. HCP has been overexpressed from a recombinant plasmid for physiological studies.

Different responses to nitrate and nitrite by the model organism Escherichia coli and the human pathogen Neisseria gonorrhoeae

2006 ◽  
Vol 34 (1) ◽  
pp. 111-114 ◽  
Author(s):  
R.N. Whitehead ◽  
J.A. Cole
Keyword(s):  
Escherichia Coli ◽  
Neisseria Gonorrhoeae ◽  
Model Organism ◽  
Anaerobic Growth ◽  
Human Pathogen ◽  
Regulate Gene Expression ◽  
E Coli ◽  
Regulatory Systems ◽  
Two Component ◽  
Nitrate And Nitrite

The ability of Escherichia coli to use both nitrate and nitrite as terminal electron acceptors during anaerobic growth is mediated by the dual-acting two-component regulatory systems NarX-NarL and NarQ-NarP. In contrast, Neisseria gonorrhoeae responds only to nitrite: it expresses only NarQ-NarP. We have shown that although N. gonorrhoeae NarQ can phosphorylate E. coli NarL and NarP, the N. gonorrhoeae NarP is unable to regulate gene expression in E. coli. Mutagenesis experiments have revealed residues in E. coli NarQ that are essential for nitrate and nitrite sensing. Chimaeric proteins revealed domains of NarQ that are important for ligand sensing.

Molecular Cloning of the gyrA andgyrB Genes of Bacteroides fragilis Encoding DNA Gyrase

1999 ◽  
Vol 43 (10) ◽  
pp. 2423-2429 ◽  
Author(s):  
Yoshikuni Onodera ◽  
Kenichi Sato
Keyword(s):  
Hot Spot ◽  
Bacteroides Fragilis ◽  
Dna Gyrase ◽  
E Coli ◽  
Important Target ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Gyrase A ◽  
Selection For

ABSTRACT The genes encoding the DNA gyrase A and B subunits ofBacteroides fragilis were cloned and sequenced. ThegyrA and gyrB genes code for proteins of 845 and 653 amino acids, respectively. These proteins were expressed inEscherichia coli, and the combination of GyrA and GyrB exhibited ATP-dependent supercoiling activity. To analyze the role of DNA gyrase in quinolone resistance of B. fragilis, we isolated mutant strains by stepwise selection for resistance to increasing concentrations of levofloxacin. We analyzed the resistant mutants and showed that Ser-82 of GyrA, equivalent to resistance hot spot Ser-83 of GyrA in E. coli, was in each case replaced with Phe. These results suggest that DNA gyrase is an important target for quinolones in B. fragilis.

scholarly journals Identification of a Streptococcus pneumoniae Gene Locus Encoding Proteins of an ABC Phosphate Transporter and a Two-Component Regulatory System

1999 ◽  
Vol 181 (4) ◽  
pp. 1126-1133 ◽  
Author(s):  
Rodger Novak ◽  
Anje Cauwels ◽  
Emmanuelle Charpentier ◽  
Elaine Tuomanen
Keyword(s):  
Response Regulator ◽  
Regulatory System ◽  
Phosphate Limitation ◽  
Pho Regulon ◽  
Loss Of Function ◽  
Gene Encoding ◽  
E Coli ◽  
Two Component ◽  
Pst System

ABSTRACT The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutantphoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

THE ROLE OF PROTON ATPase SPECIFIC INHIBITOR $N,N'$-DICYCLOHEXYLCARBODIIMIDE AND EXTERNAL FORMATE CONCENTRATION ON $E.~COLI$ GROWTH DURING MIXED CARBON SOURCES FERMENTATION AT DIFFERENT pHs.

2021 ◽  
Vol 55 (1 (254)) ◽  
pp. 67-74
Author(s):  
Heghine Kh. Gevorgyan ◽  
Anait V. Vassilian ◽  
Karen A. Trchounian
Keyword(s):  
Regulatory Protein ◽  
Carbon Sources ◽  
Specific Inhibitor ◽  
Inhibitory Effect ◽  
E Coli ◽  
Proton Atpase ◽  
Mutant Strains ◽  
Atpase Inhibition ◽  
Compensatory Effect

This research is focused on the investigation of specific growth rate changes of $E.~coli$ wild type and mutant strains with defect of Hyd, FDH enzymes and FhlA regulatory protein in the presence of $N,N'$-dicyclohexylcarbodiimide (DCCD) and external formate various concentration during co-fermentation of glucose, glycerol and formate at pHs $5.5-7.5.$ The highest value of SGR was observed at pH 7.5. It was revealed that SGR depends on external formate concentration at all pHs. DCCD inhibitory effect was shown mainly at pH 7.5 and partially at pH 6.5 and 5.5. In the case of the F0F1-ATPase inhibition FhlA compensatory effect on SGR was revealed.

scholarly journals Contribution of Fibronectin-Binding Protein to Pathogenesis of Streptococcus suis Serotype 2

2002 ◽  
Vol 70 (3) ◽  
pp. 1319-1325 ◽  
Author(s):  
Astrid de Greeff ◽  
Herma Buys ◽  
Robin Verhaar ◽  
Janny Dijkstra ◽  
Loek van Alphen ◽  
...  
Keyword(s):  
Binding Protein ◽  
Streptococcus Suis ◽  
Infection Model ◽  
Organ Cultures ◽  
Gene Encoding ◽  
E Coli ◽  
Mutant Strains ◽  
Fibronectin Binding Protein ◽  
Suis Serotype

ABSTRACT In the present study we investigated the role of the fibronectin (FN)- and fibrinogen (FGN)-binding protein (FBPS) in the pathogenesis of Streptococcus suis serotype 2 in piglets. The complete gene encoding FBPS from S. suis serotype 2 was cloned in Escherichia coli and sequenced. The occurrence of the gene in various serotypes was analyzed by hybridization studies. The FBPS protein was expressed in E. coli and purified, and binding to human FN and FGN was demonstrated. The induction of antibodies in piglets was studied upon infection. An isogenic mutant unable to produce FBPS was constructed, and the levels of virulence of the wild-type and mutant strains were compared in a competitive infection model in young piglets. Organ cultures showed that FBPS was not required for colonization of the tonsils but that FBPS played a role in the colonization of the specific organs involved in an S. suis infection. Therefore, the FBPS mutant was considered as an attenuated mutant.

scholarly journals Nasty Prophages and the Dynamics of Antibiotic-Tolerant Persister Cells

2017 ◽  
Author(s):  
Alexander Harms ◽  
Cinzia Fino ◽  
Michael A. Sørensen ◽  
Szabolcs Semsey ◽  
Kenn Gerdes
Keyword(s):  
Antibiotic Treatment ◽  
Bacterial Growth ◽  
Culture Conditions ◽  
Growth Stages ◽  
Persister Cells ◽  
E Coli ◽  
Common Basis ◽  
Mutant Strains ◽  
Future Work

AbstractBacterial persisters are phenotypic variants that survive antibiotic treatment in a dormant state and can be formed by multiple pathways. We recently proposed that the second messenger (p)ppGpp drivesEscherichia colipersister formation through protease Lon and the activation of toxin-antitoxin (TA) modules. This model found support in the field, but also generated controversy as part of recent heated debates on the validity of significant parts of the literature. In this study, we therefore used our previous work as a model to critically examine common experimental procedures in order to understand and overcome the inconsistencies often observed between results of different laboratories. Our results show that seemingly simple antibiotic killing assays are very sensitive to variation of culture conditions and bacterial growth phase. Additionally, we found that some assay conditions cause the killing of antibiotic-tolerant persisters via induction of cryptic prophages. Similarly, the inadvertent infection of mutant strains with bacteriophage φ80, a notorious laboratory contaminant, has apparently caused several phenotypes that we reported in our previous studies. We therefore reconstructed all infected mutants and probed the validity of our model of persister formation in a refined assay setup that uses robust culture conditions and unravels the dynamics of persister cells through all bacterial growth stages. Our results confirm the importance of (p)ppGpp and Lon, but do not anymore support a role of TA modules inE. colipersister formation. We anticipate that the results and approaches reported in our study will lay the ground for future work in the field.ImportanceThe recalcitrance of antibiotic-tolerant persister cells is thought to cause relapsing infections and antibiotic treatment failure in various clinical setups. Previous studies have identified multiple genetic pathways involved in persister formation, but also revealed reproducibility problems that sparked controversies about adequate tools to study persister cells. In this study we unraveled how typical antibiotic killing assays often fail to capture the biology of persisters and instead give widely different results based on ill-controlled experimental parameters and artifacts caused by cryptic as well as contaminant prophages. We therefore established a new, robust assay that enabled us to follow the dynamics of persister cells through all growth stages of bacterial cultures without distortions by bacteriophages. This system also favored adequate comparisons of mutant strains with aberrant growth phenotypes. We anticipate that our results will contribute to a robust, common basis of future studies on the formation and eradication of antibiotic-tolerant persisters.

scholarly journals Novel Pathway for Arsenic Detoxification in the Legume Symbiont Sinorhizobium meliloti

2005 ◽  
Vol 187 (20) ◽  
pp. 6991-6997 ◽  
Author(s):  
Hung-Chi Yang ◽  
Jiujun Cheng ◽  
Turlough M. Finan ◽  
Barry P. Rosen ◽  
Hiranmoy Bhattacharjee
Keyword(s):  
Sinorhizobium Meliloti ◽  
Physiological Function ◽  
Phosphate Transport ◽  
Transport Systems ◽  
Intact Cells ◽  
Arsenic Resistance ◽  
E Coli ◽  
Arsenic Detoxification ◽  
Selective Sensitivity

ABSTRACT We report a novel pathway for arsenic detoxification in the legume symbiont Sinorhizobium meliloti. Although a majority of ars operons consist of three genes, arsR (transcriptional regulator), arsB [As(OH)3/H+ antiporter], and arsC (arsenate reductase), the S. meliloti ars operon includes an aquaglyceroporin (aqpS) in place of arsB. The presence of AqpS in an arsenic resistance operon is interesting, since aquaglyceroporin channels have previously been shown to adventitiously facilitate uptake of arsenite into cells, rendering them sensitive to arsenite. To understand the role of aqpS in arsenic resistance, S. meliloti aqpS and arsC were disrupted individually. Disruption of aqpS resulted in increased tolerance to arsenite but not arsenate, while cells with an arsC disruption showed selective sensitivity to arsenate. The results of transport experiments in intact cells suggest that AqpS is the only protein of the S. meliloti ars operon that facilitates transport of arsenite. Coexpression of S. meliloti aqpS and arsC in a strain of E. coli lacking the ars operon complemented arsenate but not arsenite sensitivity. These results imply that, when S. meliloti is exposed to environmental arsenate, arsenate enters the cell through phosphate transport systems and is reduced to arsenite by ArsC. Internally generated arsenite flows out of the cell by downhill movement through AqpS. Thus, AqpS confers arsenate resistance together with ArsC-catalyzed reduction. This is the first report of an aquaglyceroporin with a physiological function in arsenic resistance.

scholarly journals The Global Regulator ArcA Modulates Expression of Virulence Factors in Vibrio cholerae

2003 ◽  
Vol 71 (10) ◽  
pp. 5583-5589 ◽  
Author(s):  
Nilanjan Sengupta ◽  
Kalidas Paul ◽  
Rukhsana Chowdhury
Keyword(s):  
Vibrio Cholerae ◽  
Mutant Strain ◽  
Virulence Factors ◽  
Response Regulator ◽  
Anaerobic Growth ◽  
Infant Mouse ◽  
Reverse Transcription Pcr ◽  
Concomitant Reduction ◽  
Aerobic And Anaerobic

ABSTRACT A Vibrio cholerae arcA mutant was constructed and used to examine the role of the global anaerobiosis response regulator ArcA in the expression of virulence factors in this important human pathogen. In V. cholerae, expression of the major virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) is regulated by the transcriptional activator ToxT. toxT expression, in turn, is controlled by the transmembrane DNA binding proteins ToxR and TcpP. In the V. cholerae arcA mutant, although ToxR and TcpP were unaffected, Northern blot and reverse transcription-PCR analyses indicated that the expression of toxT was significantly decreased with concomitant reduction in the expression of CT and TCP. CT and TCP expression was completely restored in the V. cholerae arcA mutant strain by expressing a cloned toxT gene in the mutant. These results suggest that ArcA functions as a positive regulator of toxT expression under both aerobic and anaerobic conditions, although as expected, the effect was more pronounced during anaerobic growth. This was reflected in a reduction of virulence of the V. cholerae arcA mutant strain in the infant mouse cholera model.

scholarly journals Role of rpoS in Acid Resistance and Fecal Shedding of Escherichia coli O157:H7

2000 ◽  
Vol 66 (2) ◽  
pp. 632-637 ◽  
Author(s):  
Stuart B. Price ◽  
Chorng-Ming Cheng ◽  
Charles W. Kaspar ◽  
James C. Wright ◽  
Fred J. DeGraves ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Response Regulator ◽  
Acid Resistance ◽  
Wild Type ◽  
Fecal Shedding ◽  
E Coli ◽  
Cattle Herds

ABSTRACT Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator ςS and its effect on shedding of E. coliO157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 101 to 104 CFU, we found the wild-type strain in feces of mice given lower doses (102 versus 103 CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 104 CFU. Enumeration ofE. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P ≤ 0.05). Thus, ςS appears to play a role in E. coli O157:H7 passage in mice and shedding from calves, possibly by inducing expression of the glucose-repressed RpoS-dependent AR determinant and thus increasing resistance to gastrointestinal stress. These findings may provide clues for future efforts aimed at reducing or eliminating this pathogen from cattle herds.

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