Functional analysis of sigma-70 consensus promoters in Pseudomonas aeruginosa and Escherichia coli

1997 ◽  
Vol 43 (10) ◽  
pp. 981-985 ◽  
Author(s):  
Bradley W. McLean ◽  
Shari L. Wiseman ◽  
Andrew M. Kropinski
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Promoter Sequence ◽  
Broad Host Range ◽  
E Coli ◽  
Synthetic Promoters ◽  
Range Vector ◽  
Sigma 70

A series of synthetic promoters, based upon the Escherichia coli σ70 consensus promoter sequence, was constructed upstream of the lacZ reporter gene in the modified broad-host-range vector pQF52. The role of the intervening spacer region in gene expression in Pseudomonas aeruginosa and E. coli was studied by insertions and deletions within this region. In P. aeruginosa and E. coli the patterns of gene expression were identical with maximum β-galactosidase activity being measured from promoters possessing 19 bp in their intervening regions, presumably as a result of impeded promoter clearance with the consensus 17-bp promoter. In P. aeruginosa a second occurrence of enhanced activity, which could not be attributed to the involvement of the alternative sigma factor RpoN (σ54), was evident with the promoter having a 16-bp spacer.Key words: Pseudomonas aeruginosa, promoter, RpoD, RpoN, transcription.

scholarly journals Transcription of the Subtilase Cytotoxin Gene subAB1 in Shiga Toxin-Producing Escherichia coli Is Dependent on hfq and hns

2019 ◽  
Vol 85 (20) ◽  
Author(s):  
Laura Heinisch ◽  
Katharina Zoric ◽  
Maike Krause ◽  
Herbert Schmidt
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Shiga Toxin ◽  
Promoter Activity ◽  
Deletion Mutants ◽  
Content Type ◽  
E Coli ◽  
Intensive Investigation ◽  
Subtilase Cytotoxin

ABSTRACT Certain foodborne Shiga toxin-producing Escherichia coli (STEC) strains carry genes encoding the subtilase cytotoxin (SubAB). Although the mode of action of SubAB is under intensive investigation, information about the regulation of subAB gene expression is currently not available. In this study, we investigated the regulation of the chromosomal subAB1 gene in laboratory E. coli strain DH5α and STEC O113:H21 strain TS18/08 using a luciferase reporter gene assay. Special emphasis was given to the role of the global regulatory protein genes hfq and hns in subAB1 promoter activity. Subsequently, quantitative real-time PCR was performed to analyze the expression of Shiga toxin 2a (Stx2a), SubAB1, and cytolethal distending toxin V (Cdt-V) genes in STEC strain TS18/08 and its isogenic hfq and hns deletion mutants. The deletion of hfq led to a significant increase of up to 2-fold in subAB1 expression, especially in the late growth phase, in both strains. However, deletion of hns showed different effects on the promoter activity during the early and late exponential growth phases in both strains. Furthermore, upregulation of stx2a and cdt-V was demonstrated in hfq and hns deletion mutants in TS18/08. These data showed that the expression of subAB1, stx2a, and cdt-V is integrated in the regulatory network of global regulators Hfq and H-NS in Escherichia coli. IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) strains are responsible for outbreaks of foodborne diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. The pathogenicity of those strains can be attributed to, among other factors, the production of toxins. Recently, the subtilase cytotoxin was detected in locus of enterocyte effacement (LEE)-negative STEC, and it was confirmed that it contributes to the cytotoxicity of those STEC strains. Although the mode of action of SubAB1 is under intensive investigation, the regulation of gene expression is currently not known. The global regulatory proteins H-NS and Hfq have impact on many cellular processes and have been described to regulate virulence factors as well. Here, we investigate the role of hns and hfq in expression of subAB1 as well as stx2a and cdt-V in an E. coli laboratory strain as well as in wild-type STEC strain TS18/08.

scholarly journals Bacterial Two-Hybrid Analysis of Interactions between Region 4 of the ς70 Subunit of RNA Polymerase and the Transcriptional Regulators Rsd from Escherichia coli and AlgQ from Pseudomonas aeruginosa

2001 ◽  
Vol 183 (21) ◽  
pp. 6413-6421 ◽  
Author(s):  
Simon L. Dove ◽  
Ann Hochschild
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Amino Acid Substitution ◽  
Transcriptional Regulators ◽  
Binding Motif ◽  
E Coli ◽  
Two Hybrid

ABSTRACT A number of transcriptional regulators mediate their effects through direct contact with the ς70 subunit ofEscherichia coli RNA polymerase (RNAP). In particular, several regulators have been shown to contact a C-terminal portion of ς70 that harbors conserved region 4. This region of ς contains a putative helix-turn-helix DNA-binding motif that contacts the −35 element of ς70-dependent promoters directly. Here we report the use of a recently developed bacterial two-hybrid system to study the interaction between the putative anti-ς factor Rsd and the ς70 subunit of E. coli RNAP. Using this system, we found that Rsd can interact with an 86-amino-acid C-terminal fragment of ς70 and also that amino acid substitution R596H, within region 4 of ς70, weakens this interaction. We demonstrated the specificity of this effect by showing that substitution R596H does not weaken the interaction between ς and two other regulators shown previously to contact region 4 of ς70. We also demonstrated that AlgQ, a homolog of Rsd that positively regulates virulence gene expression inPseudomonas aeruginosa, can contact the C-terminal region of the ς70 subunit of RNAP from this organism. We found that amino acid substitution R600H in ς70 fromP. aeruginosa, corresponding to the R596H substitution in E. coli ς70, specifically weakens the interaction between AlgQ and ς70. Taken together, our findings suggest that Rsd and AlgQ contact similar surfaces of RNAP present in region 4 of ς70 and probably regulate gene expression through this contact.

scholarly journals Role of Histone-Like Proteins H-NS and StpA in Expression of Virulence Determinants of Uropathogenic Escherichia coli

2006 ◽  
Vol 188 (15) ◽  
pp. 5428-5438 ◽  
Author(s):  
Claudia M. Müller ◽  
Ulrich Dobrindt ◽  
Gábor Nagy ◽  
Levente Emödy ◽  
Bernt Eric Uhlin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Gene Expression Pattern ◽  
Direct Interaction ◽  
Virulence Determinants ◽  
Dna Arrays ◽  
Infectious Dose ◽  
E Coli ◽  
Huge Impact

ABSTRACT The histone-like protein H-NS is a global regulator in Escherichia coli that has been intensively studied in nonpathogenic strains. However, no comprehensive study on the role of H-NS and its paralogue, StpA, in gene expression in pathogenic E. coli has been carried out so far. Here, we monitored the global effects of H-NS and StpA in a uropathogenic E. coli isolate by using DNA arrays. Expression profiling revealed that more than 500 genes were affected by an hns mutation, whereas no effect of StpA alone was observed. An hns stpA double mutant showed a distinct gene expression pattern that differed in large part from that of the hns single mutant. This suggests a direct interaction between the two paralogues and the existence of distinct regulons of H-NS and an H-NS/StpA heteromeric complex. hns mutation resulted in increased expression of alpha-hemolysin, fimbriae, and iron uptake systems as well as genes involved in stress adaptation. Furthermore, several other putative virulence genes were found to be part of the H-NS regulon. Although the lack of H-NS, either alone or in combination with StpA, has a huge impact on gene expression in pathogenic E. coli strains, its effect on virulence is ambiguous. At a high infection dose, hns mutants trigger more sudden lethality due to their increased acute toxicity in murine urinary tract infection and sepsis models. At a lower infectious dose, however, mutants lacking H-NS are attenuated through their impaired growth rate, which can only partially be compensated for by the higher expression of numerous virulence factors.

scholarly journals The Escherichia colirhaSR-PrhaBADInducible Promoter System Allows Tightly Controlled Gene Expression over a Wide Range in Pseudomonas aeruginosa

2016 ◽  
Vol 82 (22) ◽  
pp. 6715-6727 ◽  
Author(s):  
Jeffrey Meisner ◽  
Joanna B. Goldberg
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Catabolite Repression ◽  
Inducible Promoter ◽  
Translational Initiation ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Promoter System

ABSTRACTThearaC-ParaBADinducible promoter system is tightly controlled and allows gene expression to be modulated over a wide range inEscherichia coli, which has led to its widespread use in other bacteria. Although anecdotal evidence suggests thataraC-ParaBADis leaky inPseudomonas aeruginosa, neither a thorough analysis of this inducible promoter system inP. aeruginosanor a concerted effort to identify alternatives with improved functionality has been reported. Here, we evaluated the functionality of thearaC-ParaBADsystem inP. aeruginosa. Using transcriptional fusions to alacZreporter gene, we determined that the noninduced expression fromaraC-ParaBADis high and cannot be reduced by carbon catabolite repression as it can inE. coli. Modulating translational initiation by altering ribosome-binding site strength reduced the noninduced activity but also decreased the maximal induced activity and narrowed the induction range. Integrating the inducible promoter system into the posttranscriptional regulatory network that controls catabolite repression inP. aeruginosasignificantly decreased the noninduced activity and increased the induction range. In addition to these improvements in the functionality of thearaC-ParaBADsystem, we found that thelacIq-PtacandrhaSR-PrhaBADinducible promoter systems had significantly lower noninduced expression and were inducible over a broader range thanaraC-ParaBAD. We demonstrated that noninduced expression from thearaC-ParaBADsystem supported the function of genes involved in antibiotic resistance and tryptophan biosynthesis inP. aeruginosa, problems that were avoided withrhaSR-PrhaBAD. rhaSR-PrhaBADis tightly controlled, allows gene expression over a wide range, and represents a significant improvement overaraC-ParaBADinP. aeruginosa.IMPORTANCEWe report the shortcomings of the commonly usedEscherichia coli araC-ParaBADinducible promoter system inPseudomonas aeruginosa, successfully reengineered it to improve its functionality, and show that theE. colirhaSR-PrhaBADsystem is tightly controlled and allows inducible gene expression over a wide range inP. aeruginosa.

scholarly journals Interactions of the Hindgut Mucosa-Associated Microbiome with Its Host Regulate Shedding of Escherichia coli O157:H7 by Cattle

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Ou Wang ◽  
Tim A. McAllister ◽  
Graham Plastow ◽  
Kim Stanford ◽  
Brent Selinger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Differentially Expressed Genes ◽  
Host Gene ◽  
Host Gene Expression ◽  
Differential Abundance ◽  
Content Type ◽  
E Coli ◽  
Differential Abundance Analysis

ABSTRACTCattle are the primary carrier ofEscherichia coliO157:H7, a foodborne human pathogen, and those shedding >104CFU/gram of feces ofE. coliO157:H7 are defined as supershedders (SS). This study investigated the rectoanal junction (RAJ) mucosa-associated microbiota and its relationship with host gene expression in SS and in cattle from whichE. coliO157:H7 was not detected (nonshedders [NS]), aiming to elucidate the mechanisms involved in supershedding. In total, 14 phyla, 66 families, and 101 genera of RAJ mucosa-associated bacteria were identified andFirmicutes(61.5 ± 7.5%),Bacteroidetes(27.9 ± 6.4%), andProteobacteria(5.5 ± 2.1%) were the predominant phyla. Differential abundance analysis of operational taxonomic units (OTUs) identified 2 OTUs unique to SS which were members ofBacteroidesandClostridiumand 7 OTUs unique to NS which were members ofCoprococcus,Prevotella,Clostridium, andPaludibacter. Differential abundance analysis of predicted microbial functions (using PICRUSt [phylogenetic investigation of communities by reconstruction of unobserved states]) revealed that 3 pathways had higher abundance (log2fold change, 0.10 to 0.23) whereas 12 pathways had lower abundance (log2fold change, −0.36 to −0.20) in SS. In addition, we identified significant correlations between expression of 19 differentially expressed genes and the relative abundance of predicted microbial functions, including nucleic acid polymerization and carbohydrate and amino acid metabolism. Our findings suggest that differences in RAJ microbiota at both the compositional and functional levels may be associated withE. coliO157:H7 supershedding and that certain microbial groups and microbial functions may influence RAJ physiology of SS by affecting host gene expression.IMPORTANCECattle with fecalE. coliO157:H7 at >104CFU per gram of feces have been defined as the supershedders, and they are responsible for the most of theE. coliO157:H7 spread into farm environment. Currently, no method is available for beef producers to eliminate shedding ofE. coliO157:H7 in cattle, and the lack of information about the mechanisms of supershedding greatly impedes the development of effective methods. This study investigated the role of the rectoanal junction (RAJ) mucosa-associated microbiome inE. coliO157:H7 shedding, and our results indicated that the compositions and functions of RAJ microbiota differed between supershedders and nonshedders. The identified relationship between the differentially abundant microbes and 19 previously identified differentially expressed genes suggests the role of host-microbial interactions involved inE. coliO157:H7 supershedding. Our findings provide a fundamental understanding of the supershedding phenomenon which is essential for the development of strategies, such as the use of directly fed microbials, to reduceE. coliO157:H7 shedding in cattle.

scholarly journals Susceptibility to beta-lactam antibiotics of Pseudomonas aeruginosa overproducing penicillin-binding protein 3.

1997 ◽  
Vol 41 (5) ◽  
pp. 1158-1161 ◽  
Author(s):  
X Liao ◽  
R E Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Protein ◽  
Broad Host Range ◽  
Penicillin Binding Protein ◽  
Beta Lactam ◽  
Beta Lactams ◽  
E Coli ◽  
Beta Lactam Antibiotics ◽  
Range Vector ◽  
Cell Inhibition

By using a broad-host-range vector, pUCP27, the Pseudomonas aeruginosa and Escherichia coli pbpB genes, which encode penicillin-binding protein 3 (PBP3), were separately overexpressed in a P. aeruginosa strain, PAO4089, that is deficient in producing chromosomal beta-lactamase. Susceptibility studies indicated that overproduction of the P. aeruginosa PBP3 in PAO4089 resulted in twofold-increased resistance to aztreonam, fourfold-increased resistance to cefepime and cefsulodin, and eightfold-increased resistance to ceftazidime, whereas overproduction of the P. aeruginosa PBP3 in PAO4089 did not affect susceptibility to PBP1-targeted cephaloridine or PBP2-targeted imipenem. Similar results were obtained with PAO4089 overproducing E. coli PBP3, with the exception that there was no influence on the MICs or minimal bactericidal concentrations of cefsulodin and cefepime, which have very low affinities for E. coli PBP3. These data are consistent with the conclusion that PBP3 has to achieve a certain level of saturation, with beta-lactams targeted to this protein, to result in cell inhibition or death.

Антимікробна резистентність провідних збудників інфекцій сечових шляхів у Києві (Україна)

2017 ◽  
Vol 1 (2) ◽  
pp. 48-60
Author(s):  
A.G. Salmanov ◽  
A.V. Rudenko
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Klebsiella Pneumoniae ◽  
Staphylococcus Epidermidis ◽  
Klebsiella Oxytoca ◽  
Enterobacter Aerogenes ◽  
Enterobacter Cloacae ◽  
Proteus Vulgaris ◽  
Providencia Rettgeri ◽  
E Coli

Мета роботи — вивчити резистентність до антибіотиків бактеріальних збудників інфекцій сечових шляхів (ІСШ), виділених у пацієнтів урологічного стаціонару в м. Києві. Матеріали і методи. Досліджено 1612 штамів бактерій, виділених із сечі хворих з ІСШ (цистит, уретрит, пієлонефрит), госпіталізованих в урологічне відділення ДУ «Інститут урології НАМН України» у м. Києві протягом 2016 р. Серед пацієнтів переважали жінки — 1201 (74,5 %). Вік хворих становив від 17 до 74 років. Для збору даних використано медичну документацію лікарні. Мікробіологічні дослідження виконано у лабораторії мікробіології ДУ «Інститут урології НАМН України». Аналізували результати культурального дослідження зразків сечі, зібраних за наявності клінічних ознак ІСШ. Дослідження клінічного матеріалу та інтерпретацію отриманих результатів проводили загальноприйнятими методами. Вивчено чутливість уропатогенів до 31 антибіотика дискодифузійним методом відповідно до рекомендацій Інституту клінічних та лабораторних стандартів США (Clinical and Laboratory Standards Institute (CLSI)). Результати та обговорення. Аналіз мікробного спектра сечі виявив домінування серед уропатогенів штамів Escherichia coli (32,0 %), Enterococcus faecalis (19,5 %), Klebsiella pneumoniae (10,9 %), Staphylococcus epidermidis (8,9 %), S. haemolyticus (6,5 %) та Pseudomonas aeruginosa (6,4 %). Частка Enterococcus faecium, Enterobacter aerogenes і Streptococcus viridans становила відповідно 2,5, 2,2 і 1,6 %, Enterobacter cloacae, Klebsiella oxytoca, Acinetobacter baumannii, Proteus vulgaris та Providencia rettgeri — менше 1,0 %. У більшості випадків (69,7 %) мікроорганізми виділено у монокультурі, у решті випадків — у мікробних асоціа- ціях. Високу резистентність до тестованих антибіотиків виявили штами E. aerogenes (45,1 %), E. cloacae (45,7 %), E. faecium (40,9 %), E. faecalis (40,7 %), E. coli (39,9 %), P. aeruginosa (34,0 %), K. pneumoniae (28,6 %). Найбільш активними до уропатогенів були іміпенем (E. coli — 87,6 %, P. aeruginosa — 75,7 %, E. cloacae — 67,3 %, E. aerogenes — 72,6 %, K. pneumoniae — 93,2 %), меропенем (E. coli — 89,1 %, P. aeruginosa — 76,7 %, K. pneumoniae — 82,6 %), лефлоцин (E. coli — 74,5 %, ентерококи — 78,7 %, P. aeruginosa — 76,7 %, E. cloacae — 73,9 %, E. aerogenes — 80,4 %, K. pneumoniae — 83,5 %), амоксицилін/клавуланат (ентерококи — 84,6 %), фурагін (ентерококи — 82,6 %), цефоперазон (K. pneumoniae — 89,2 %, P. aeruginosa — 73,8 %), цефтріаксон (K. pneumoniae — 80,1 %). Висновки. Антибіотикорезистентність збудників ІСШ — важлива терапевтична проблема. Найбільшою активністю до уропатогенів характеризуються іміпенем, меропенем, лефлоцин, амоксицилін/ клавуланат, фурагін, цефоперазон, цефтріаксон, які можна розглядати як препарат вибору для призначення стартової терапії ІСШ. Необхідно здійснювати постійний моніторинг за резистентністю до дії антибіотиків. Політику використання антибіотиків у кожному стаціонарі слід визначати залежно від локальних даних щодо резистентності до протимікробних препаратів.

scholarly journals MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5328
Author(s):  
Miao Ma ◽  
Margaux Lustig ◽  
Michèle Salem ◽  
Dominique Mengin-Lecreulx ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Cell Division ◽  
Membrane Proteins ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Active Efflux

One of the major families of membrane proteins found in prokaryote genome corresponds to the transporters. Among them, the resistance-nodulation-cell division (RND) transporters are highly studied, as being responsible for one of the most problematic mechanisms used by bacteria to resist to antibiotics, i.e., the active efflux of drugs. In Gram-negative bacteria, these proteins are inserted in the inner membrane and form a tripartite assembly with an outer membrane factor and a periplasmic linker in order to cross the two membranes to expulse molecules outside of the cell. A lot of information has been collected to understand the functional mechanism of these pumps, especially with AcrAB-TolC from Escherichia coli, but one missing piece from all the suggested models is the role of peptidoglycan in the assembly. Here, by pull-down experiments with purified peptidoglycans, we precise the MexAB-OprM interaction with the peptidoglycan from Escherichia coli and Pseudomonas aeruginosa, highlighting a role of the peptidoglycan in stabilizing the MexA-OprM complex and also differences between the two Gram-negative bacteria peptidoglycans.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials

2020 ◽  
Vol 367 (22) ◽  
Author(s):  
Chris Coward ◽  
Gopujara Dharmalingham ◽  
Omar Abdulle ◽  
Tim Avis ◽  
Stephan Beisken ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Pathogenic Bacteria ◽  
Selective Advantage ◽  
Mechanisms Of Action ◽  
Over Expression ◽  
E Coli ◽  
Synthase Inhibitor ◽  
Established Technique ◽  
Bacterial Transposon

ABSTRACT The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon–phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria.

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