scholarly journals Evaluation of the effects of sdiA, a luxR homologue, on adherence and motility of Escherichia coli O157 : H7

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1303-1312 ◽  
Author(s):  
Vijay K. Sharma ◽  
Shawn M. D. Bearson ◽  
Bradley L. Bearson
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Mutant Strain ◽  
Regulatory Networks ◽  
Double Mutant ◽  
Negative Regulator ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Genes Encoding

Quorum-sensing (QS) signalling pathways are important regulatory networks for controlling the expression of genes promoting adherence of enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 to epithelial cells. A recent study has shown that EHEC O157 : H7 encodes a luxR homologue, called sdiA, which upon overexpression reduces the expression of genes encoding flagellar and locus of enterocyte effacement (LEE) proteins, thus negatively impacting on the motility and intimate adherence phenotypes, respectively. Here, we show that the deletion of sdiA from EHEC O157 : H7 strain 86-24, and from a hha (a negative regulator of ler) mutant of this strain, enhanced bacterial adherence to HEp-2 epithelial cells of the sdiA mutant strains relative to the strains containing a wild-type copy of sdiA. Quantitative reverse transcription PCR showed that the expression of LEE-encoded genes ler, espA and eae in strains with the sdiA deletions was not significantly different from that of the strains wild-type for sdiA. Similarly, no additional increases in the expression of LEE genes were observed in a sdiA hha double mutant strain relative to that observed in the hha deletion mutant. While the expression of fliC, which encodes flagellin, was enhanced in the sdiA mutant strain, the expression of fliC was reduced by several fold in the hha mutant strain, irrespective of the presence or absence of sdiA, indicating that the genes sdiA and hha exert opposing effects on the expression of fliC. The strains with deletions in sdiA or hha showed enhanced expression of csgA, encoding curlin of the curli fimbriae, with the expression of csgA highest in the sdiA hha double mutant, suggesting an additive effect of these two gene deletions on the expression of csgA. No significant differences were observed in the expression of the genes lpfA and fimA of the operons encoding long polar and type 1 fimbriae in the sdiA mutant strain. These data indicate that SdiA has no significant effect on the expression of LEE genes, but that it appears to act as a strong repressor of genes encoding flagella and curli fimbriae, and the alleviation of the SdiA-mediated repression of these genes in an EHEC O157 : H7 sdiA mutant strain contributes to enhanced bacterial motility and increased adherence to HEp-2 epithelial cells.

scholarly journals Sexual Differentiation Is Coordinately Regulated by Cryptococcus neoformans CRK1 and GAT1

Genes ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. 669
Author(s):  
Kuang-Hung Liu ◽  
Wei-Chiang Shen
Keyword(s):  
Cryptococcus Neoformans ◽  
Sexual Differentiation ◽  
Regulatory Networks ◽  
Genetic Interaction ◽  
Negative Regulator ◽  
Wild Type ◽  
Basidiomycetous Fungus ◽  
Regulatory Circuit ◽  
Expression Of Genes ◽  
In The Wild

The heterothallic basidiomycetous fungus Cryptococcus neoformans has two mating types, MATa and MATα. Morphological progression of bisexual reproduction in C. neoformans is as follows: yeast to hyphal transition, filament extension, basidium formation, meiosis, and sporulation. C. neoformans Cdk-related kinase 1 (CRK1) is a negative regulator of bisexual mating. In this study, we characterized the morphological features of mating structures in the crk1 mutant and determined the genetic interaction of CRK1 in the regulatory networks of sexual differentiation. In the bilateral crk1 mutant cross, despite shorter length of filaments than in the wild-type cross, dikaryotic filaments and other structures still remained intact during bisexual mating, but the timing of basidium formation was approximately 18 h earlier than in the cross between wild type strains. Furthermore, gene expression analyses revealed that CRK1 modulated the expression of genes involved in the progression of hyphal elongation, basidium formation, karyogamy and meiosis. Phenotypic results showed that, although deletion of C. neoformans CRK1 gene increased the efficiency of bisexual mating, filamentation in the crk1 mutant was blocked by MAT2 or ZNF2 mutation. A bioinformatics survey predicted the C. neoformans GATA transcriptional factor Gat1 as a potential substrate of Crk1 kinase. Our genetic and phenotypic findings revealed that C. neoformans GAT1 and CRK1 formed a regulatory circuit to negatively regulate MAT2 to control filamentation progression and transition during bisexual mating.

scholarly journals ClpP of Streptococcus mutans Differentially Regulates Expression of Genomic Islands, Mutacin Production, and Antibiotic Tolerance

2009 ◽  
Vol 192 (5) ◽  
pp. 1312-1323 ◽  
Author(s):  
Partho Chattoraj ◽  
Anirban Banerjee ◽  
Saswati Biswas ◽  
Indranil Biswas
Keyword(s):  
Streptococcus Mutans ◽  
Mutant Strain ◽  
Protein Profile ◽  
Vital Role ◽  
Genomic Islands ◽  
Wild Type ◽  
Proteomic Data ◽  
Altered Protein ◽  
Expression Of Genes ◽  
Genes Encoding

ABSTRACT Streptococcus mutans is the primary etiological agent of human dental caries and, at times, of infective endocarditis. Within the oral cavity, the pathogen is subjected to conditions of stress. A well-conserved protein complex named ClpP (caseinolytic protease) plays a vital role in adaptation under stress conditions. To gain a better understanding of the global role of the ClpP protease in cellular homeostasis, a transcriptome analysis was performed using a ΔclpP mutant strain. The expression levels of more than 100 genes were up- or downregulated in the ΔclpP mutant compared to the wild type. Notably, the expression of genes in several genomic islands, such as TnSmu1 and TnSmu2, was differentially modulated in the ΔclpP mutant strain. ClpP deficiency also increased the expression of genes associated with a putative CRISPR locus. Furthermore, several stress-related genes and genes encoding bacteriocin-related peptides and many transcription factors were also found to be altered in the ΔclpP mutant strain. A comparative analysis of the two-dimensional protein profile of the wild type and the ΔclpP mutant strains showed altered protein profiles. Comparison of the transcriptome data with the proteomic data identified four common gene products, suggesting that the observed altered protein expression of these genes could be due to altered transcription. The results presented here indicate that ClpP-mediated proteolysis plays an important global role in the regulation of several important traits in this pathogen.

scholarly journals An msbB Homologue Carried in Plasmid pO157 Encodes an Acyltransferase Involved in Lipid A Biosynthesis in Escherichia coli O157:H7

2004 ◽  
Vol 72 (2) ◽  
pp. 1174-1180 ◽  
Author(s):  
Sang-Hyun Kim ◽  
Wenyi Jia ◽  
Russell E. Bishop ◽  
Carlton Gyles
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Double Mutant ◽  
Lipid A ◽  
Escherichia Coli O157 ◽  
Wild Type ◽  
Single Mutant ◽  
Double Mutant Strain ◽  
Mutant Strains ◽  
Lipid A Biosynthesis

ABSTRACT Escherichia coli O157:H7 carries a chromosomal msbB1 and a plasmid-encoded msbB2 gene. We characterized msbB2 function as a homologue of msbB1 by examination of wild-type organisms and mutant strains that lacked functional msbB1, msbB2, and both msbB1 and msbB2. The msbB double-mutant strain generated pentaacyl lipid A, while the single-mutant strains synthesized hexaacyl lipid A. Complementation with overexpressed msbB2 converted pentaacyl into hexaacyl lipid A in the double-mutant strain. The transcription of both msbB genes occurred simultaneously. Lack of MsbB2 activity slightly increased the microheterogeneity of the lipid A species. These results suggest that the msbB2 gene plays a role not only in the routine generation of fully hexaacylated lipid A but also in suppressing the microheterogeneity of lipid A species, the endotoxic determinant of the organism.

scholarly journals Role of the Extracytoplasmic Function Protein Family Sigma Factor RpoE in Metal Resistance of Escherichia coli

2005 ◽  
Vol 187 (7) ◽  
pp. 2297-2307 ◽  
Author(s):  
Monique Egler ◽  
Cornelia Grosse ◽  
Gregor Grass ◽  
Dietrich H. Nies
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Sigma Factor ◽  
Double Mutant ◽  
Expression Profiles ◽  
Protein Family ◽  
Wild Type ◽  
Qrt Pcr ◽  
Metal Treatment

ABSTRACT RpoE of Escherichia coli is a sigma factor of the extracytoplasmic function protein family and is required for the expression of proteins involved in maintaining the integrity of periplasmic and outer membrane components. RpoE of E. coli was needed for full resistance to Zn(II), Cd(II), and Cu(II). Promoter gene fusion and quantitative real time reverse transcription (RT)-PCR (qRT-PCR) assays demonstrated that expression of RpoE was induced by metals. Global gene expression profiles upon metal treatment of a ΔrpoE mutant strain and its wild-type strain were analyzed with microarrays, and selected genes were confirmed by qRT-PCR. The absolute number of genes that were changed in their expression upon metal stress was similar in both strains, but the increase or decrease in transcript levels upon metal treatment was smaller in the ΔrpoE mutant strain than in the wild type. Genes showing increased expression in the ΔrpoE mutant strain encoded proteins that belong to general defense systems against protein-denaturing agents. Genes showing decreased expression were part of the RpoE modulon itself plus the ompC gene, encoding a major outer membrane protein. A ΔompC deletion strain was as sensitive to Cu(II) and Cd(II) as the ΔrpoE mutant or a ΔrpoE ΔompC double mutant strain. In the case of Zn(II), the double mutant was more sensitive than either single mutant. This indicates that increased expression of OmpC contributes to the RpoE modulon-mediated response to metals.

scholarly journals Alopecia in Harlequin mutant mice is associated with reduced AIF protein levels and expression of retroviral elements

2020 ◽  
Author(s):  
Maik Hintze ◽  
Sebastian Griesing ◽  
Marion Michels ◽  
Birgit Blanck ◽  
Lena Wischhof ◽  
...  
Keyword(s):  
Hair Growth ◽  
Transcriptional Regulators ◽  
Mutant Mice ◽  
Wild Type ◽  
Protein Levels ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Keratinocyte Cell ◽  
Apoptosis Inducing Factor ◽  
Hair Cortex

AbstractWe investigated the contribution of apoptosis-inducing factor (AIF), a key regulator of mitochondrial biogenesis, in supporting hair growth. We report that pelage abnormalities developed during hair follicle (HF) morphogenesis in Harlequin (Hq) mutant mice. Fragility of the hair cortex was associated with decreased expression of genes encoding structural hair proteins, though key transcriptional regulators of HF development were expressed at normal levels. Notably, Aifm1 (R200 del) knockin males and Aifm1(R200 del)/Hq females showed minor hair defects, despite substantially reduced AIF levels. Furthermore, we cloned the integrated ecotropic provirus of the Aifm1Hq allele. We found that its overexpression in wild-type keratinocyte cell lines led to down-regulation of HF-specific Krt84 and Krtap3-3 genes without altering Aifm1 or epidermal Krt5 expression. Together, our findings imply that pelage paucity in Hq mutant mice is mechanistically linked to severe AIF deficiency and is associated with the expression of retroviral elements that might potentially influence the transcriptional regulation of structural hair proteins.

scholarly journals Variable Expressions of Staphylococcus aureus Bicomponent Leucotoxins Semiquantified by Competitive Reverse Transcription-PCR

2000 ◽  
Vol 66 (9) ◽  
pp. 3931-3938 ◽  
Author(s):  
St�phane Bronner ◽  
Patricia Stoessel ◽  
Alain Gravet ◽  
Henri Monteil ◽  
Gilles Pr�vost
Keyword(s):  
Staphylococcus Aureus ◽  
Reverse Transcription ◽  
Regulatory System ◽  
Optimization Procedure ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Specific Mrnas ◽  
Casamino Acids ◽  
Global Regulatory System

ABSTRACT A competitive reverse transcription-PCR method was developed for the semiquantitation of the expression of genes encoding bicomponent leucotoxins of Staphylococcus aureus, e.g., Panton-Valentine leucocidin (lukPV), gamma-hemolysin (hlgA and hlgCB), and LukE-LukD (lukED). The optimization procedure included RNA preparation; reverse transcription; the use of various amounts of enzymes, antisense primer, and RNA; and the final amplification chain reaction. Reproducible results were obtained, with sensitivity for detection of cDNA within the range of 1 mRNA/104 CFU to 102 mRNA/CFU, depending on the gene. Both specific mRNAs were more significantly expressed at the late-exponential phase of growth. Expression was about 100-fold higher in yeast extract-Casamino Acids-pyruvate medium than in heart infusion medium. Expression of the widely distributed gamma-hemolysin locus in the NTCC 8178 strain was around 10-fold diminished compared with that in the ATCC 49775 strain. Because of the lower level of hlgA expression, the corresponding protein, which is generally not abundant in culture supernatant, should be investigated for its contribution to the leucotoxin-associated virulence. The agr, sar, and agr sar mutant strains revealed a great dependence with regard to leucotoxin expression on the global regulatory system inS. aureus, except that expression of hlgA was not affected in the agr mutant.

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.

scholarly journals An Epistasis Analysis of recA and recN in Escherichia coli K-12

Genetics ◽  
2020 ◽  
Vol 216 (2) ◽  
pp. 381-393
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Double Mutant ◽  
Strand Break ◽  
Wild Type ◽  
Epistasis Analysis ◽  
Chromatid Cohesion ◽  
K 12 ◽  
Structural Maintenance Of Chromosomes ◽  
Sos Protein

RecA is essential for double-strand-break repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and a member of the Structural Maintenance of Chromosomes family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ∆recN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact, and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ∆recN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆recN were Rec+ as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆recN cells were very UVS (sensitive), Rec−, had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆recN remove functions in genetically distinct pathways important for DNA repair, and that RecA Q300 was not important for an interaction between RecN and RecA in vivo. recA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain, reflecting greater stringency of RecA’s role in that background.

scholarly journals Inactivation of Genes Encoding MutL and MutS Proteins Influences Adhesion and Biofilm Formation by Neisseria gonorrhoeae

2019 ◽  
Vol 7 (12) ◽  
pp. 647 ◽  
Author(s):  
Jagoda Płaczkiewicz ◽  
Monika Adamczyk-Popławska ◽  
Robert Lasek ◽  
Pawel Bącal ◽  
Agnieszka Kwiatek
Keyword(s):  
Epithelial Cells ◽  
Neisseria Gonorrhoeae ◽  
Biofilm Formation ◽  
Dna Microarrays ◽  
Wild Type ◽  
Human Genes ◽  
Genes Encoding ◽  
Differential Gene ◽  
Bacterial Genes ◽  
Muts Gene

Neisseria gonorrhoeae is an etiological agent of gonorrhea, which remains a global health problem. This bacterium possesses MutL and MutS DNA repair proteins encoded by mutL and mutS genes, whose inactivation causes a mutator phenotype. We have demonstrated the differential gene expression in N. gonorrhoeae mutL and mutS mutants using DNA microarrays. A subset of differentially expressed genes encodes proteins that can influence adhesion and biofilm formation. Compared to the wild-type strain, N. gonorrhoeae mutL and mutS mutants formed denser biofilms with increased biofilm-associated biomass on the abiotic surface. The N. gonorrhoeae mutS::km, but not the mutL mutant, was also more adherent and invasive to human epithelial cells. Further, during infection of epithelial cells with N. gonorrhoeae mutS::km, the expression of some bacterial genes encoding proteins that can influence gonococcal adhesion was changed compared with their expression in cells infected with the wild-type gonococcus, as well as of human genes’ encoding receptors utilized by N. gonorrhoeae (CD46, CEACAM 1, HSPG 2). Thus, deficiency in the mutS gene resulting in increased mutation frequency in singular organisms can be beneficial in populations because these mutants can be a source of features linked to microbial fitness.

scholarly journals Interaction of an Outer Membrane Protein of Enterotoxigenic Escherichia coli with Cell Surface Heparan Sulfate Proteoglycans

2002 ◽  
Vol 70 (3) ◽  
pp. 1530-1537 ◽  
Author(s):  
James M. Fleckenstein ◽  
James T. Holland ◽  
David L. Hasty
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Heparan Sulfate Proteoglycans ◽  
Eukaryotic Cells ◽  
Heparin Binding ◽  
Wild Type ◽  
E Coli

ABSTRACT We have previously shown that enterotoxigenic invasion protein A (Tia), a 25-kDa outer membrane protein encoded on an apparent pathogenicity island of enterotoxigenic Escherichia coli (ETEC) strain H10407, mediates attachment to and invasion into cultured human gastrointestinal epithelial cells. The epithelial cell receptor(s) for Tia has not been identified. Here we show that Tia interacts with cell surface heparan sulfate proteoglycans. Recombinant E. coli expressing Tia mediated invasion into wild-type epithelial cell lines but not invasion into proteoglycan-deficient cells. Furthermore, wild-type eukaryotic cells, but not proteoglycan-deficient eukaryotic cells, attached to immobilized polyhistidine-tagged recombinant Tia (rTia). Binding of epithelial cells to immobilized rTia was inhibited by exogenous heparan sulfate glycosaminoglycans but not by hyaluronic acid, dermatan sulfate, or chondroitin sulfate. Similarly, pretreatment of eukaryotic cells with heparinase I, but not pretreatment of eukaryotic cells with chrondroitinase ABC, inhibited attachment to rTia. In addition, we also observed heparin binding to both immobilized rTia and recombinant E. coli expressing Tia. Heparin binding was inhibited by a synthetic peptide representing a surface loop of Tia, as well as by antibodies directed against this peptide. Additional studies indicated that Tia, as a prokaryotic heparin binding protein, may also interact via sulfated proteoglycan molecular bridges with a number of mammalian heparan sulfate binding proteins. These findings suggest that the binding of Tia to host epithelial cells is mediated at least in part through heparan sulfate proteoglycans and that ETEC belongs on the growing list of pathogens that utilize these ubiquitous cell surface molecules as receptors.

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