Effects of the twin-arginine translocase on the structure and antimicrobial susceptibility of Escherichia coli biofilms

2005 ◽  
Vol 51 (8) ◽  
pp. 671-683 ◽  
Author(s):  
Joe J Harrison ◽  
Howard Ceri ◽  
Erin A Badry ◽  
Nicole J Roper ◽  
Kerry L Tomlin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Planktonic Cells ◽  
E Coli ◽  
Minimal Media ◽  
Twin Arginine Translocase ◽  
Biofilm Structure

In this descriptive study, we used Escherichia coli twin-arginine translocase (tat) mutants to distinguish antibiotic tolerance from the formation of mature biofilm structure. Biofilm formation by wild-type and Δtat strains of E. coli was evaluated using viable cell counts, scanning electron microscopy, and confocal laser-scanning microscopy. Escherichia coli Δtat mutants had an impaired ability to form biofilms when grown in rich or minimal media. These mutants produced disorganized layers and cell aggregates with significantly decreased cell density relative to the wild-type strain. In contrast, wild-type E. coli grown under similar test conditions formed highly structured, surface-adherent communities. We thus determined if this decreased biofilm formation by E. coli Δtat mutants may result in lowered tolerance to antimicrobials. When grown in rich media, planktonic Δtat mutants were hypersensitive to some metals, detergents, and antibiotics. However, the corresponding biofilms were about as resilient as the wild-type strain. In contrast, both planktonic cells and biofilms of the ΔtatABC strain grown in minimal media were hypersensitive to many antimicrobials. Remarkably, these biofilms remained up to 365 times more tolerant to β-lactams than corresponding planktonic cells. Our data suggest that the twin-arginine translocase may play a contributing role in the antimicrobial tolerance, structural organization, and formation of mature E. coli biofilms under nutrient-limited conditions. However, the high tolerance of the ΔtatABC strain to bactericidal concentrations of antimicrobials indicates that mature biofilm structure may not be required for surface-adherent E. coli to survive exposure to these lethal factors.Key words: biofilm structure, twin-arginine translocase (tat), Escherichia coli, antimicrobial susceptibility/tolerance.

scholarly journals Escherichia coliO157:H7 responds to phosphate starvation by modifying LPS involved in biofilm formation

2019 ◽  
Author(s):  
Philippe Vogeleer ◽  
Antony T. Vincent ◽  
Samuel M. Chekabab ◽  
Steve J. Charette ◽  
Alexey Novikov ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Inorganic Phosphate ◽  
Type Strain ◽  
Wild Type Strain ◽  
Pho Regulon ◽  
Wild Type ◽  
E Coli ◽  
Pi Starvation ◽  
Pst System

ABSTRACTIn open environments such as water, enterohemorrhagicEscherichia coliO157:H7 responds to inorganic phosphate (Pi) starvation by inducing the Pho regulon controlled by PhoB. The phosphate-specific transport (Pst) system is the high-affinity Pi transporter. In the Δpstmutant, PhoB is constitutively activated and regulates the expression of genes from the Pho regulon. InE. coliO157:H7, the Δpstmutant, biofilm, and autoagglutination were increased. In the double-deletion mutant ΔpstΔphoB, biofilm and autoagglutination were similar to the wild-type strain, suggesting that PhoB is involved. We investigated the relationship between PhoB activation and enhanced biofilm formation by screening a transposon mutant library derived from Δpstmutant for decreased autoagglutination and biofilms mutants. Lipopolysaccharide (LPS) genes involved in the synthesis of the LPS core were identified. Transcriptomic studies indicate the influence of Pi-starvation andpstmutation on LPS biosynthetic gene expression. LPS analysis indicated that the O-antigen was deficient in the Δpstmutant. Interestingly,waaH, encoding a glycosyltransferase associated with LPS modifications inE. coliK-12, was highly expressed in the Δpstmutant ofE. coliO157:H7. Deletion ofwaaHfrom the Δpstmutant and from the wild-type strain grown in Pi-starvation conditions decreased the biofilm formation but without affecting LPS. Our findings suggest that LPS core is involved in the autoagglutination and biofilm phenotypes of the Δpstmutant and that WaaH plays a role in biofilm in response to Pi-starvation. This study highlights the importance of Pi-starvation in biofilm formation of E. coli O157:H7, which may affect its transmission and persistence.IMPORTANCEEnterohemorrhagicEscherichia coliO157:H7 is a human pathogen responsible for bloody diarrhea and renal failures. In the environment, O157:H7 can survive for prolonged periods of time under nutrient-deprived conditions. Biofilms are thought to participate in this environmental lifestyle. Previous reports have shown that the availability of extracellular inorganic phosphate (Pi) affected bacterial biofilm formation; however, nothing was known about O157:H7 biofilm formation. Our results show that O157:H7 membrane undergoes modifications upon PhoB activation leading to increased biofilm formation. A mutation in the Pst system results in reduced amount of the smooth type LPS and that this could influence the biofilm composition. This demonstrates how theE. coliO157:H7 adapts to Pi starvation increasing its ability to occupy different ecological niches.

scholarly journals Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic Escherichia coli O157:H7

2001 ◽  
Vol 183 (17) ◽  
pp. 5187-5197 ◽  
Author(s):  
Vanessa Sperandio ◽  
Alfredo G. Torres ◽  
Jorge A. Girón ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Quorum Sensing ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Mechanism ◽  
Sos Response ◽  
Enterohemorrhagic Escherichia Coli ◽  
Trna Genes ◽  
Wild Type ◽  
E Coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

scholarly journals Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 774
Author(s):  
Virginio Cepas ◽  
Victoria Ballén ◽  
Yaiza Gabasa ◽  
Miriam Ramírez ◽  
Yuly López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
De Novo ◽  
Wild Type ◽  
Planktonic Cells ◽  
E Coli ◽  
Qrt Pcr ◽  
Transposon Insertion ◽  
De Novo Purine Biosynthesis

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

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.

scholarly journals Expression of Different-Size Transcripts from theclpP-clpX Operon of Escherichia coli during Carbon Deprivation

2000 ◽  
Vol 182 (23) ◽  
pp. 6630-6637 ◽  
Author(s):  
Chin Li ◽  
Yi Ping Tao ◽  
Lee D. Simon
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Type Strain ◽  
Wild Type Strain ◽  
Transcription Termination ◽  
Carbon Starvation ◽  
Lower Percentage ◽  
Wild Type ◽  
Lower Efficiency ◽  
E Coli

ABSTRACT Transcription of the clpP-clpX operon ofEscherichia coli leads to the production of two different sizes of transcripts. In log phase, the level of the longer transcript is higher than the level of the shorter transcript. Soon after the onset of carbon starvation, the level of the shorter transcript increases significantly, and the level of the longer transcript decreases. The longer transcript consists of the entireclpP-clpX operon, whereas the shorter transcript contains the entire clpP gene but none of the clpXcoding sequence. The RpoH protein is required for the increase in the level of the shorter transcript during carbon starvation. Primer extension experiments suggest that there is increased usage of the ς32-dependent promoter of the clpP-clpXoperon within 15 min after the start of carbon starvation. Expression of the clpP-clpX operon from the promoters upstream of theclpP gene decreases to a very low level by 20 min after the onset of carbon starvation. Various pieces of evidence suggest, though they do not conclusively prove, that production of the shorter transcript may involve premature termination of the longer transcript. The half-life of the shorter transcript is much less than that of the longer transcript during carbon starvation. E. coli rpoBmutations that affect transcription termination efficiency alter the ratio of the shorter clpP-clpX transcript to the longer transcript. The E. coli rpoB3595 mutant, with an RNA polymerase that terminates transcription with lower efficiency than the wild type, accumulates a lower percentage of the shorter transcript during carbon starvation than does the isogenic wild-type strain. In contrast, the rpoB8 mutant, with an RNA polymerase that terminates transcription with higher efficiency than the wild type, produces a higher percentage of the shorter clpP-clpXtranscript when E. coli is in log phase. These and other data are consistent with the hypothesis that the shorter transcript results from premature transcription termination during production of the longer transcript.

scholarly journals DOSEc, a Heme-Regulated Phosphodiesterase, Plays an Important Role in the Regulation of the Cyclic AMP Level in Escherichia coli

2005 ◽  
Vol 187 (19) ◽  
pp. 6678-6682 ◽  
Author(s):  
Tokiko Yoshimura-Suzuki ◽  
Ikuko Sagami ◽  
Nao Yokota ◽  
Hirofumi Kurokawa ◽  
Toru Shimizu
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Type Strain ◽  
Redox State ◽  
Wild Type Strain ◽  
Functional Domain ◽  
Wild Type ◽  
Aerobic Conditions ◽  
E Coli ◽  
Knockout Strain

ABSTRACT Heme-regulated phosphodiesterase from Escherichia coli (DOSEc) catalyzes the hydrolysis of cyclic AMP (cAMP) in vitro and is regulated by the redox state of the bound heme. Changes in the redox state result in alterations in the three-dimensional structure of the enzyme, which is then transmitted to the functional domain to switch catalysis on or off. Because DOSEc was originally cloned from E. coli genomic DNA, it has not been known whether it is actually expressed in wild-type E. coli. In addition, the turnover number of DOSEc using cAMP as a substrate is only 0.15 min−1, which is relatively low for a physiologically relevant enzyme. In the present study, we demonstrated for the first time that the DOSEc gene and protein are expressed in wild-type E. coli, especially under aerobic conditions. We also developed a DOSEc gene knockout strain (Δdos). Interestingly, the knockout of dos caused excess accumulation of intracellular cAMP (26-fold higher than in the wild-type strain) under aerobic conditions, whereas accumulation of cAMP was not observed under anaerobic conditions. We also found differences in cell morphology and growth rate between the mutant cells and the wild-type strain. The changes in the knockout strain were partially complemented by introducing an expression plasmid for dos. Thus, the present study revealed that expression of DOSEc is regulated according to environmental O2 availability at the transcriptional level and that the concentration of cAMP in cells is regulated by DOSEc expression.

scholarly journals RstA, a two-component response regulator, plays important roles in multiple virulence-associated processes in enterohemorrhagic Escherichia coli O157:H7

Gut Pathogens ◽  
2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Yutao Liu ◽  
Shujie Li ◽  
Wendi Li ◽  
Peisheng Wang ◽  
Peng Ding ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Acid Tolerance ◽  
Enterohemorrhagic Escherichia Coli ◽  
Regulatory Function ◽  
Wild Type ◽  
Two Component

Abstract Background Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC O157 encounters varied microenvironments during infection, and can efficiently adapt to these using the two-component system (TCS). Recently, a functional TCS, RstAB, has been implicated in the regulation of virulence of several bacterial pathogens. However, the regulatory function of RstAB in EHEC O157 is poorly understood. This study aimed at providing insights into the global effects of RstA on gene expression in EHEC O157. Results In the present study, we analyzed gene expression differences between the EHEC O157 wild-type strain and a ΔrstA mutant using RNA-seq technology. Genes with differential expression in the ΔrstA mutant compared to that in the wild-type strain were identified and grouped into clusters of orthologous categories. RstA promoted EHEC O157 LEE gene expression, adhesion in vitro, and colonization in vivo by indirect regulation. We also found that RstA could bind directly to the promoter region of hdeA and yeaI to enhance acid tolerance and decrease biofilm formation by modulating the concentration of c-di-GMP. Conclusions In summary, the RstAB TCS in EHEC O157 plays a major role in the regulation of virulence, acid tolerance, and biofilm formation. We clarified the regulatory function of RstA, providing an insight into mechanisms that may be potential drug targets for treatment of EHEC O157-related infections.

scholarly journals The Transcriptional Antiterminator RfaH Represses Biofilm Formation in Escherichia coli

2006 ◽  
Vol 188 (4) ◽  
pp. 1316-1331 ◽  
Author(s):  
Christophe Beloin ◽  
Kai Michaelis ◽  
Karin Lindner ◽  
Paolo Landini ◽  
Jörg Hacker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Wild Type Strain ◽  
Wild Type ◽  
Strain Mg1655 ◽  
Upec Strain ◽  
E Coli ◽  
Initial Adhesion ◽  
State Production ◽  
K 12

ABSTRACT We investigated the influence of regulatory and pathogenicity island-associated factors (Hha, RpoS, LuxS, EvgA, RfaH, and tRNA5 Leu) on biofilm formation by uropathogenic Escherichia coli (UPEC) strain 536. Only inactivation of rfaH, which encodes a transcriptional antiterminator, resulted in increased initial adhesion and biofilm formation by E. coli 536. rfaH inactivation in nonpathogenic E. coli K-12 isolate MG1655 resulted in the same phenotype. Transcriptome analysis of wild-type strain 536 and an rfaH mutant of this strain revealed that deletion of rfaH correlated with increased expression of flu orthologs. flu encodes antigen 43 (Ag43), which mediates autoaggregation and biofilm formation. We confirmed that deletion of rfaH leads to increased levels of flu and flu-like transcripts in E. coli K-12 and UPEC. Supporting the hypothesis that RfaH represses biofilm formation through reduction of the Ag43 level, the increased-biofilm phenotype of E. coli MG1655rfaH was reversed upon inactivation of flu. Deletion of the two flu orthologs, however, did not modify the behavior of mutant 536rfaH. Our results demonstrate that the strong initial adhesion and biofilm formation capacities of strain MG1655rfaH are mediated by both increased steady-state production of Ag43 and likely increased Ag43 presentation due to null rfaH-dependent lipopolysaccharide depletion. Although the roles of rfaH in the biofilm phenotype are different in UPEC strain 536 and K-12 strain MG1655, this study shows that RfaH, in addition to affecting the expression of bacterial virulence factors, also negatively controls expression and surface presentation of Ag43 and possibly another Ag43-independent factor(s) that mediates cell-cell interactions and biofilm formation.

The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein

1978 ◽  
Vol 24 (5) ◽  
pp. 629-631 ◽  
Author(s):  
La Verne Russell ◽  
Hiroshi Yamazaki
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Type Strain ◽  
Wild Type Strain ◽  
Anaerobic Growth ◽  
Receptor Protein ◽  
Wild Type ◽  
Cyclic Amp Receptor Protein ◽  
E Coli ◽  
Cyclic Amp Synthesis

The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.

scholarly journals Genetic Analysis of the Role ofyfiRin the Ability of Escherichia coli CFT073 To Control Cellular Cyclic Dimeric GMP Levels and To Persist in the Urinary Tract

2013 ◽  
Vol 81 (9) ◽  
pp. 3089-3098 ◽  
Author(s):  
Erica L. Raterman ◽  
Daniel D. Shapiro ◽  
Daniel J. Stevens ◽  
Kevin J. Schwartz ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Cellulose Production ◽  
Content Type ◽  
E Coli ◽  
Successful Infection ◽  
Tract Infections

ABSTRACTDuring urinary tract infections (UTIs), uropathogenicEscherichia colimust maintain a delicate balance between sessility and motility to achieve successful infection of both the bladder and kidneys. Previous studies showed that cyclic dimeric GMP (c-di-GMP) levels aid in the control of the transition between motile and nonmotile states inE. coli. TheyfiRNBlocus inE. coliCFT073 contains genes for YfiN, a diguanylate cyclase, and its activity regulators, YfiR and YfiB. Deletion ofyfiRyielded a mutant that was attenuated in both the bladder and the kidneys when tested in competition with the wild-type strain in the murine model of UTI. A doubleyfiRNmutant was not attenuated in the mouse model, suggesting that unregulated YfiN activity and likely increased cytoplasmic c-di-GMP levels cause a survival defect. Curli fimbriae and cellulose production were increased in theyfiRmutant. Expression ofyhjH, a gene encoding a proven phosphodiesterase, in CFT073 ΔyfiRsuppressed the overproduction of curli fimbriae and cellulose and further verified that deletion ofyfiRresults in c-di-GMP accumulation. Additional deletion ofcsgDandbcsA, genes necessary for curli fimbriae and cellulose production, respectively, returned colonization levels of theyfiRdeletion mutant to wild-type levels. Peroxide sensitivity assays and iron acquisition assays displayed no significant differences between theyfiRmutant and the wild-type strain. These results indicate that dysregulation of c-di-GMP production results in pleiotropic effects that disableE. coliin the urinary tract and implicate the c-di-GMP regulatory system as an important factor in the persistence of uropathogenicE. coli in vivo.

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