Related Enterobacteria Research Articles

A molecular comparison of [Fe-S] cluster-based homeostasis in Escherichia coli and Pseudomonas aeruginosa.

ISC and SUF molecular systems build and transfer Fe-S cluster to cellular apo protein clients. The model Escherichia coli has both ISC and SUF and study of the interplay between the two systems established that the ISC system is the house-keeping one and SUF the stress-responding one. Unexpectedly, our recent phylogenomic analysis revealed that in contrast to E. coli (and related enterobacteria such as Salmonella), most bacteria have only one system, and, in most cases, it is SUF. Pseudomonas aeruginosa fits the general rule of having only one system but stands against the rule by having ISC. This study aims at engineering P. aeruginosa harboring E. coli systems and vice versa. Comparison of the recombinants allowed to assess the functional versatility of each system while appreciating their contribution to cellular homeostasis in different species context.

A two-step activation mechanism enables mast cells to differentiate their response between extracellular and invasive enterobacterial infection

Mast cells localize to mucosal tissues and contribute to innate immune defense against infection. How mast cells sense, differentiate between, and respond to bacterial pathogens remains a topic of ongoing debate. Using the prototype enteropathogen Salmonella Typhimurium (S.Tm) and other related enterobacteria, here we show that mast cells can regulate their cytokine secretion response to distinguish between extracellular and invasive bacterial infection. Tissue-invasive S.Tm and mast cells colocalize in the mouse gut during acute Salmonella infection. Toll-like Receptor 4 (TLR4) sensing of extracellular S.Tm, or pure lipopolysaccharide, causes a modest induction of cytokine transcripts and proteins, including IL-6, IL-13, and TNF. By contrast, type-III-secretion-system-1 (TTSS-1)-dependent S.Tm invasion of both mouse and human mast cells triggers rapid and potent inflammatory gene expression and >100-fold elevated cytokine secretion. The S.Tm TTSS-1 effectors SopB, SopE, and SopE2 here elicit a second activation signal, including Akt phosphorylation downstream of effector translocation, which combines with TLR activation to drive the full-blown mast cell response. Supernatants from S.Tm-infected mast cells boost macrophage survival and maturation from bone-marrow progenitors. Taken together, this study shows that mast cells can differentiate between extracellular and host-cell invasive enterobacteria via a two-step activation mechanism and tune their inflammatory output accordingly.

Topoisomerase I Essentiality, DnaA-Independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli.

Topoisomerase I (Topo I) of Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here, we show that several topA alleles, including ΔtopA, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in the absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain (MDS42) or (ii) by an RNA polymerase (RNAP) mutation, rpoB*35, that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) one of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn leads to transcription-replication conflicts and to cSDR. IMPORTANCE In all life forms, double-helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli. That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability and suggests that in its absence, aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.

IgaA negatively regulates the Rcs Phosphorelay via contact with the RcsD Phosphotransfer Protein.

Two-component systems and phosphorelays play central roles in the ability of bacteria to rapidly respond to changing environments. In E. coli and related enterobacteria, the complex Rcs phosphorelay is a critical player in the bacterial response to antimicrobial peptides, beta-lactam antibiotics, and other disruptions at the cell surface. The Rcs system is unusual in that an inner membrane protein, IgaA, is essential due to its negative regulation of the RcsC/RcsD/RcsB phosphorelay. While it is known that IgaA transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the phosphorelay has remained unknown. Here we performed in vivo interaction assays and genetic dissection of the critical proteins and found that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation. Interactions between IgaA and RcsD within their respective periplasmic domains of these two proteins anchor repression of signaling. However, the signaling response depends on a second interaction between cytoplasmic loop 1 of IgaA and a truncated Per-Arndt-Sim (PAS-like) domain in RcsD. A single point mutation in the PAS-like domain increased interactions between the two proteins and blocked induction of the phosphorelay. IgaA may regulate RcsC, the histidine kinase that initiates phosphotransfer through the phosphorelay, indirectly, via its contacts with RcsD. Unlike RcsD, and unlike many other histidine kinases, the periplasmic domain of RcsC is dispensable for the response to signals that induce the Rcs phosphorelay system. The multiple contacts between IgaA and RcsD constitute a poised sensing system, preventing potentially toxic over-activation of this phosphorelay while enabling it to rapidly and quantitatively respond to signals.

Duplex PCR for specific detection of Escherichia coli and its differentiation from other Enterobacteriaceae

Escherichia coli is a normal inhabitant of gastrointestinal tract of humans and animals and one of the most important causes of bovine mastitis. Definitive identification of E. coli from other members of Enterobacteriaceae remains ambiguous. The present study describes a duplex PCR, targeting 2 housekeeping genes, the lacy (lactose permease) and phoA (alkaline phosphatase) for the reliable detection of E. coli that enables its differentiation from biochemically and phylogenetically related bacteria. The assay was evaluated employing 19 ATCC (American type culture collection) reference strains of Enterobacteriaceae family. Validation of the assay with E. coli (154) isolated from milk and faeces rendered the assay to be specific. The results suggest that the technique can be used for accurate detection of E. coli and thus can be adapted for testing bacteriological safety of milk, for field applications, and in laboratories handling clinical samples. This PCR (polymerase chain reaction) can successfully distinguish E. coli including E. coli O157 from Shigella spp and other related enterobacteria, emphasizing its relevance and utility in studies related to E. coli infection.

Monovalent Cation Dependence on the Kinase Activity of Salmonella Typhimurium CheA: Experiment and Modeling

CheA is a multi-domain histidine kinase which has been demonstrated to play a role in the chemosensory array in Escherichia coli, Salmonella typhimurium, Thermotoga maritima and related enterobacteria. The P4 domain of CheA is of particular interest due to its role in catalyzing ATP/ADP conversion, a process which involves Mg2+ in the ATP-binding pocket of the enzyme. Recent studies in GHKL superfamily members MutL (from E coli) and BCK (from Rattus norvegicus) demonstrate improved activity in the presence of monovalent cations Na+ and K+. Here we present results from a combined experimental and computational analysis to assess the effect of monovalent cations on the activity of Salmonella typhimurium CheA and subsequent phosphor-transfer to response regulator CheY.

Comparative study ofSoxRactivation by redox‐active compounds

SoxR from Escherichia coli and related enterobacteria is activated by a broad range of redox-active compounds through oxidation or nitrosylation of its [2Fe-2S] cluster. Activated SoxR then induces SoxS, which subsequently activates more than 100 genes in response. In contrast, non-enteric SoxRs directly activate their target genes in response to redox-active compounds that include endogenously produced metabolites. We compared the responsiveness of SoxRs from Streptomyces coelicolor (ScSoxR), Pseudomonas aeruginosa (PaSoxR) and E. coli (EcSoxR), all expressed in S. coelicolor, towards natural or synthetic redox-active compounds. EcSoxR responded to all compounds examined, whereas ScSoxR was insensitive to oxidants such as paraquat (Eh -440 mV) and menadione sodium bisulphite (Eh -45 mV) and to NO generators. PaSoxR was insensitive only to some NO generators. Whole-cell EPR analysis of SoxRs expressed in E. coli revealed that the [2Fe-2S](1+) of ScSoxR was not oxidizable by paraquat, differing from EcSoxR and PaSoxR. The mid-point redox potential of purified ScSoxR was determined to be -185 ± 10 mV, higher by approximately 100 mV than those of EcSoxR and PaSoxR, supporting its limited response to paraquat. The overall sensitivity profile indicates that both redox potential and kinetic reactivity determine the differential responses of SoxRs towards various oxidants.

Benzimidazole analogs of l‐tryptophan are substrates and inhibitors of tryptophan indole lyase from Escherichia coli

Tryptophan indole lyase (TIL), an enzyme found in Escherichia coli and related enterobacteria, produces indole from l-tryptophan (l-Trp). Indole is a signaling molecule in bacteria, affecting biofilm formation, pathogenicity and antibiotic resistance. β-(Benzimidazol-1-yl)-l-alanine (BZI-Ala), 2-amino-4-(benzimidazol-1-yl)butyric acid (homo-BZI-Ala) and 2-amino-5-(benzimidazol-1-yl)pentanoic acid (bishomo-BZI-Ala) were synthesized and tested as substrates and inhibitors of TIL. BZI-Ala is a good substrate of TIL, with Km = 300 μm, kcat = 5.6 s(-1) and kcat /Km = 1.9 × 10(4) , similar to l-Trp. BZI-Ala is also a good substrate for H463F mutant TIL, which has very low activity with l-Trp. In contrast, homo-BZI-Ala was found to be a potent competitive inhibitor of TIL, with a Ki of 13.4 μm. However, the higher homolog, bishomo-BZI-Ala, was inactive as an inhibitor of TIL at a concentration of 600 μm, and is thus a much weaker inhibitor. The reaction of TIL with BZI-Ala was too fast to be observed in the stopped-flow spectrophotometer, and shows an aldimine intermediate in the steady state. However, H463F TIL shows equilibrating mixtures of aldimine and quinonoid complexes in the steady state. The spectra of the reaction of TIL with homo-BZI-Ala show a rapidly formed intermediate absorbing at 340 nm, probably a gem-diamine, that decays slowly to form a quinonoid complex absorbing at 494 nm. The potent binding of homo-BZI-Ala may be due to it being a 'bi-product' analog of the indole-α-aminoacrylate complex. These results demonstrate that an amino acid substrate may be converted to a potent inhibitor of TIL simply by homologation, which may be useful in the design of other potent TIL inhibitors.

Microcins in action: amazing defence strategies of Enterobacteria

Probably the oldest and most widespread antimicrobial strategy in living organisms is the use of antimicrobial peptides. Bacteria secrete such defence peptides, termed bacteriocins, that they use for microbial competitions. Microcins are bacteriocins of less than 10 kDa produced by Escherichia coli and related enterobacteria through the ribosomal pathway. They are synthesized as linear precursors, which can further undergo complex post-translational modifications resulting from dedicated maturation enzymes encoded in the microcin gene clusters, and are processed by proteolytic cleavage. Microcins exert potent bactericidal activities that use subtle and clever mechanisms to cross outer and inner membranes of Gram-negative bacteria. To cross the outer membrane, siderophore-microcins hijack receptors involved in iron acquisition. The lasso-peptide microcin J25, which is characterized by a knotted arrangement where the C-terminal tail is threaded through an N-terminal macrolactam ring, uses a hydroxamate siderophore receptor and the inner-membrane protein SbmA for import in sensitive bacteria, where it inhibits bacterial transcription through binding to RNAP (RNA polymerase). Microcin C produced as a heptapeptide adenylate, requires an outer-membrane porin and an inner-membrane ABC (ATP-binding-cassette) transporter to reach the cytoplasm of target bacteria, where it is processed by proteases into a non-hydrolysable aspartyl-adenylate analogue. Therefore, despite showing different killing mechanisms and the absence of any structural homology, microcins have the common characteristic to use Trojan horse strategies to destroy their competitors. They offer new and promising tracks for further design and engineering of novel efficient antibiotics.

STM2209-STM2208 (opvAB): A Phase Variation Locus of Salmonella enterica Involved in Control of O-Antigen Chain Length

STM2209 and STM2208 are contiguous loci annotated as putative protein-coding genes in the chromosome of Salmonella enterica. Lack of homologs in related Enterobacteria and low G+C content suggest that S. enterica may have acquired STM2209-STM2208 by horizontal transfer. STM2209 and STM2208 are co-transcribed from a promoter upstream STM2209, and their products are inner (cytoplasmic) membrane proteins. Analysis with the bacterial adenylate cyclase two-hybrid system suggests that STM2209 and STM2208 may interact. Expression of STM2209-STM2208 is subjected to phase variation in wild type Salmonella enterica serovar Typhimurium. Switching frequencies in LB medium are 6.1×10−5 (OFF→ON) and 3.7×10−2 (ON→OFF) per cell and generation. Lack of DNA adenine methylation locks STM2209-STM2208 in the ON state, and lack of the LysR-type factor OxyR locks STM2209-STM2208 in the OFF state. OxyR-dependent activation of STM2209-STM2208 expression is independent of the oxidation state of OxyR. Salmonella cultures locked in the ON state show alteration of O-antigen length in the lipopolysaccharide, reduced absorption of bacteriophage P22, impaired resistance to serum, and reduced proliferation in macrophages. Phenotypic heterogeneity generated by STM2209-STM2208 phase variation may thus provide defense against phages. In turn, formation of a subpopulation unable to proliferate in macrophages may restrain Salmonella spread in animal organs, potentially contributing to successful infection.

The Critical Roles of Polyamines in Regulating ColE7 Production and Restricting ColE7 Uptake of the Colicin-producing Escherichia coli

The ColE7 operon is an SOS response regulon, which encodes bacteriocin ColE7 to kill susceptible Escherichia coli and its related enterobacteria under conditions of stress. We have observed for the first time that polyamines confer limited resistance against ColE7 on E. coli cells. Thus, this study aims to investigate the role of polyamines in modulating the protective effect of the E. coli cells against colicin. In the experiments, we surprisingly found that endogenous polyamines are also essential for ColE7 production, and the rate of polyamine synthesis is directly related to the SOS response. Our experimental results further indicated that exogenous polyamines suppress the expression of TolA, BtuB, OmpF, and OmpC proteins that are responsible for ColE7 uptake. Moreover, two-dimensional gel electrophoresis revealed that the production of two periplasmic proteins, PotD and OppA, is increased in E. coli cells under ColE7 exposure. Based on these observations, we propose that endogenous polyamines may play a dual role in the ColE7 system. Polyamines may participate in initiating the expression of the SOS response of the ColE7 operon and simultaneously down-regulate proteins that are essential for colicin uptake, thus conferring a survival advantage on colicin-producing E. coli under stress conditions in the natural environment.

The roles of positive and negative selection in the molecular evolution of insect endosymbionts

The evolutionary rate acceleration observed in most endosymbiotic bacteria may be explained by higher mutation rates, changes in selective pressure, and increased fixation of deleterious mutations by genetic drift. Here, we explore the forces influencing molecular evolution in Blochmannia, an obligate endosymbiont of Camponotus and related ant genera. Our goals were to compare rates of sequence evolution in Blochmannia with related bacteria, to explore variation in the strength and efficacy of negative (purifying) selection, and to evaluate the effect of positive selection. For six Blochmannia pairs, plus Buchnera and related enterobacteria, estimates of sequence divergence at four genes confirm faster rates of synonymous evolution in the ant mutualist. This conclusion is based on higher d S between Blochmannia lineages despite their more recent divergence. Likewise, generally higher dN in Blochmannia indicates faster rates of nonsynonymous substitution in this group. One exception is the groEL gene, for which lower d N and d N / d S compared to Buchnera indicate exceptionally strong negative selection in Blochmannia. In addition, we explored evidence for positive selection in Blochmannia using both site-and lineage-based maximum likelihood models. These approaches confirmed heterogeneity of d N / d S among codon sites and revealed significant variation in d N / d S across Blochmannia lineages for three genes. Lineage variation affected genes independently, with no evidence of parallel changes in d N / d S across genes along a given branch. Our data also reveal instances of d N / d S greater than one; however, we do not interpret these large d N / d S ratios as evidence for positive selection. In sum, while drift may contribute to an overall rate acceleration at nonsynonymous sites in Blochmannia, variable selective pressures best explain the apparent gene-specific changes in d N / d S across lineages of this ant mutualist. In the course of this study, we reanalyzed variation at Buchnera groEL and found no evidence of positive selection that was previously reported.

Complete genome sequence of Salmonella enterica serovar Typhimurium LT2.

Salmonella enterica subspecies I, serovar Typhimurium (S. typhimurium), is a leading cause of human gastroenteritis, and is used as a mouse model of human typhoid fever. The incidence of non-typhoid salmonellosis is increasing worldwide, causing millions of infections and many deaths in the human population each year. Here we sequenced the 4,857-kilobase (kb) chromosome and 94-kb virulence plasmid of S. typhimurium strain LT2. The distribution of close homologues of S. typhimurium LT2 genes in eight related enterobacteria was determined using previously completed genomes of three related bacteria, sample sequencing of both S. enterica serovar Paratyphi A (S. paratyphi A) and Klebsiella pneumoniae, and hybridization of three unsequenced genomes to a microarray of S. typhimurium LT2 genes. Lateral transfer of genes is frequent, with 11% of the S. typhimurium LT2 genes missing from S. enterica serovar Typhi (S. typhi), and 29% missing from Escherichia coli K12. The 352 gene homologues of S. typhimurium LT2 confined to subspecies I of S. enterica-containing most mammalian and bird pathogens-are useful for studies of epidemiology, host specificity and pathogenesis. Most of these homologues were previously unknown, and 50 may be exported to the periplasm or outer membrane, rendering them accessible as therapeutic or vaccine targets.

Genome sequence of E. coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 is the agent responsible for several outbreaks of bloody diarrhea in the USA, UK, Japan and many other countries. Recently Perna and collaborators 1xGenome sequence of enterohaemorrhagic Escherichia coli O157:H7. Perna, N.T. Nature. 2001; 409: 529–533Crossref | PubMed | Scopus (1329)See all References1 reported the genome sequence of this pathogen and compared it with the already known sequence of the non-pathogenic E. coli K-12. The availability of this genome sequence will help the identification of genes responsible for pathogenesis, help develop better methods for detection of this strain and also enlighten our knowledge of E. coli evolution. These authors observed that E. coli O157:H7 has 1.34 Mb of DNA that is absent in K-12, and K-12 has 0.53 Mb of DNA that is absent in O157:H7. These strains share a common ‘backbone’ sequence of about 4.1 Mb that is co-linear except for one 422 kb inversion spanning the replication terminus. The ‘backbone’ is punctuated by hundreds of islands of apparently introgressed DNA designated ‘K-islands’ for the DNA segments present only in E. coli K-12 and ‘O-islands’ for DNA segments present only in E. coli O157:H7. These results suggest that lateral gene transfer is far more extensive than previously anticipated. Of the O-islands, only 40% could be assigned a function and many classifiable proteins are related to known virulence-associated proteins from other E. coli strains or related enterobacteria. Together, these findings reveal a great level of diversity between two members of the E. coli species. Most differences are attributable to horizontal transfer and offer a wealth of candidate virulence genes.

Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria

Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria

Phosphatase-mediated heavy metal accumulation by a Citrobacter sp. and related enterobacteria.

A Citrobacter sp. was reported previously to accumulate heavy metals as cell-bound heavy metal phosphates. Metal uptake is mediated by the activity of a periplasmic acid-type phosphatase that liberates inorganic phosphate to provide the precipitant ligand for heavy metals presented to the cells. Amino acid sequencing of peptide fragments of the purified enzyme revealed significant homology to the phoN product (acid phosphatase) of some other enterobacteria. These organisms, together with Klebsiella pneumoniae, previously reported to produce acid phosphatase, were tested for their ability to remove uranium and lanthanum from challenge solutions supplemented with phosphatase substrate. The coupling of phosphate liberation to metal bioaccumulation was limited to the metal accumulating Citrobacter sp.; therefore the participation of species-specific additional factors in metal bioaccumulation was suggested.