Showing papers on "Escherichia coli published in 2003"
TL;DR: Resistance to silver compounds as determined by bacterial plasmids and genes has been defined by molecular genetics and the use of molecular epidemiological tools will establish the range and diversity of such resistance systems in clinical and non-clinical sources.
Abstract: Resistance to silver compounds as determined by bacterial plasmids and genes has been defined by molecular genetics. Silver resistance conferred by the Salmonella plasmid pMGH100 involves nine genes in three transcription units. A sensor/responder (SilRS) two-component transcriptional regulatory system governs synthesis of a periplasmic Ag(I)-binding protein (SilE) and two efflux pumps (a P-type ATPase (SilP) plus a three-protein chemiosmotic RND Ag(I)/H+ exchange system (SilCBA)). The same genes were identified on five of 19 additional IncH incompatibility class plasmids but thus far not on other plasmids. Of 70 random enteric isolates from a local hospital, isolates from catheters and other Ag-exposed sites, and total genomes of enteric bacteria, 10 have recognizable sil genes. The centrally located six genes are found and functional in the chromosome of Escherichia coli K-12, and also occur on the genome of E. coli O157:H7. The use of molecular epidemiological tools will establish the range and diversity of such resistance systems in clinical and non-clinical sources. Silver compounds are used widely as effective antimicrobial agents to combat pathogens (bacteria, viruses and eukaryotic microorganisms) in the clinic and for public health hygiene. Silver cations (Ag+) are microcidal at low concentrations and used to treat burns, wounds and ulcers. Ag is used to coat catheters to retard microbial biofilm development. Ag is used in hygiene products including face creams, ‘alternative medicine’ health supplements, supermarket products for washing vegetables, and water filtration cartridges. Ag is generally without adverse effects for humans, and argyria (irreversible discoloration of the skin resulting from subepithelial silver deposits) is rare and mostly of cosmetic concern.
1,257 citations
TL;DR: The gram-negative bacterial species Salmonella enterica and Escherichia coli are members of the family Enterobacteriaceae that spend a good part of their lives as residents of animal hosts.
Abstract: The gram-negative bacterial species Salmonella enterica and Escherichia coli are members of the family Enterobacteriaceae that spend a good part of their lives as residents of animal hosts. S. enterica is the etiologic agent of gastroenteritis and typhoid fever in humans ([88][1]), whereas E. coli
732 citations
TL;DR: Escherichia coli is equipped with multiple systems to ensure safe copper handling under varying environmental conditions, but pathways of copper uptake and intracellular copper handling are still not identified in E. coli.
Abstract: Escherichia coli is equipped with multiple systems to ensure safe copper handling under varying environmental conditions. The Cu(I)-translocating P-type ATPase CopA, the central component in copper homeostasis, is responsible for removing excess Cu(I) from the cytoplasm. The multi-copper oxidase CueO and the multi-component copper transport system CusCFBA appear to safeguard the periplasmic space from copper-induced toxicity. Some strains of E. coli can survive in copper-rich environments that would normally overwhelm the chromosomally encoded copper homeostatic systems. Such strains possess additional plasmid-encoded genes that confer copper resistance. The pco determinant encodes genes that detoxify copper in the periplasm, although the mechanism is still unknown. Genes involved in copper homeostasis are regulated by MerR-like activators responsive to cytoplasmic Cu(I) or two-component systems sensing periplasmic Cu(I). Pathways of copper uptake and intracellular copper handling are still not identified in E. coli.
675 citations
TL;DR: Detailed examination demonstrated that in experimentally infected calves the majority of tissue-associated bacteria were adherent to mucosal epithelium within a defined region extending up to 5 cm proximally from the recto-anal junction, a novel tropism that may enhance dissemination both between animals and from animals to humans.
Abstract: Escherichia coli O157:H7 causes bloody diarrhea and potentially fatal systemic sequelae in humans. Cattle are most frequently identified as the primary source of infection, and E. coli O157:H7 generally colonizes the gastrointestinal tracts of cattle without causing disease. In this study, persistence and tropism were assessed for four different E. coli O157:H7 strains. Experimentally infected calves shed the organism for at least 14 days prior to necropsy. For the majority of these animals, as well as for a naturally colonized animal obtained from a commercial beef farm, the highest numbers of E. coli O157:H7 were found in the feces, with negative or significantly lower levels detected in lumen contents taken from the gastrointestinal tract. Detailed examination demonstrated that in these individuals the majority of tissue-associated bacteria were adherent to mucosal epithelium within a defined region extending up to 5 cm proximally from the recto-anal junction. The tissue targeted by E. coli O157:H7 was characterized by a high density of lymphoid follicles. Microcolonies of the bacterium were readily detected on the epithelium of this region by immunofluorescence microscopy. As a consequence of this specific distribution, E. coli O157:H7 was present predominately on the surface of the fecal stool. In contrast, other E. coli serotypes were present at consistent levels throughout the large intestine and were equally distributed in the stool. This is a novel tropism that may enhance dissemination both between animals and from animals to humans. The accessibility of this site may facilitate simple intervention strategies.
529 citations
TL;DR: It is shown that the expression of stress envelope response genes, such as the psp operon or elements of the cpx and rpoE pathways, is a general feature of E. coli mature biofilms.
Abstract: The formation of biofilm results in a major lifestyle switch that is thought to affect the expression of multiple genes and operons. We used DNA arrays to study the global effect of biofilm formation on gene expression in mature Escherichia coli K-12 biofilm. We show that, when biofilm is compared with the exponential growth phase, 1.9% of the genes showed a consistent up- or downregulation by a factor greater than two, and that 10% of the E. coli genome is significantly differentially expressed. The functions of the genes induced in these conditions correspond to stress response as well as energy production, envelope biogenesis and unknown functions. We provide evidence that the expression of stress envelope response genes, such as the psp operon or elements of the cpx and rpoE pathways, is a general feature of E. coli mature biofilms. We also compared biofilm with the stationary growth phase and showed that the biofilm lifestyle, although sharing similarities with the stationary growth phase, triggers the expression of specific sets of genes. Using gene disruption of 54 of the most biofilm-induced genes followed by a detailed phenotypic study, we validated the biological relevance of our analysis and showed that 20 of these genes are required for the formation of mature biofilm. This group includes 11 genes of previously unknown function. These results constitute a comprehensive analysis of the global transcriptional response triggered in mature E. coli biofilms and provide insights into its physiological signature.
476 citations
TL;DR: A macroarray-based global analysis of iron and Fur-dependent gene expression in E. coli has revealed several novel Fur-repressed genes likely to specify at least three additional iron-transport pathways as mentioned in this paper.
468 citations
TL;DR: The results demonstrate the effectiveness of the current interventions used by the industry and highlight the significance of hides as a major source of pathogens on beef carcasses.
464 citations
TL;DR: Transferable plasmid-mediated quinolone resistance associated with qnr is prevalent in quinOLone-resistant clinical strains of E. coli from Shanghai and may contribute to the rapid increase in bacterial resistance to qu inolones in China.
Abstract: Although quinolone resistance usually results from chromosomal mutations, recent studies indicate that quinolone resistance can also be plasmid mediated. The gene responsible, qnr, is distinct from the known quinolone resistance genes and in previous studies seemed to be restricted to Klebsiella pneumoniae and Escherichia coli isolates from the University of Alabama in Birmingham, where this resistance was discovered. In Shanghai, the frequency of ciprofloxacin resistance in E. coli has exceeded 50% since 1993. Seventy-eight unique ciprofloxacin-resistant clinical isolates of E. coli from Shanghai hospitals were screened for the qnr gene by colony blotting and Southern hybridization of plasmid DNA. Conjugation experiments were done with azide-resistant E. coli J53 as a recipient with selection for plasmid-encoded antimicrobial resistance (chloramphenicol, gentamicin, or tetracycline) and azide counterselection. qnr genes were sequenced, and the structure of the plasmid DNA adjacent to qnr was analyzed by primer walking with a sequential series of outward-facing sequencing primers with plasmid DNA templates purified from transconjugants. Six (7.7%) of 78 strains gave a reproducible hybridization signal with a qnr gene probe on colony blots and yielded strong signals on plasmid DNA preparations. Quinolone resistance was transferred from all six probe-positive strains. Transconjugants had 16- to 250-fold increases in the MICs of ciprofloxacin relative to that of the recipient. All six strains contained qnr with a nucleotide sequence identical to that originally reported, except for a single nucleotide change (CTA→CTG at position 537) encoding the same amino acid. qnr was located in complex In4 family class 1 integrons. Two completely sequenced integrons were designated In36 and In37. Transferable plasmid-mediated quinolone resistance associated with qnr is thus prevalent in quinolone-resistant clinical strains of E. coli from Shanghai and may contribute to the rapid increase in bacterial resistance to quinolones in China.
460 citations
TL;DR: The complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp) was determined and it was found that the strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences.
Abstract: We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism9s distinctive lifestyle that have yet to be explained.
419 citations
TL;DR: The F sex factor of Escherichia coli is a paradigm for bacterial conjugation and its transfer (tra) region represents a subset of the type IV secretion system (T4SS) family, especially F- and P-like systems, and summarizes the properties of the F transfer region gene products.
Abstract: The F sex factor of Escherichia coli is a paradigm for bacterial conjugation and its transfer (tra) region represents a subset of the type IV secretion system (T4SS) family. The F tra region encodes eight of the 10 highly conserved (core) gene products of T4SS including TraAF (pilin), the TraBF, -KF (secretin-like), -VF (lipoprotein) and TraCF (NTPase), -EF, -LF and TraGF (N-terminal region) which correspond to TrbCP, -IP, -GP, -HP, -EP, -JP, DP and TrbLP, respectively, of the P-type T4SS exemplified by the IncP plasmid RP4. F lacks homologs of TrbBP (NTPase) and TrbFP but contains a cluster of genes encoding proteins essential for F conjugation (TraFF, -HF, -UF, -WF, the C-terminal region of TraGF, and TrbCF) that are hallmarks of F-like T4SS. These extra genes have been implicated in phenotypes that are characteristic of F-like systems including pilus retraction and mating pair stabilization. F-like T4SS systems have been found on many conjugative plasmids and in genetic islands on bacterial chromosomes. Although few systems have been studied in detail, F-like T4SS appear to be involved in the transfer of DNA only whereas P- and I-type systems appear to transport protein or nucleoprotein complexes. This review examines the similarities and differences among the T4SS, especially F- and P-like systems, and summarizes the properties of the F transfer region gene products.
403 citations
TL;DR: Data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane.
Abstract: The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), have been reported to bind lipopolysaccharide (LPS), opsonize microorganisms, and enhance the clearance of lung pathogens. In this study, we examined the effect of SP-A and SP-D on the growth and viability of Gram-negative bacteria. The pulmonary clearance of Escherichia coli K12 was reduced in SP-A–null mice and was increased in SP-D–overexpressing mice, compared with strain-matched wild-type controls. Purified SP-A and SP-D inhibited bacterial synthetic functions of several, but not all, strains of E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes. In general, rough E. coli strains were more susceptible than smooth strains, and collectin-mediated growth inhibition was partially blocked by coincubation with rough LPS vesicles. Although both SP-A and SP-D agglutinated E. coli K12 in a calcium-dependent manner, microbial growth inhibition was independent of bacterial aggregation. At least part of the antimicrobial activity of SP-A and SP-D was localized to their C-terminal domains using truncated recombinant proteins. Incubation of E. coli K12 with SP-A or SP-D increased bacterial permeability. Deletion of the E. coli OmpA gene from a collectin-resistant smooth E. coli strain enhanced SP-A and SP-D–mediated growth inhibition. These data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane.
TL;DR: The λ Red recombineering technology has been optimized for use in pathogenic species of E. coli, namely EHEC and EPEC and should be encouraged for faster identification of virulence factors and the speedy generation of bacterial mutants for vaccine development.
Abstract: The λ Red recombineering technology has been used extensively in Escherichia coli and Salmonella typhimurium for easy PCR-mediated generation of deletion mutants, but less so in pathogenic species of E. coli such as EHEC and EPEC. Our early experiments with the use of λ Red in EHEC and EPEC have led to sporadic results, leading to the present study to identify factors that might improve the efficiency of Red recombineering in these pathogenic strains of E. coli. In this report, we have identified conditions that optimize the use of λ Red for recombineering in EHEC and EPEC. Using plasmids that contain a Ptac-red-gam operon and a temperature-sensitive origin of replication, we have generated multiple mutations (both marked and unmarked) in known virulence genes. In addition, we have easily deleted five O157-specific islands (O-islands) of EHEC suspected of containing virulence factors. We have examined the use of both PCR-generated substrates (40 bp of flanking homology) and plasmid-derived substrates (~1 kb of flanking homology); both work well and each have their own advantages. The establishment of the hyper-rec phenotype requires only a 20 minute IPTG induction period of red and gam. This recombinogenic window is important as constitutive expression of red and gam induces a 10-fold increase in spontaneous resistance to rifampicin. Other factors such as the orientation of the drug marker in recombination substrates and heat shock effects also play roles in the success of Red-mediated recombination in EHEC and EPEC. The λ Red recombineering technology has been optimized for use in pathogenic species of E. coli, namely EHEC and EPEC. As demonstration of this technology, five O-islands of EHEC were easily and precisely deleted from the chromosome by electroporation with PCR-generated substrates containing drug markers flanked with 40 bp of target DNA. These results should encourage the use of λ Red recombineering in these and other strains of pathogenic bacteria for faster identification of virulence factors and the speedy generation of bacterial mutants for vaccine development.
TL;DR: It is suggested that E. coli K‐12 biofilm development and maturation is dependent on cell‐cell adhesion factors, which may act as inducers of self‐assembly processes that result in differently structured biofilms depending on the adhesive properties on the cell surface.
Abstract: The development and maturation of E. coli biofilms in flow-chambers was investigated. We found that the presence of transfer constitutive IncF plasmids induced biofilm development forming structures resembling those reported for Pseudomonas aeruginosa. The development occurred in a step-wise process: (i). attachment of cells to the substratum, (ii). clonal growth and microcolony formation, and (iii). differentiation into expanding structures rising 70-100 microm into the water phase. The first two steps were the same in the plasmid-carrying and plasmid-free strains, whereas the third step only occurred in conjugation pilus proficient plasmid-carrying strains. The final shapes of the expanding structures in the mature biofilm seem to be determined by the pilus configuration, as various mutants affected in the processing and activity of the transfer pili displayed differently structured biofilms. We further provide evidence that flagella, type 1 fimbriae, curli and Ag43 are all dispensable for the observed biofilm maturation. In addition, our results indicate that cell-to-cell signalling mediated by autoinducer 2 (AI-2) is not required for differentiation of E. coli within a biofilm community. We suggest on the basis of these results that E. coli K-12 biofilm development and maturation is dependent on cell-cell adhesion factors, which may act as inducers of self-assembly processes that result in differently structured biofilms depending on the adhesive properties on the cell surface.
TL;DR: Escherichia coli strains from patients with uncomplicated urinary tract infections were examined by DNA sequencing for fluoroquinolone resistance-associated mutations in six genes, suggesting that an increased general mutation rate may play a significant role in the development of high-level resistance to fluoroquolones by increasing the rate of accumulation of rare new mutations.
Abstract: Escherichia coli strains from patients with uncomplicated urinary tract infections were examined by DNA sequencing for fluoroquinolone resistance-associated mutations in six genes: gyrA, gyrB, parC, parE, marOR, and acrR. The 54 strains analyzed had a susceptibility range distributed across 15 dilutions of the fluoroquinolone MICs. There was a correlation between the fluoroquinolone MIC and the number of resistance mutations that a strain carried, with resistant strains having mutations in two to five of these genes. Most resistant strains carried two mutations in gyrA and one mutation in parC. In addition, many resistant strains had mutations in parE, marOR, and/or acrR. No (resistance) mutation was found in gyrB. Thus, the evolution of fluoroquinolone resistance involves the accumulation of multiple mutations in several genes. The spontaneous mutation rate in these clinical strains varied by 2 orders of magnitude. A high mutation rate correlated strongly with a clinical resistance phenotype. This correlation suggests that an increased general mutation rate may play a significant role in the development of high-level resistance to fluoroquinolones by increasing the rate of accumulation of rare new mutations.
TL;DR: It is found that both AmiA and AmiC are routed to the periplasm via Tat, providing an explanation for the Tat– division phenotype and is the first entirelyPeriplasmic component to be localized.
Abstract: The N-acetylmuramoyl-l-alanine amidases of Escherichia coli (AmiA, B and C) are periplasmic enzymes that remove murein cross-links by cleaving the peptide moiety from N-acetylmuramic acid. Ami- cells form chains, indicating that the amidases help to split the septal murein. Interestingly, cells defective in the twin-arginine protein transport (Tat) pathway show a similar division defect. We find that both AmiA and AmiC are routed to the periplasm via Tat, providing an explanation for the Tat- division phenotype. Taking advantage of the ability of Tat to export prefolded (fluorescent) green fluorescent protein (GFP) to the periplasm, we sublocalized AmiA and AmiC in live cells using functional fusions to GFP. Interestingly, the periplasmic localization of the fusions differed markedly. AmiA-GFP appeared to be dispersed throughout the periplasm in all cells. AmiC-GFP similarly appeared throughout the periplasm in small cells, but was concentrated almost exclusively at the septal ring in constricting cells. Recruitment of AmiC to the ring was mediated by an N-terminal non-amidase targeting domain and required the septal ring component FtsN. AmiC therefore replaces FtsN as the latest known recruit to the septal ring and is the first entirely periplasmic component to be localized.
TL;DR: Members of a family of catecholate siderophores, called salmochelins, were isolated by reversed-phase HPLC from Salmonella enterica serotype Typhimurium and structurally characterized and indicated that the IroB protein might be responsible for the glucosyl transfer predicted by sequence similarities to known glycosyltransferases.
Abstract: Members of a family of catecholate siderophores, called salmochelins, were isolated by reversed-phase HPLC from Salmonella enterica serotype Typhimurium and structurally characterized by Fourier transform ion cyclotron resonance–MS/MS and GC–MS. The tentative structure of salmochelin 1 contained two 2,3- dihydroxybenzoylserine moieties bridged by a glucose residue, bound to the serine hydroxyl group of one moiety and the carboxylate of the second moiety. Salmochelin 2 contained in addition a second glucose residue linked to a third 2,3-dihydroxybenzoylserine moiety. Salmochelins were not produced by an iroBC mutant, which indicated that the IroB protein might be responsible for the glucosyl transfer predicted by sequence similarities to known glycosyltransferases. Uptake experiments with radiolabeled 55Fe-salmochelin and growth promotion tests with salmochelins showed that the IroN outer membrane receptor, encoded in the iroA locus of S. enterica and uropathogenic Escherichia coli strains, was the main receptor for ferric salmochelin transport.
TL;DR: The authors' experiments monitor directly a dramatic decrease of the stress inside the unopened phage capsid upon addition of polyvalent cations to the host solution, in agreement with many recent theories of DNA interactions.
Abstract: Bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. We demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. In the particular case of bacteriophage λ, the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by a pressure of 20 atmospheres. Furthermore, our experiments monitor directly a dramatic decrease of the stress inside the unopened phage capsid upon addition of polyvalent cations to the host solution, in agreement with many recent theories of DNA interactions.
TL;DR: The similarity of the Nal-resistant and -susceptible populations suggests that they derive from the same source population, presumably the avian fecal flora, with Nal resistance emerging by spontaneous mutation as a result of fluoroquinolone exposure.
Abstract: Fluoroquinolone use in poultry production may select for resistant Escherichia coli that can be transmitted to humans. To define the prevalence and virulence potential of poultry-associated, quinolone-resistant E. coli in the United States, 169 retail chicken products from the Minneapolis-St. Paul area (1999 to 2000) were screened for nalidixic acid (Nal)-resistant E. coli. Sixty-two (37%) products yielded Nal-resistant E. coli. From 55 products that yielded both Nal-resistant and susceptible E. coli, two isolates (one resistant, one susceptible) per sample were further characterized. Twenty-three (21%) of the 110 E. coli isolates (13 resistant, 10 susceptible) satisfied criteria for extraintestinal pathogenic E. coli (ExPEC), i.e., exhibited ≥2 of pap (P fimbriae), sfa/foc (S/F1C fimbriae), afa/dra (Dr binding adhesins), iutA (aerobactin receptor), and kpsMT II (group 2 capsule synthesis). Compared with other isolates, ExPEC isolates more often derived from virulence-associated E. coli phylogenetic groups B2 or D (74% versus 32%; P < 0.001) and exhibited more ExPEC-associated virulence markers (median, 10.0 versus 4.0; P < 0.001). In contrast, the Nal-resistant and -susceptible populations were indistinguishable according to all characteristics analyzed, including pulsed-field gel electrophoresis profiles. These findings indicate that Nal-resistant E. coli is prevalent in retail poultry products and that a substantial minority of such strains represent potential human pathogens. The similarity of the Nal-resistant and -susceptible populations suggests that they derive from the same source population, presumably the avian fecal flora, with Nal resistance emerging by spontaneous mutation as a result of fluoroquinolone exposure.
TL;DR: A new gene, sul3, which specifies a 263-amino-acid protein similar to a dihydropteroate synthase encoded by the 54-kb conjugative plasmid pVP440 from Escherichia coli was characterized and conferred resistance to sulfamethoxazole on E. coli.
Abstract: A new gene, sul3, which specifies a 263-amino-acid protein similar to a dihydropteroate synthase encoded by the 54-kb conjugative plasmid pVP440 from Escherichia coli was characterized. Expression of the cloned sul3 gene conferred resistance to sulfamethoxazole on E. coli. Two copies of the insertion element IS15Delta/26 flanked the region containing sul3. The sul3 gene was detected in one-third of the sulfonamide-resistant pathogenic E. coli isolates from pigs in Switzerland.
TL;DR: Root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition, and Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae.
Abstract: Enteric pathogens, such as Salmonella enterica and Escherichia coli O157:H7, have been shown to contaminate fresh produce. Under appropriate conditions, these bacteria will grow on and invade the plant tissue. We have developed Arabidopsis thaliana (thale cress) as a model system with the intention of studying plant responses to human pathogens. Under sterile conditions and at 100% humidity, S. enterica serovar Newport and E. coli O157:H7 grew to 10(9) CFU g(-1) on A. thaliana roots and to 2 x 10(7) CFU g(-1) on shoots. Furthermore, root inoculation led to contamination of the entire plant, indicating that the pathogens are capable of moving on or within the plant in the absence of competition. Inoculation with green fluorescent protein-labeled S. enterica and E. coli O157:H7 showed invasion of the roots at lateral root junctions. Movement was eliminated and invasion decreased when nonmotile mutants of S. enterica were used. Survival of S. enterica serovar Newport and E. coli O157:H7 on soil-grown plants declined as the plants matured, but both pathogens were detectable for at least 21 days. Survival of the pathogen was reduced in unautoclaved soil and amended soil, suggesting competition from indigenous epiphytes from the soil. Enterobacter asburiae was isolated from soil-grown A. thaliana and shown to be effective at suppressing epiphytic growth of both pathogens under gnotobiotic conditions. Seed and chaff harvested from contaminated plants were occasionally contaminated. The rate of recovery of S. enterica and E. coli O157:H7 from seed varied from undetectable to 19% of the seed pools tested, depending on the method of inoculation. Seed contamination by these pathogens was undetectable in the presence of the competitor, Enterobacter asburiae. Sampling of 74 pools of chaff indicated a strong correlation between contamination of the chaff and seed (P = 0.025). This suggested that contamination of the seed occurred directly from contaminated chaff or by invasion of the flower or silique. However, contaminated seeds were not sanitized by extensive washing and chlorine treatment, indicating that some of the bacteria reside in a protected niche on the seed surface or under the seed coat.
TL;DR: Cryo-EM density maps showing the 70S ribosome of E. coli in two different functional states related by a ratchet-like motion were analyzed using real-space refinement, suggesting an important role of ribosomal proteins in facilitating the dynamics of translation.
TL;DR: A completely autonomous bacterium with a 21 amino acid genetic code that can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon is generated.
Abstract: We have generated a completely autonomous bacterium with a 21 amino acid genetic code. This bacterium can biosynthesize a nonstandard amino acid from basic carbon sources and incorporate this amino acid into proteins in response to the amber nonsense codon. The biosynthetic pathway for the amino acid p-aminophenylalanine (pAF) as well as a unique pAF synthetase and cognate tRNA were added to Escherichia coli. Denaturing gel electrophoresis and mass spectrometric analysis show that pAF is incorporated into myoglobin with fidelity and efficiency rivaling those of the common 20 amino acids. This and other such organisms may provide an opportunity to examine the evolutionary consequences of adding new amino acids to the genetic repertoire, as well as generate proteins with new or enhanced biological functions.
TL;DR: It is demonstrated that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis.
Abstract: Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudogene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis.
TL;DR: The news article pointed out that not only could the immunogenic reaction be triggered by a change in formulation—as in the Eprex case—but also by variations in amino acid sequence, glycosylation or even by impurities cropping up during manufacturing or administration of the drug.
Abstract: Sabine Louët responds: In researching the news story, I had several interviews with Huub Schellekens, who explained to me the key findings of his laboratory’s research on Eprex. At no point during these interviews did he strongly underline the fact that there was such a level of uncertainty regarding the findings of his study. However, it is clear that the activity of a therapeutic protein is likely to depend on many factors; indeed, the news article pointed out this fact: “Not only could the immunogenic reaction be triggered by a change in formulation—as in the Eprex case—but also by variations in amino acid sequence, glycosylation or even by impurities cropping up during manufacturing or administration of the drug.” The adverse events associated with the manufacture, formulation and administration of Ortho Biotech’s (a Johnson & Johnson affiliate) erythropoietin alpha (Eprex) exemplify the difficulties faced by companies that seek to manufacture and formulate generic biopharmaceuticals. control the Plac promoter in E. coli strain XLOLR and examined the growth characteristics of the transgene after induction of expression with isopropyl-Dgalactopyranoside (IPTG; Fig. 1a). The strain bearing the construct grows much faster than the parental strain at low temperatures: 3-fold faster than the parental strain at 15 °C, 36-fold faster at 10 °C and 141-fold faster at 8 °C (growth rate of parental E. coli ∼ 0.002 h–1; that of the transgenic strain ∼ 0.282 h–1). No growth of the parental E. coli was detected below 8 °C, whereas the transgenic strain grew at temperatures below 4 °C As determined using the square-root growth model of Ratkowsky et al.6, the theoretical minimum temperatures for the parental and transgenic E. coli would be 7.5 °C and –13.7 °C, respectively (see Supplementary Methods online). To rule out the possibility that hyperexpression of chaperones per se lowers the growth limit of E. coli, we also expressed the GroEL and GroES chaperonins to similar cellular levels— 160 μg GroEL/ES per milligram of protein versus 120 μg Cpn60/10 per milligram of protein, using plasmids pBB528 and pBB541 (kindly provided by E. Betiku and U. Rinas (GBF)), in which the chaperonins are expressed from the same Plac promoter (for details, see Supplementary Fig. 1 online). The growth characteristics of E. coli at temperatures below 15 °C were not influenced by hyperexpression of the homologous chaperonins (data not shown). This demonstrates that the depression of the lower limit of growth of E. coli by Cpn60 and Cpn10 is due to a
TL;DR: The results demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.
Abstract: Genomes of prokaryotes differ significantly in size and DNA composition. Escherichia coli is considered a model organism to analyze the processes involved in bacterial genome evolution, as the species comprises numerous pathogenic and commensal variants. Pathogenic and nonpathogenic E. coli strains differ in the presence and absence of additional DNA elements contributing to specific virulence traits and also in the presence and absence of additional genetic information. To analyze the genetic diversity of pathogenic and commensal E. coli isolates, a whole-genome approach was applied. Using DNA arrays, the presence of all translatable open reading frames (ORFs) of nonpathogenic E. coli K-12 strain MG1655 was investigated in 26 E. coli isolates, including various extraintestinal and intestinal pathogenic E. coli isolates, 3 pathogenicity island deletion mutants, and commensal and laboratory strains. Additionally, the presence of virulence-associated genes of E. coli was determined using a DNA "pathoarray" developed in our laboratory. The frequency and distributional pattern of genomic variations vary widely in different E. coli strains. Up to 10% of the E. coli K-12-specific ORFs were not detectable in the genomes of the different strains. DNA sequences described for extraintestinal or intestinal pathogenic E. coli are more frequently detectable in isolates of the same origin than in other pathotypes. Several genes coding for virulence or fitness factors are also present in commensal E. coli isolates. Based on these results, the conserved E. coli core genome is estimated to consist of at least 3,100 translatable ORFs. The absence of K-12-specific ORFs was detectable in all chromosomal regions. These data demonstrate the great genome heterogeneity and genetic diversity among E. coli strains and underline the fact that both the acquisition and deletion of DNA elements are important processes involved in the evolution of prokaryotes.
TL;DR: In this article, the authors constructed derivatives of Escherichia coli W3110 (prototrophic) as new biocatalysts for the production of d-lactic acid.
Abstract: The resistance of polylactide to biodegradation and the physical properties of this polymer can be controlled by adjusting the ratio of l-lactic acid to d-lactic acid. Although the largest demand is for the l enantiomer, substantial amounts of both enantiomers are required for bioplastics. We constructed derivatives of Escherichia coli W3110 (prototrophic) as new biocatalysts for the production of d-lactic acid. These strains (SZ40, SZ58, and SZ63) require only mineral salts as nutrients and lack all plasmids and antibiotic resistance genes used during construction. d-Lactic acid production by these new strains approached the theoretical maximum yield of two molecules per glucose molecule. The chemical purity of this d-lactic acid was ∼98% with respect to soluble organic compounds. The optical purity exceeded 99%. Competing pathways were eliminated by chromosomal inactivation of genes encoding fumarate reductase (frdABCD), alcohol/aldehyde dehydrogenase (adhE), and pyruvate formate lyase (pflB). The cell yield and lactate productivity were increased by a further mutation in the acetate kinase gene (ackA). Similar improvements could be achieved by addition of 10 mM acetate or by an initial period of aeration. All three approaches reduced the time required to complete the fermentation of 5% glucose. The use of mineral salts medium, the lack of antibiotic resistance genes or plasmids, the high yield of d-lactate, and the high product purity should reduce costs associated with nutrients, purification, containment, biological oxygen demand, and waste treatment.
TL;DR: Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity ofDJ-1 and the proteolytic activity of Hsp30, and the possible role of DJs in the pathogenesis of Parkinson's disease is discussed.
TL;DR: This work has identified homologues of the FadA, FadB and FadD proteins required for aerobic fatty acid utilization called YfcY, YfcX and YdiD, respectively, which are involved in anaerobic growth on fatty acids.
Abstract: Escherichia coli uses fatty acids as a sole carbon and energy source during aerobic growth by means of the enzymes encoded by the fad regulon. We report that this bacterium can also grow on fatty acids under anaerobic conditions provided that a terminal respiratory electron acceptor such as nitrate is available. This anaerobic utilization pathway is distinct from the well-studied aerobic pathway in that (i). it proceeds normally in mutant strains lacking various enzymes of the aerobic pathway; (ii). it functions with fatty acids (octanoate and decanoate) that cannot be used by wild-type E. coli strains under aerobic conditions; and (iii). super-repressor mutants of the fadR regulatory locus that block aerobic growth on fatty acids fail to block the anaerobic pathway. We have identified homologues of the FadA, FadB and FadD proteins required for aerobic fatty acid utilization called YfcY, YfcX and YdiD, respectively, which are involved in anaerobic growth on fatty acids. A strong FadR binding site was detected upstream of the yfcY gene consistent with microarray analyses, indicating that yfcYX expression is negatively regulated by FadR under aerobic growth conditions. In contrast, transcriptional regulation of ydiD appears to be independent of FadR, and anaerobic growth on fatty acids is not under FadR control. These three genes are conserved in the available genome sequences of pathogenic E. coli, Shigella and Salmonella strains.
TL;DR: It is suggested that the RcsC sensor kinase may play an important role in the remodelling of the bacterial surface during growth on a solid surface and biofilm formation.
Abstract: Summary
Bacteria are often found associated with surfaces as sessile bacterial communities called biofilms, and the formation of a biofilm can be split up into different stages each requiring the expression of specific genes. The production of extracellular polysaccharides (EPS) is important for the maturation of biofilms and is controlled by the Rcs two-component pathway in Escherichia coli (and other Gram-negative bacteria). In this study, we show, for the first time, that the RcsC sensor kinase is required for normal biofilm development in E. coli. Moreover, using a combination of DNA macroarray technology and transcriptional fusion analysis, we show that the expression of > 150 genes is controlled by RcsC in E. coli. In silico analyses of the RcsC regulon predicts that 50% of the genes encode proteins that are either localized to the envelope of E. coli or have activities that affect the structure/properties of the bacterial surface, e.g. the production of colanic acid. Moreover, we also show that RcsC is activated during growth on a solid surface. Therefore, we suggest that the RcsC sensor kinase may play an important role in the remodelling of the bacterial surface during growth on a solid surface and biofilm formation.
TL;DR: This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube and selected targets selected for each category were eae for enteropathogenic E coli, stx for Shiga toxin-producing E. bacteria, and aggR for enteroaggregative E coli.
Abstract: A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.