Chaperone Hsp31 contributes to acid resistance in stationary-phase Escherichia coli

Hsp31, the product of the sigmaS - and sigmaD -dependent hchA gene, is a heat-inducible chaperone implicated in the management of protein misfolding at high temperatures. We show here that Hsp31 plays an important role in the acid resistance of starved Escherichia coli but that it has little influence on oxidative-stress survival.     

Cytotoxic necrotizing factor-1 contributes to Escherichia coli K1 invasion of the central nervous system

Escherichia coli K1 invasion of brain microvascular endothelial cells (BMECs) is a prerequisite for penetration into the central nervous system and requires actin cytoskeletal rearrangements. Here, we demonstrate that E. coli K1 invasion of BMECs requires RhoA activation. In addition, we show that cytotoxic necrotizing factor-1 (CNF1) contributes to E. coli K1 invasion of brain endothelial cells in vitro and traversal of the blood-brain barrier in the experimental hematogenous meningitis animal model. These in vitro and in vivo effects of CNF1 were dependent upon RhoA activation as shown by (a) decreased invasion and RhoA activation with the Delta cnf1 mutant of E. coli K1 and (b) restoration of invasion frequency of the Delta cnf1 mutant to the level of the parent E. coli K1 strain in BMECs with constitutively active RhoA. In addition, CNF1-enhanced E. coli invasion of brain endothelial cells and stress fiber formation were independent of focal adhesion kinase and phosphatidylinositol 3-kinase activation. This is the first demonstration that CNF1 contributes to E. coli K1 invasion of BMECs.     

Prevalence of antibiotic resistance profile in relation to phylogenetic background among commensal Escherichia coli derived from various mammals.

The paper describes the prevalence of resistant strains within the genetic structure of E. coli (phylogenetic group A, B1, B2 and D). A total of 200 commensal E. coli strains have been derived from 10 species of healthy animals residing on ZOO Safari Park area, in Swierkocin, Poland. The phylogenetic structure of E. coli has been analysed with the use of a PCR-based method. The strains were tested in terms of their susceptibility to eight classes of antibiotics: aminoglycosides, penicillins, cephalosporins, tetracyclines, nitrofurans, sulphonamides, phinicols, and quinolones. The genetic structure of E. coli revealed a not uniform distribution of strains among the four phylogenetic groups with significantly numerous representation of groups A and B1. Resistant E. coli were found within each of the phylogenetic groups. Strains resistant to one class of antibiotics occurred significantly more frequently in phylogenetic groups B2 and D (potential pathogens), whereas strains resistant to more than one class of antibiotics belonged to phylogenetic groups A and B1 (typical commensals) in a prevailing number of cases.     

Aberrant O‐glycosylation contributes to tumorigenesis in human colorectal cancer

Aberrant O‐glycosylation is frequently observed in colorectal cancer (CRC) patients, but it is unclear if it contributes intrinsically to tumorigenesis. Here, we investigated the biological consequences of aberrant O‐glycosylation in CRC. We first detected the expression profile of Tn antigen in a serial of human CRC tissues and then explored the genetic and biosynthetic mechanisms. Moreover, we used a human CRC cell line (LS174T), which express Tn antigen, to assess whether aberrant O‐glycosylation can directly promote oncogenic properties. It showed that Tn antigen was detected in around 86% human primary and metastatic CRC tissues. Bio‐functional investigations showed that T‐synthase and Cosmc were both impaired in cancer tissues. A further analysis detected an occurrence of hypermethylation of Cosmc gene, which possibly caused its loss‐of‐function and a consequent inactive T‐synthase. Transfection of LS174T cells with WT Cosmc restored mature O‐glycosylation, which subsequently down‐regulated cancer cell proliferation, migration and apoptotic‐resistant ability. Significantly, the expression of MUC2, a heavily O‐glycosylated glycoprotein that plays an essential role in intestinal function, was uniformly reduced in human CRC tissues as well as in LS174T cells. These data suggest that aberrant O‐glycosylation contributes to the development of CRC through direct induction of oncogenic properties in cancer cells.     

Mutations to nitrofurantoin and nitrofurazone resistance in Escherichia coli K12

The isolation and properties of nitrofurantoin- and nitrofurazone-resistant mutants have been compared. Nitrofurantoin-resistant mutants were easier to obtain and showed a higher resistance level. There was some cross-resistance at lower drug concentrations but not at higher levels. There was no difference in the UV resistance of most of the mutants obtained although a recB strain, AB2470, did yield nitrofurantoin-resistant mutants with increased UV resistance. This was however the only repair-defective strain which could be mutated to nitrofurantoin resistance. It is suggested that there is a mechanism for nitrofurantoin resistance in Escherichia coli K12 which does not confer resistance to nitrofurazone and which may be associated with the repair of damaged DNA. This mechanism is in addition to that which also confers resistance to nitrofurazone.     

Genetic control of resistance of Escherichia coli K12 to phage C1

The bacteriophage C1 isolated in production cycle lysis belongs to the most common group of bacteriophages wide spread in nature and having the long noncontractile motile tail. Bacterial cells sensitivity to bacteriophage C1 is determined by functioning of the three different loci mapped in different regions of Escherichia coli map at 89, 75 and 61 min. The possibility of existence of a complex receptor for bacteriophage C1 is discussed.     

Strength of Cu-efflux response in Escherichia coli coordinates metal resistance in Caenorhabditis elegans and contributes to the severity of environmental toxicity

Without effective homeostatic systems in place, excess copper (Cu) is universally toxic to organisms. While increased utilization of anthropogenic Cu in the environment has driven the diversification of Cu-resistance systems within enterobacteria, little research has focused on how this change in bacterial architecture impacts host organisms that need to maintain their own Cu homeostasis. Therefore, we utilized a simplified host–microbe system to determine whether the efficiency of one bacterial Cu-resistance system, increasing Cu-efflux capacity via the ubiquitous CusRS two-component system, contributes to the availability and subsequent toxicity of Cu in host Caenorhabditis elegans nematode. We found that a fully functional Cu-efflux system in bacteria increased the severity of Cu toxicity in host nematodes without increasing the C. elegans Cu-body burden. Instead, increased Cu toxicity in the host was associated with reduced expression of a protective metal stress-response gene, numr-1, in the posterior pharynx of nematodes where pharyngeal grinding breaks apart ingested bacteria before passing into the digestive tract. The spatial localization of numr-1 transgene activation and loss of bacterially dependent Cu-resistance in nematodes without an effective numr-1 response support the hypothesis that numr-1 is responsive to the bacterial Cu-efflux capacity. We propose that the bacterial Cu-efflux capacity acts as a robust spatial determinant for a host’s response to chronic Cu stress.     

Genotype‐by‐environment interactions due to antibiotic resistance and adaptation in Escherichia coli

Mutations that are beneficial in one environment can have different fitness effects in other environments. In the context of antibiotic resistance, the resulting genotype‐by‐environment interactions potentially make selection on resistance unpredictable in heterogeneous environments. Furthermore, resistant bacteria frequently fix additional mutations during evolution in the absence of antibiotics. How do these two types of mutations interact to determine the bacterial phenotype across different environments? To address this, I used Escherichia coli as a model system, measuring the effects of nine different rifampicin resistance mutations on bacterial growth in 31 antibiotic‐free environments. I did this both before and after approximately 200 generations of experimental evolution in antibiotic‐free conditions (LB medium), and did the same for the antibiotic‐sensitive wild type after adaptation to the same environment. The following results were observed: (i) bacteria with and without costly resistance mutations adapted to experimental conditions and reached similar levels of competitive fitness; (ii) rifampicin resistance mutations and adaptation to LB both indirectly altered growth in other environments; and (iii) resistant‐evolved genotypes were more phenotypically different from the ancestor and from each other than resistant‐nonevolved and sensitive‐evolved genotypes. This suggests genotype‐by‐environment interactions generated by antibiotic resistance mutations, observed previously in short‐term experiments, are more pronounced after adaptation to other types of environmental variation, making it difficult to predict long‐term selection on resistance mutations from fitness effects in a single environment.     

glnA mutations conferring resistance to methylammonium in Escherichia coli K12

Cells of Escherichia coli K12 were sensitive to 100 mM-methylammonium when cultured under nitrogen limitation, and resistant when grown with an excess of either NH4Cl or glutamine. Glutamine synthetase activity was required for expression of the methylammonium-sensitive phenotype. Mutants were isolated which were resistant to 100 mM-methylammonium, even when grown under nitrogen limitation. P1 bacteriophage transduction and F' complementation analysis revealed that the resistance-conferring mutations mapped either inside the glnA structural gene and/or elsewhere in the E. coli chromosome. Glutamine synthetase was purified from the wild-type and from some of the mutant strains. Strains carrying glnA-linked mutations that were solely responsible for the methylammonium-resistant phenotype yielded an altered enzyme, which was less active biosynthetically with either ammonium or methylammonium as substrate. Sensitivity to methylammonium appeared to be due to synthesis of gamma-glutamylmethylamide by glutamine synthetase, which was synthesized poorly, if at all, by mutants carrying an altered glutamine synthetase enzyme.     

Spermidine acetyltransferase is required to prevent spermidine toxicity at low temperatures in Escherichia coli

Polyamines are required for optimal growth in most cells; however, polyamine accumulation leads to inhibition of cellular growth. To reduce intracellular polyamine levels, spermidine is monoacetylated in both prokaryotes and eukaryotes. In Escherichia coli, the speG gene encodes the spermidine acetyltransferase, which transfers the acetyl group to either the N-1 or N-8 position. In addition to polyamine accumulation, stress conditions, such as cold shock, cause an increase in the level of spermidine acetylation, suggesting an adaptive role for reduced polyamine levels under stressful growth conditions. The effect of spermidine accumulation on the growth of E. coli at low temperature was examined using a speG mutant. At 37 degrees C, growth of the speG mutant was normal in the presence of 0. 5 or 1 mM spermidine. However, following a shift to 7 degrees C, the addition of 0.5 or 1 mM spermidine resulted in inhibition of cellular growth or cell lysis, respectively. Furthermore, at 7 degrees C, spermidine accumulation resulted in a decrease in total protein synthesis accompanied by an increase in the synthesis of the major cold shock proteins CspA, CspB, and CspG. However, the addition of 50 mM Mg(2+) restored growth and protein synthesis in the presence of 0.5 mM spermidine. The results indicate that the level of spermidine acetylation increases at low temperature to prevent spermidine toxicity. The data suggest that the excess spermidine replaces the ribosome-bound Mg(2+), resulting in ribosome inactivation at low temperatures.     

Escherichia coli K1's capsule is a barrier to bacteriophage T7

Escherichia coli strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cell's invasiveness, allowing the bacteria to avoid phagocytosis and inactivation by complement. It is also recognized as a receptor by some phages, such as K1F and K1-5, which have virion-associated enzymes that degrade the polysaccharide. In this report we show that expression of the K1 capsule in E. coli physically blocks infection by T7, a phage that recognizes lipopolysaccharide as the primary receptor. Enzymatic removal of the K1 antigen from the cell allows T7 to adsorb and replicate. This observation suggests that the capsule plays an important role as a defense against some phages that recognize structures beneath it and that the K1-specific phages evolved to counter this physical barrier.     

Modulation of O‐antigen chain length by the wzz gene in Escherichia coli O157 influences its sensitivities to serum complement

Escherichia coli O157 strains belonging to a distinct lineage and expressing different O‐antigen (Oag) lengths were isolated. Although the function of wzz in E. coli has not been adequately investigated, this gene is known to be associated with regulation of Oag length. Using E. coli O157:H7 ATCC43888 (wild‐type), several wzz mutants of E. coli O157, including a wzz deletion mutant, were generated and the relationship between the length of Oag modulated by the wzz gene and sensitivities to serum complement investigated. SDS–PAGE, immunoblot analyses and sensitivity tests to human serum complement were performed on these strains. The lengths of the O157‐antigen could be modulated by the wzz gene mutations and were classified into long, intermediate and short groups. The short chain mutant was more serum sensitive than the wild‐type strain and the other wzz mutants (P < 0.001). In conclusion, Oag chain length modulated by the wzz gene in E. coli O157 influences its sensitivities to serum complement. The present findings suggest that E. coli O157 strains with intermediate or long length Oag chains might show greater resistance to serum complement than those with short chains.     

Oligopeptide uptake and aminoglycoside resistance in Escherichia coli K12

Previous reports have suggested that Escherichia coli K12 mutants defective in the expression of oligogopeptide permease protein A (OppA) exhibit reduced sensitivity to aminoglycosides due to altered permeability of the cell envelope. In this work, the role of the OppA protein, and the oligogopeptide permease (Opp) transport system has been evaluated, in the resistance to aminoglycosides using derivatives of the E. coli K12 SS320 strain selected for triornithine resistance or with a deletion of the complete opp operon. All tested mutants were defective in the uptake of tri- and tetra-peptides but did not expressed resistance to aminoglycosides. Additionally, complementation tests carried out with a plasmid encoding the OppA protein did not affect the sensitivity of the strains to these antibiotics. Taken together, these evidences indicate that the Opp uptake system, as well as the OppA protein, does not play a direct role in the sensitivity to aminoglycosides in E. coli K12.     

The P1 plasmid is segregated to daughter cells by a 'capture and ejection' mechanism coordinated with Escherichia coli cell division

The fate of the P1 plasmid of Escherichia coli was followed by time-lapse photomicroscopy. A GFP-ParB fusion marked the plasmid during partition (segregation) to daughter cells at slow growth rate. The process differs from that previously inferred from statistical analysis of fixed cells. A focus of plasmid copies is captured at the cell centre. Immediately before cell division, the copies eject bidirectionally along the long axis of the cell. Cell division traps one or more plasmid copies in each daughter. They are not directed to a prescribed position but are free to move, associate and disassociate. Later, they are captured to the new cell centre to restart the cycle. A null P1 par mutant associates to form a focus, but it is neither captured nor ejected. A dominant negative ParB protein forms a plasmid focus that attaches to the cell centre but never ejects. It remains captive at the centre and blocks host cell division. The cells elongate. Eventually the intact focus is pushed to one side and the cells divide simultaneously in several places at the same time. This suggests that the wild-type plasmid imposes a regulatory node on the host cell cycle, preventing cell division until its own segregation is completed.     

Sequence and expression of the Escherichia coli K1 neuC gene product.

The nucleotide sequence of the neuC gene of the Escherichia coli K1 capsule gene cluster encodes a protein with a predicted molecular weight of 44,210 containing 391 amino acids. A chimeric protein with beta-galactosidase fused to the carboxy terminus of the neuC gene product (P7) was constructed and purified. Its amino-terminal sequence confirmed the prediction from the nucleotide sequence that the neuC gene overlaps the distal end of the neuA gene by a single base pair. Both the neuA and neuC genes are coexpressed under the control of a single upstream T7 or tac promoter, suggesting that neuA and neuC are part of an operon.     

Detection of the mcr‐1 gene in colistin‐resistant Escherichia coli from retail meat in Japan

In this study, the presence of the mcr‐1 gene in Escherichia coli from retail meat in Japan was investigated. Nine E. coli isolates (eight from chickens and one from pork) carried the mcr‐1 gene on the plasmid. In six isolates from domestic chickens, mcr‐1 was located on the IncI2 plasmid, which is approximately 60 kb in size. In the remaining three isolates from imported chicken and pork, mcr‐1 was located on the IncX4 plasmid (30 kb).

     

Expression and regulation of protein K, an Escherichia coli K1 porin, in Escherichia coli K-12

Using a modified lambda phage as a vector and a procedure developed in Dr. C. Schnaitman's laboratory, we have cloned the structural gene for protein K from an Escherichia coli K1 strain to an E coli K-12 strain. The cloned inserts consist of two HindIII fragments, 4 kb and 6.5 kb in size. The protein produced by the insert is nearly identical to "authentic" protein K when chymotryptic peptides of 125I-labeled proteins are compared. Protein K was found to respond to changes in the osmolarity of the medium, being favored in trypticase soy broth (high osmolarity). This fluctuation was not dependent on a functional ompR gene. However, protein K was not expressed in strains carrying the envZ-473 mutation. Thus, protein K appears to be within a class of exported proteins whose expression is regulated by the envZ gene independent of the ompR gene.