Involvement of a Cell Envelope Component of Escherichia coli in the Early Stages of Infection with Bacteriophage ϕX174

Envelope fraction I prepared from a ?X174 sensitive host, KD4301, showed a strong eclipsing activity, while the lipopolysaccharide (LPS) fraction showed a weak activity. The eclipsing activity in envelope fraction I was sensitive to heat treatment, while that in the LPS fraction was insensitive. When the complete phage particles (114S) were treated with envelope fraction I, the eclipsed particles (70S) and a rapidly sedimenting component were obtained, but when they were treated with LPS, only 70S eclipsed particles were obtained. Electron microscopic observation showed that there were two types of eclipsed particles formed on treatment with fraction I; in one of them phage DNA was extruded from the phage particles as a thick bundle, and in the other more than 95% of the phage DNA was extruded from the phage particles. The rapidly sedimenting component was the membrane-eclipsed particle complex. LPS gave only one type of eclipsed particles in which DNA was extruded as a thick bundle. These results indicate that a heat labile component in the cell envelopes other than LPS is involved in the extrusion of ?X174 DNA.     

MreB-Dependent Organization of the E.?coli Cytoplasmic Membrane Controls Membrane Protein Diffusion

The functional organization of prokaryotic cell membranes, which is essential for many cellular processes, has been challenging to analyze due to the small size and nonflat geometry of bacterial cells. Here, we use single-molecule fluorescence microscopy and three-dimensional quantitative analyses in live Escherichia coli to demonstrate that its cytoplasmic membrane contains microdomains with distinct physical properties. We show that the stability of these microdomains depends on the integrity of the MreB cytoskeletal network underneath the membrane. We explore how the interplay between cytoskeleton and membrane affects trans-membrane protein (TMP) diffusion and reveal that the mobility of the TMPs tested is subdiffusive, most likely caused by confinement of TMP mobility by the submembranous MreB network. Our findings demonstrate that the dynamic architecture of prokaryotic cell membranes is controlled by the MreB cytoskeleton and regulates the mobility of TMPs.     

Crystal structure of Escherichia coli BamB, a lipoprotein component of the β-barrel assembly machinery complex

In Gram-negative bacteria, the BAM (β-barrel assembly machinery) complex catalyzes the essential process of assembling outer membrane proteins. The BAM complex in Escherichia coli consists of five proteins: one β-barrel membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their role in outer membrane protein biogenesis, there is currently a lack of functional and structural information on the lipoprotein components of the BAM complex. Here, we report the first crystal structure of BamB, the largest and most functionally characterized lipoprotein component of the BAM complex. The crystal structure shows that BamB has an eight-bladed β-propeller structure, with four β-strands making up each blade. Mapping onto the structure the residues previously shown to be important for BamA interaction reveals that these residues, despite being far apart in the amino acid sequence, are localized to form a continuous solvent-exposed surface on one side of the β-propeller. Found on the same side of the β-propeller is a cluster of residues conserved among BamB homologs. Interestingly, our structural comparison study suggests that other proteins with a BamB-like fold often participate in protein or ligand binding, and that the binding interface on these proteins is located on the surface that is topologically equivalent to where the conserved residues and the residues that are important for BamA interaction are found on BamB. Our structural and bioinformatic analyses, together with previous biochemical data, provide clues to where the BamA and possibly a substrate interaction interface may be located on BamB.     

Packing Density of the Amyloid Precursor Protein in the Cell Membrane

Plasma membrane proteins organize into structures named compartments, microdomains, rafts, phases, crowds, or clusters. These structures are often smaller than 100 nm in diameter. Despite their importance in many cellular functions, little is known about their inner organization. For instance, how densely are molecules packed? Being aware of the protein compaction may contribute to our general understanding of why such structures exist and how they execute their functions. In this study, we have investigated plasma membrane crowds formed by the amyloid precursor protein (APP), a protein well known for its involvement in Alzheimer’s disease. By combining biochemical experiments with conventional and super-resolution stimulated emission depletion microscopy, we quantitatively determined the protein packing density within APP crowds. We found that crowds occurring with reasonable frequency contain between 20 and 30 molecules occupying a spherical area with a diameter between 65 and 85 nm. Additionally, we found the vast majority of plasmalemmal APP residing in these crowds. The model suggests a high molecular density of protein material within plasmalemmal APP crowds. This should affect the protein’s biochemical accessibility and processing by nonpathological α-secretases. As clustering of APP is a prerequisite for endocytic entry into the pathological processing pathway, elucidation of the packing density also provides a deeper understanding of this part of APP’s life cycle.     

Structural characterization of Escherichia coli BamE, a lipoprotein component of the β-barrel assembly machinery complex

In Escherichia coli, the BAM complex catalyzes the essential process of assembling outer membrane proteins (OMPs). This complex consists of five proteins: one membrane-bound protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Despite their importance in OMP biogenesis, there is currently a lack of functional and structural information on the BAM complex lipoproteins. BamE is the smallest but most conserved lipoprotein in the complex. The structural and dynamic properties of monomeric BamE (residues 21-133) were determined by NMR spectroscopy. The protein folds as two α-helices packed against a three-stranded antiparallel β-sheet. The N-terminal (Ser21-Thr39) and C-terminal (Pro108-Asn113) residues, as well as a β-hairpin loop (Val76-Gln89), are highly flexible on the subnanosecond time scale. BamE expressed and purified from E. coli also exists in a kinetically trapped dimeric state that has dramatically different NMR spectra, and hence structural features, relative to its monomeric form. The functional significance of the BamE dimer remains to be established. Structural comparison to proteins with a similar architecture suggests that BamE may play a role in mediating the association of the BAM complex or with the BAM complex substrates.     

Impact of the Use of β-Lactam Antimicrobials on the Emergence of Escherichia coli Isolates Resistant to Cephalosporins under Standard Pig-Rearing Conditions

The aim of this study was to evaluate if the treatments with ceftiofur and amoxicillin are risk factors for the emergence of cephalosporin resistant (CR) E. coli in a pig farm during the rearing period. One hundred 7-day-old piglets were divided into two groups, a control (n = 50) group and a group parenterally treated with ceftiofur (n = 50). During the fattening period, both groups were subdivided in two. A second treatment with amoxicillin was administered in feed to two of the four groups, as follows: group 1 (untreated, n = 20), group 2 (treated with amoxicillin, n = 26), group 3 (treated with ceftiofur, n = 20), and group 4 (treated with ceftiofur and amoxicillin, n = 26). During treatment with ceftiofur, fecal samples were collected before treatment (day 0) and at days 2, 7, 14, 21, and 42 posttreatment, whereas with amoxicillin, the sampling was extended 73 days posttreatment. CR E. coli bacteria were selected on MacConkey agar with ceftriaxone (1 mg/liter). Pulsed-field gel electrophoresis (PFGE), MICs of 14 antimicrobials, the presence of cephalosporin resistance genes, and replicon typing of plasmids were analyzed. Both treatments generated an increase in the prevalence of CR E. coli, which was statistically significant in the treated groups. Resistance diminished after treatment. A total of 47 CR E. coli isolates were recovered during the study period; of these, 15 contained bla_CTX-M-1, 10 contained bla_CTX-M-14, 4 contained bla_CTX-M-9, 2 contained bla_CTX-M-15, and 5 contained bla_SHV-12. The treatment with ceftiofur and amoxicillin was associated with the emergence of CR E. coli during the course of the treatment. However, by the time of finishing, CR E. coli bacteria were not recovered from the animals.     

Folding of the β-Barrel Membrane Protein OmpA into Nanodiscs

Nanodiscs (NDs) are an excellent alternative to small unilamellar vesicles (SUVs) for studies of membrane protein structure, but it has not yet been shown that membrane proteins are able to spontaneously fold and insert into a solution of freely diffusing NDs. In this article, we present SDS-PAGE differential mobility studies combined with fluorescence, circular dichroism, and ultraviolet resonance Raman spectroscopy to confirm the spontaneous folding of outer membrane protein A (OmpA) into preformed NDs. Folded OmpA in NDs was incubated with Arg-C protease, resulting in the digestion of OmpA to membrane-protected fragments with an apparent molecular mass of ∼26 kDa (major component) and ∼24 kDa (minor component). The OmpA folding yields were greater than 88% in both NDs and SUVs. An OmpA adsorbed intermediate on NDs could be isolated at low temperature and induced to fold via an increase in temperature, analogous to the temperature-jump experiments on SUVs. The circular dichroism spectra of OmpA in NDs and SUVs were similar and indicated β-barrel secondary structure. Further evidence of OmpA folding into NDs was provided by ultraviolet resonance Raman spectroscopy, which revealed the intense 785 cm⁻¹ structural marker for folded OmpA in NDs. The primary difference between folding in NDs and SUVs was the kinetics; the rate of folding was two- to threefold slower in NDs compared to in SUVs, and this decreased rate can tentatively be attributed to the properties of NDs. These data indicate that NDs may be an excellent alternative to SUVs for folding experiments and offer benefits of optical clarity, sample homogeneity, control of ND:protein ratios, and greater stability.     

Impact of Therapeutic Treatment with β-Lactam on Transfer of the blaCTX-M-9 Resistance Gene from Salmonella enterica Serovar Virchow to Escherichia coli in Gnotobiotic Rats

The conjugative transfer of the plasmid carrying the bla_CTX-M-9 gene from Salmonella enterica serovar Virchow isolated from a chicken farm to a recipient Escherichia coli strain was evaluated in vitro and in axenic rats inoculated with both strains, with or without selective pressure due to therapeutic doses of cefixime. The transfer of the bla_CTX-M-9 gene of S. enterica serovar Virchow to E. coli was confirmed in vitro, at a low frequency of 5.9 × 10⁻⁸ transconjugants/donors. This transfer rate was higher in gnotobiotic rats and reached ∼10⁻⁵ transconjugants/donors without selective pressure. This frequency was not affected by the addition of therapeutic doses of cefixime. Thus, estimates of in vitro transfer underestimated potential transfer in the digestive tract, and therapeutic doses of cefixime did not increase the selection for transconjugants.β-Lactams and extended-spectrum cephalosporins are widely used in human and veterinary medicine to treat severe infections in humans and animals caused by Enterobacteriaceae and other gram-negative pathogens (7). However, extended-spectrum β-lactamases (ESBLs) have emerged and become the major mechanism of resistance to β-lactam antibiotics. Until the late 1990s, TEM and SHV enzymes were the predominant ESBLs. However, over the last decade, CTX-M-type enzymes have become the most prevalent extended-spectrum β-lactamases worldwide. Currently, there have been a number of reports documenting an increasing prevalence of enteric pathogens that produce these plasmid-mediated CTX-M enzymes (4, 8, 9, 39).Although originally confined to hospitals, ESBL-producing strains are now emerging in the community. Several investigations have shown that the rate of fecal carriage of ESBL-positive isolates (especially Escherichia coli) in humans is increasing, with CTX-M type enzymes found in most isolates (14, 17, 25, 28, 30, 36). This alarming phenomenon may have serious economic consequences and implications for treatment.bla_CTX-M genes spread throughout the community, mostly through the transmission of plasmids, and some studies have reported that animals may serve as a possible source for the dissemination of ESBL-encoding genes to humans. Indeed, common conjugative resistance plasmids and resistant clones have been found in animals, food products, and humans, suggesting that the transfer of extended-spectrum cephalosporin resistance between animals and humans is possible (17, 18, 21, 31, 40).In France, the emergence of the CTX-M-9 enzyme in Salmonella enterica serovar Virchow strains recovered from poultry, poultry products, and one human patient was reported between 2002 and 2003 (5, 31, 38, 40, 41). A comparative analysis of the bla_CTX-M-2 and bla_CTX-M-9 plasmids from S. enterica serovar Virchow isolates from human and poultry sources demonstrated a close relationship between the plasmids (16).To examine the hypothesis that antimicrobial resistance in humans could partly be attributed to food products, a number of studies have investigated the transfer of antibiotic resistance from bacteria originating in animal hosts or food products. Previously, Lester et al. (24) demonstrated the transfer of the vanA resistance gene from Enterococcus faecium isolated from animals to E. faecium isolated from human volunteers during transient intestinal colonization in humans in the absence of selective pressure. In a gnotobiotic mouse model, Feld et al. demonstrated the evidence of spread of a small plasmid pLFE1 harboring the erythromycin-resistant gene erm(B) from Lactobacillus plantarum isolated from raw-milk cheese to exogenous Enterococcus faecalis inoculated into the intestinal flora of mice (16). The transfer rate was enhanced when erythromycin was coadministered. Improvements in our understanding of the in vivo transmissibility and stability of antimicrobial resistance markers and the mechanisms driving horizontal transmission may help to predict the outcome of different strategies for controlling epidemic plasmids.The aim of the present study was to determine whether the bla_CTX-M-9 resistance gene could be transferred from an animal S. enterica serovar Virchow strain to a commensal E. coli strain that originated from the human intestinal tract while under selective pressure from a therapeutic dose of β-lactam. The transfer was investigated by using in vitro mating and a germfree rat model. We also evaluated the impact of the concentration of cefixime, an expanded-spectrum cephalosporin, on the transfer rate.     

Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the β-oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012, http://dx.doi.org/10.1021/sb3000782). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a β-oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled ΔfabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the β-oxidation reversal in E. coli.     

The expression of β-galactosidase by Escherichia coli during continuous culture

We have used the technique of continuous culture to study the expression of β-galactosidase in Escherichia coli. In these experiments the cultures were grown on carbon-limited media in which half of the available carbon was supplied as glycerol, glucose, or glucose 6-phosphate, and the other half as lactose. Lactose itself provided the sole source of inducer for the lac operon. The steady-state specific activity of the enzyme passed through a maximal value as a function of dilution rate. Moreover, the rate at which activity was maximal (0.40 h^?1) and the observed specific activity of the enzyme at a given growth rate were found to be identical in each of the three media tested. This result was unexpected, since the steady-state specific activity can be shown to be equal to the differential rate of enzyme synthesis, and since it is known that glycerol, glucose, and glucose-6-P-cause different degrees of catabolite repression in batch culture. The differential rate of β-galactosidase synthesis was an apparently linear function of the rate of lactose utilization per milligram protein regardless of the composition of the input medium. That is, it is independent of the rate of metabolism of substrates other than lactose which are concurrently being utilized and the enzyme level appears to be matched to the metabolic requirement for it. If this relationship is taken to indicate the existence of a fundamental control mechanism, it may represent a form of attenuation of the rate of β-galactosidase synthesis which is independent of cyclic AMP levels.     

Native Electrophoresis-Coupled Activity Assays Reveal Catalytically-Active Protein Aggregates of Escherichia coli β-Glucuronidase

β-glucuronidase is found as a functional homotetramer in a variety of organisms, including humans and other animals, as well as a number of bacteria. This enzyme is important in these organisms, catalyzing the hydrolytic removal of a glucuronide moiety from substrate molecules. This process serves to break down sugar conjugates in animals and provide sugars for metabolism in bacteria. While β-glucuronidase is primarily found as a homotetramer, previous studies have indicated that the human form of the protein is also catalytically active as a dimer. Here we present evidence for not only an active dimer of the E. coli form of the protein, but also for several larger active complexes, including an octomer and a 16-mer. Additionally, we propose a model for the structures of these large complexes, based on computationally-derived molecular modeling studies. These structures may have application in the study of human disease, as several diseases have been associated with the aggregation of proteins.     

The β-Barrel Outer Membrane Protein Assembly Complex of Neisseria meningitidis

The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the β-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components in Neisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As in E. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by its E. coli homolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. A bamC mutant showed only marginal OMP assembly defects, but the impossibility of creating a bamC bamE double mutant further indicated the function of BamC in OMP assembly. An mlp mutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex in N. meningitidis consists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly.Membrane-embedded β-barrel proteins are found in the outer membranes (OMs) of gram-negative bacteria, mitochondria, and chloroplasts. Only in recent years have cellular components required for the assembly and insertion of these OM proteins (OMPs) into the OM been identified. Omp85, which was first characterized in Neisseria meningitidis, is the key protein of the OMP assembly machinery (41). The function of Omp85 has been preserved during evolution, not only in gram-negative bacteria (8, 37, 44, 46) but also in mitochondria, where an Omp85 homolog, also known as Tob55 or Sam50, was shown to mediate the assembly of β-barrel proteins into the OM (15, 23, 27). Accordingly, bacterial OMPs are still recognized by the eukaryotic assembly machinery: when expressed in yeast, bacterial OMPs were found to be assembled into the mitochondrial OM in a Tob55-dependent manner (43). Omp85 in Escherichia coli, which was recently renamed BamA, for β-barrel assembly machinery (Bam) component A, is associated with at least four lipoproteins: BamB (formerly known as YfgL), BamC (NlpB), BamD (YfiO), and BamE (SmpA) (32, 46). In E. coli, BamB, BamC, and BamE are not essential, but the phenotypes of deletion mutants suggest that these proteins contribute to the efficiency of OMP assembly. Like BamA, BamD is an essential protein in E. coli (24, 26), involved in OMP assembly (24). These lipoproteins are evolutionarily less well conserved; the mitochondrial Tob55 protein is associated with two accessory proteins, but they do not show any sequence similarity with the lipoproteins of the E. coli Bam complex (14).Besides E. coli, N. meningitidis is one of the major bacterial model organisms for studies of OM assembly. As mentioned above, it was the first organism in which the function of Omp85 was identified (41), and also, the role of an integral OMP, designated LptD (formerly Imp or OstA), in the transport of lipopolysaccharide (LPS) to the cell surface was first established in N. meningitidis (3). With regard to OM biogenesis, N. meningitidis has several features that distinguish it from E. coli. For example, in contrast to E. coli (13), N. meningitidis mutants defective in LPS synthesis or transport are viable (3, 34), and OMPs are assembled perfectly well in such mutants (33). Furthermore, in OMP assembly mutants of E. coli, the periplasmic accumulation of unassembled OMPs is limited due to the induction of the σ^E extracytoplasmic stress response, which results in the degradation of unfolded OMPs (30) and the inhibition of their synthesis by small regulatory RNAs (20). In contrast, in N. meningitidis, most of the components involved in this response are absent (4), and unassembled OMPs continue to accumulate as periplasmic aggregates when OMP assembly is halted (41). However, the composition of the Bam complex and the role of accessory components in OMP assembly have not so far been studied in this organism. Therefore, to further understand the OMP assembly process in N. meningitidis, we have now analyzed the composition of the Bam complex and addressed the roles of the different components.     

A Small Heat Shock Protein Enables Escherichia coli To Grow at a Lethal Temperature of 50°C Conceivably by Maintaining Cell Envelope Integrity

It is essential for organisms to adapt to fluctuating growth temperatures. Escherichia coli, a model bacterium commonly used in research and industry, has been reported to grow at a temperature lower than 46.5°C. Here we report that the heterologous expression of the 17-kDa small heat shock protein from the nematode Caenorhabditis elegans, CeHSP17, enables E. coli cells to grow at 50°C, which is their highest growth temperature ever reported. Strikingly, CeHSP17 also rescues the thermal lethality of an E. coli mutant deficient in degP, which encodes a protein quality control factor localized in the periplasmic space. Mechanistically, we show that CeHSP17 is partially localized in the periplasmic space and associated with the inner membrane of E. coli, and it helps to maintain the cell envelope integrity of the E. coli cells at the lethal temperatures. Together, our data indicate that maintaining the cell envelope integrity is crucial for the E. coli cells to grow at high temperatures and also shed new light on the development of thermophilic bacteria for industrial application.     

Factors That Affect Transfer of the IncI1 β-Lactam Resistance Plasmid pESBL-283 between E. coli Strains

The spread of antibiotic resistant bacteria worldwide presents a major health threat to human health care that results in therapy failure and increasing costs. The transfer of resistance conferring plasmids by conjugation is a major route by which resistance genes disseminate at the intra- and interspecies level. High similarities between resistance genes identified in foodborne and hospital-acquired pathogens suggest transmission of resistance conferring and transferrable mobile elements through the food chain, either as part of intact strains, or through transfer of plasmids from foodborne to human strains. To study the factors that affect the rate of plasmid transfer, the transmission of an extended-spectrum β-lactamase (ESBL) plasmid from a foodborne Escherichia coli strain to the β-lactam sensitive E. coli MG1655 strain was documented as a function of simulated environmental factors. The foodborne E. coli isolate used as donor carried a CTX-M-1 harboring IncI1 plasmid that confers resistance to β-lactam antibiotics. Cell density, energy availability and growth rate were identified as factors that affect plasmid transfer efficiency. Transfer rates were highest in the absence of the antibiotic, with almost every acceptor cell picking up the plasmid. Raising the antibiotic concentrations above the minimum inhibitory concentration (MIC) resulted in reduced transfer rates, but also selected for the plasmid carrying donor and recombinant strains. Based on the mutational pattern of transconjugant cells, a common mechanism is proposed which compensates for fitness costs due to plasmid carriage by reducing other cell functions. Reducing potential fitness costs due to maintenance and expression of the plasmid could contribute to persistence of resistance genes in the environment even without antibiotic pressure. Taken together, the results identify factors that drive the spread and persistence of resistance conferring plasmids in natural isolates and shows how these can contribute to transmission of resistance genes through the food chain.