The outer membrane permeability mutation of the virulence-associated plasmid of Salmonella typhimurium is located in a traT-like gene

Abstract The SS-A mutation carried by the virulence-as-associated plasmid of Salmonella typhimurium results in increased outer membrane permeability to hydrophobic compounds. A 7.8-kilobase pair Bam HI- Sal I fragment containing the SS-A mutation was cloned from the virulence-associated plasmid into the cloning vector pACYC184. The cloned DNA segment hybridized with a radioactive probed prepared from the traT gene of R6-5. A similar DNA fragment, cloned from the wild-type virulence-associated plasmid, complemented the SS-A mutant phenotype. Both clones produced a protein that immunologically resembled the R6-5 TraT protein; however, the protein produced by the SS-A containing clone appeared truncated by approximately M _r 1000 indicating an alteration in the primary structure or processing of the protein. We conclude that the mutation producing the SS-A phenotype has occured in a traT -like gene of the Salmonella plasmid.     

The EnvZ protein of Salmonella typhimurium LT-2 and Escherichia coli K-12 is located in the cytoplasmic membrane

Abstract The osmoregulated expression of the porin proteins OmpC and OmpF in S. typhimurium and E. coli is dependent on the regulatory proteins OmpR and EnvZ. The function of the EnvZ protein is not clear. In order to establish the cellular location of EnvZ two different methods of buoyant sucrose density centrifugation was employed. The presence of EnvZ in the different fractions was visualised by immunoblotting. It was conclusively shown that the EnvZ protein is located in the cytoplasmic membrane fraction. The result is in agreement with the available sequence data which shows that the EnvZ polypeptide contains two long hydrophobic stretches.     

Molecular organization of the outer membrane of Salmonella typhimurium different release of lipopolysaccharide from wild type and lipopolysaccharide mutant cells by EDTA treatment

Cells of Salmonella typhimurium wild type and of several well defined lipopolysaccharide mutants were treated with EDTA. The percentage release of lipopolysaccharide and phospholipid was determined. The results obtained show that the release of lipopolysaccharide by EDTA declines along with the gradually diminishing chain length of the lipopolysaccharide, althought the total amount of lipopolysaccharide was found to increase at the same time in the respective mutants. Implications of these findings for the organization of the outer membrane are discussed.     

Reconstitution of binding protein dependent ribose transport in spheroplasts derived from a binding protein negative escherichia coli K12 mutant and from salmonella typhimurium

Highly purified ribose-binding protein from Escherichia coli has been used to reconstitute a binding-protein-dependent ribose transport in spheroplasts derived from a binding-protein-deficient mutant of E coli K 12, and in spheroplasts derived from Salmonella typhimurium. The cross-species reconstitution was nearly as efficient as the reconstitution of the E coli strain from which the binding protein was derived. Antibody raised against the ribose binding protein completely prevented reconstitution, whereas it had no effect on whole cells. The reconstitution procedure has been improved by generating spheroplasts from cells grown in a rich medium and by reducing the background uptake in spheroplasts through a special washing procedure. Rapid purification of ribose binding protein by high pressure liquid chromatography is also described.     

The outer membrane protein of enteropathogenic Escherichia coli, described as the ''localised adherence factor'', is OmpF and probably not involved in adhesion to HEp-2 cells

Strains of enteropathogenic Escherichia coli (EPEC) were examined for a factor, described as an outer membrane protein (OMP) of 32 kilodaltons (kDa) and reported to be involved in the adhesion of EPEC to HeLa cells. A comparable OMP of 35 kDa was detected in strains of EPEC, although expression of this protein was not related to the ability of strains to adhere to HEp-2 cells. The 35 kDa OMP was found to be heat-modifiable and peptidoglycan associated, and considered to be the porin protein OmpF.     

Energy-coupled colicin transport through the outer membrane of Escherichia coli K-12: mutated TonB proteins alter receptor activities and colicin uptake

Abstract The current model of TonB-dependent colicin transport through the outer membrane of Escherichia coli proposes initial binding to receptor proteins, vectorial release from the receptors and uptake into the periplasm from where the colicins, according to their action, insert into the cytoplasmic membrane or enter the cytoplasm. The uptake is energy-dependent and the TonB protein interacts with the receptors as well as with the colicins. In this paper we have studied the uptake of colicins B and Ia, both pore-forming colicins, into various tonB point mutants. Colicin Ia resistance of the tonB mutant (G186D, R204H) was consistent with a defective Cir receptor-TonB interaction while colicin Ia resistance of E. coli expressing TonB of Serratia marcescens , or TonB of E. coli carrying a C-terminal fragment of the S. marcescens TonB, seemed to be caused by an impaired colicin Ia-TonB interaction. In contrast, E. coli tonB (G174R, V178I) was sensitive to colicin Ia and resistant to colicin B unless TonB, ExbB and ExbD were overproduced which resulted in colicin B sensitivity. The differential effects of tonB mutations indicate differences in the interaction of TonB with receptors and colicins.     

Some safety aspects of Salmonella vaccines for poultry: in vivo study of the genetic stability of three Salmonella typhimurium live vaccines

Live vaccine strains of Salmonella should be avirulent, immunogenic and genetically stable. Some isolates of three commercially available live vaccine strains of Salmonella typhimurium, sampled during a study on their persistence in a vaccinated flock of chickens, were analyzed for genetic stability using macrorestriction analysis of their genome. Two out of the three vaccine strains showed genetic instabilities. Two of the 51 isolates of Zoosaloral vaccine strain and nine of the 32 analyzed isolates of chi(3985), a genetically modified organism, were variants and showed different macrorestriction profiles.     

Incompatibility of outer membrane proteins OmpA and OmpF of Escherichia coli with secretion in Bacillus subtilis: Fusions with secretable peptides

The secretion of the outer membrane proteins OmpA and OmpF of Escherichia coli has previously been found to be blocked at an early intracellular step, when these proteins were fused to a bacillar signal sequence and expressed in Bacillus subtilis. We have now fused these proteins to long secretable polypeptides, the amino-terminal portions of alpha-amylase or beta-lactamase. In spite of this, no secretion of the fusion proteins was detected in B. subtilis. With the exception of a small fraction of the beta-lactamase fusion, the proteins were cell-bound with uncleaved signal sequences. Protease accessibility indicated that the fusion proteins were not even partially exposed on the outer surface of the cytoplasmic membrane. Thus there was no change of the location compared to the OmpA or OmpF fused to the signal sequence only. We conclude that, like OmpA and OmpF, the fusion proteins fold into an export-incompatible conformation in B. subtilis before the start of translocation, which we postulate to be a late post-translational event.     

Antigenic cross-reactions between fimbriae expressed by Salmonella enteritidis, S. dublin and an 18 kDa outer membrane associated protein expressed by Escherichia coli O126: H27

Abstract A commercial kit (SEFEX), designed to detect strains of Salmonella enteritidis , was used to demonstrate antigenic cross-reactions between the fimbriae of S. enteritidis and an 18 kDa outer membrane protein expressed by enteroaggregative strains of E. coli O126: H27.     

Outer membrane permeability of Escherichia coli K12: isolation, cloning and mapping of suppressors of a defined antibiotic-hypersensitive mutant

Summary We have previously described defined mutants of the TraT protein, an outer membrane lipoprotein specified by F-like plasmids, which sensitize Escherichia coli and Salmonella typhimurium to antibiotics that are normally excluded from the cell. In this paper, the isolation, characterization and molecular cloning of suppressors of one such mutant (pDOC40) is reported. The suppressors, which were isolated by selection for vancomycin-resistant revertants, also restored resistance to several hydrophobic antibiotics although there were no detectable changes in lipopolysaccharides (LPS), phospholipids or outer membrane proteins. Three suppressor loci, provisionally designated sip, for suppression of increased permeability, were cloned in cosmids and mapped by a novel approach involving random sequencing of cloned DNA to identify flanking genes with known map positions. Our results indicate that the sipB locus is located in the 11 min region (485–510 kb) whereas sipC and sipD both map to 82 min (3850–3885 kb). Additionally, the previously sequenced nlpA gene was also mapped to the 82 min region. The cloned suppressor loci were specific for the permeability phenotype caused by the mutant R6-5 TraT protein and had no effect on the permeability phenotype caused by a related TraT mutant of S. typhimurium.     

Combined effect of mild heat and acetic acid treatment for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an asparagus puree

AIMS: This study was conducted to validate combined heat and acid treatments for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an acidified brine containing, or pickled, asparagus model food. METHODS AND RESULTS: A mixture of three strains of E. coli O157:H7, L. monocytogenes and S. typhimurium were inoculated onto pickled asparagus samples. Combinations of various concentrations of acetic acid [0%, 0.25%, 0.5%, 0.75%, 1%, 1.5% and 2% (v/v)] and various temperatures (40 degrees C, 50 degrees C, 60 degrees C and 75 degrees C) were investigated. Following treatment, asparagus samples were stored at room temperature and enumerated at 0, 0.5, 1, 2 and 3 days. Heat and acetic acid treatments were synergistic. The inhibitory effects of these combined treatments on the tested foodborne pathogens were also effective during storage. Loss of green colour in the pickled asparagus significantly increased with increasing concentrations of acetic acid. CONCLUSIONS: Using a combination of mild heat and acetic acid treatments can successfully control E. coli O157:H7, L. monocytogenes and S. typhimurium in pickled asparagus, combinations of heat and acid are synergistic and effective treatments can be selected to reduce adverse effect on colour which occur during product storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Mild heating plus acetic acid treatment are synergistic, so combined treatments can be developed, which would lower the temperature and amount of acetic acid required for minimally processed vegetables while maintaining pathogen control.     

Transport across the outer membrane of Escherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane

Summary Point mutations in the “TonB box” offhuA were suppressed by point mutations in thetonB gene, suggesting both a functional and physical interaction between the FhuA receptor protein in the outer membrane and the TonB protein in the cytoplasmic membrane ofEscherichia coli K12. Mutations influA were classified into four types according to the extent by which they impaired mutant cells in their growth on ferrichrome as sole iron source and in their sensitivity to the antibiotic albomycin and to colicin M. ThetonB mutation with a glutamine to leucine replacement at position 165 was less efficient in restoring the FhuA functions than the glutamine to lysine exchange at the same position. The better the coupling between FhuA and TonB the poorer was the inhibition of phage T1 binding to FhuA by ferrichrome. A working model is proposed in which the TonB protein assumes different conformations in response to the energized state of the cytoplasmic membrane and thereby allosterically regulates the activity of the FhuA receptor. This model implies an intermembrane coupling between two proteins in adjacent membranes.     

Epitope mapping by cysteine mutagenesis: Identification of residues involved in recognition by three monoclonal antibodies directed against LamB glycoporin in the outer membrane of Escherichia coli

Abstract Site-directed mutagenesis of the lamB gene was used to introduce individual cysteine substitutions at 20 sites in two regions (surface loops L7 and L8) of LamB protein significant in antibody recognition. Characterisation of cysteine mutants involved immunoblotting with three surface-specific monoclonal antibodies (mAb72, mAb302, mAb347) before and after incubation with thiol-specific reagents. In contrast to an earlier study that showed no amino acid changes affecting recognition by all three antibodies, changes at six amino acids were found to influence a common core epitope. These core sites included one residue (T336) in the predicted loop L7 containing amino acids 329–342 and four (Y379, N387, N389, K392, F398) in the large surface loop involving residues 370–412. Individual antibodies made additional but distinct contacts within the two studied regions, with mAb347 binding the most different and affected by seven substitutions in the 328–338 regions. The lamB mutants were also tested for phage λ receptor activity and starch binding before and after thiol modification and were useful in extending previous maps of these ligand binding sites.     

The role of the carboxy-terminal membrane-spanning fragment in the biogenesis of Escherichia coli K12 outer membrane protein PhoE

Summary PhoE protein of Escherichia coli K12 is an outer membrane protein which is supposed to span the membrane sixteen times. By creating a deletion which removes the last membrane-spanning fragment and studying the localization of the truncated PhoE, we show that this fragment is indispensable for trimerization and outer membrane localization. In addition, circumstantial evidence for the proposed topology model of the protein was obtained. An insertion mutation in a region supposed to be cell surface-exposed, interferes with the binding of a monoclonal antibody which recognizes a cell surface-exposed epitope of the protein.     

Nature of the regions involved in the insertion of newly synthesized protein into the outer membrane of Escherichia coli

Outer membrane proteins are synthesized by cytoplasmic membrane-bound polysomes, and inserted at insertion sites which cover about 10% of the total outer membrane when cells grow with a generation time of 1 h. A membrane fraction enriched in outer membrane insertion regions was isolated and partly characterized. The rate at which newly inserted proteins are transferred from such insertion regions into the rest of the outer membrane was found to be very fast; the new protein content of insertion regions and that of the remaining outer membrane equilibrate completely within about 20 s at 25°C.Given the rather rigid structure of the outer membrane and the multiple interactions between outer membane components and the murein layer, lateral diffusion of newly inserted proteins from insertion sites to the remaining outer membrane is not likely to explain this rapid equilibration. Instead, the data support a model in which mobile insertion regions move along the cell surface, leaving behind stationary, newly inserted outer membrane proteins.