Lipopolysaccharide core mutants of Salmonella typhimurium containing D-glycero-D-manno-heptose

Mutants resistant to several hydrophobic membrane antagonists were isolated from a "deep rough" (rfaC) mutant of Salmonella typhimurium. The resistance was due to an alteration in the core region lipopolysaccharide composition as evidenced by altered bacteriophage and complement sensitivity and by compositional analysis. The principal change in carbohydrate composition was the predominance of the unusual heptose isomer D-glycero-D-manno-heptose. The unusually wide pleiotropic phenotype of this organism is suggested to be due to a fundamental change in the properties of the bacterial outer membrane.     

嗜肺巴氏杆菌外膜蛋白和脂多糖抗原在血清学诊断中的意义

目的评价嗜肺巴氏杆菌外膜蛋白(OMP)和脂多糖(LPs)作为血清学诊断抗原的敏感性和特异性.方法用OMP、LPS和全菌(WC)作为Western blot和ELISA的诊断抗原检测自然感染和实验感染嗜肺巴氏杆菌小鼠相应的IgG抗体滴度,同时测定3种抗原与实验动物常见致病菌的交叉反应.结果与嗜肺巴氏杆菌自然感染和实验感染小鼠血清的ELISA反应中,不同时期,LPS作为诊断抗原时血清抗体阳性率最高,WC次之,OMP最低.自然感染小鼠群中,出生4周LPS抗体阳性率即可达80%,而同期的WC和OMP仅为25%和20%,故LPS敏感性最高.与实验动物常见致病菌免疫血清和阴性种鼠血清的ELISA反应中,WC抗原表现出较高的吸光度(A)值,经Western blot证实,其反应为非特异性反应,LPS抗原特异性最强,OMP抗原次之.结论混合多株具有型或种特异性的OMP或LPS作为ELISA的诊断抗原,无论从特异性和敏感性上均高于全菌抗原.     

UGA Nonsense Mutations in Salmonella typhimurium

Salmonella typhimurium strain LT-2 carries a weak UGA suppressor activity. This activity prevents the detection of some UGA mutants as auxotrophs and probably accounts for the rarity of his UGA mutants in this strain. A selection method is described which permits the isolation of these rare his UGA mutants. Map distribution of his UGA mutations is normal, and their polarity effects are indistinguishable from the polarity effects of amber and ochre mutations at similar locations. Isolation and properties of a prototrophic his UGA mutant are described. UGA mutants are common among lac mutants isolated from Salmonella strains carrying an F'lac episome. Apparently the suppressor activity is insufficient to prevent detection of lac UGA mutants. It is not yet clear whether the suppressor activity plays an important role in normal cell physiology.     

Methionine Mutations of Outer Membrane Protein X Influence Structural Stability and Beta-Barrel Unfolding

We report the biochemical and biophysical characterization of outer membrane protein X (OmpX), an eight-stranded transmembrane β-barrel from E. coli, and compare the barrel behavior with a mutant devoid of methionine residues. Transmembrane outer membrane proteins of bacterial origin are known to display high tolerance to sequence rearrangements and mutations. Our studies with the triple mutant of OmpX that is devoid of all internal methionine residues (M18L; M21L; M118L) indicate that Met replacement has no influence on the refolding efficiency and structural characteristics of the protein. Surprisingly, the conserved substitution of Met→Leu leads to barrel destabilization and causes a lowering of the unfolding free energy by a factor of ∼8.5 kJ/mol, despite the mutations occurring at the loop regions. We report that the barrel destabilization is accompanied by a loss in cooperativity of unfolding in the presence of chemical denaturants. Furthermore, we are able to detect an unfolding intermediate in the Met-less barrel, whereas the parent protein exhibits a classic two-state unfolding. Thermal denaturation measurements also suggest a greater susceptibility of the OmpX barrel to heat, in the Met-less construct. Our studies reveal that even subtle variations in the extra-membrane region of rigid barrel structures such as OmpX, may bear severe implications on barrel stability. We propose that methionines contribute to efficient barrel structuring and protein-lipid interactions, and are therefore important elements of OmpX stability.     

An Extracellular Protein of Propionic Acid Bacteria Inhibits Induced Mutations in Salmonella typhimurium Strains

A culture of propionic acid bacteria grown in a glucose-containing minimal medium, as well as the culture liquid and logarithmic-phase cells obtained from this culture, were found to inhibit the base pair substitution mutations induced by 4-nitroquinoline N-oxide, N-methyl-N"-nitro-N-nitrosoguanidine, and sodium azide and the frameshift mutations induced by 9-aminoacridine. The antimutagenic activity of the culture liquid (CL) was presumably due to the presence of an extracellular thermolabile protein with a molecular mass of no more than 12 kDa, as evidenced by the facts that this activity considerably decreased after the treatment of the CL with pronase, its heating at 92°C, and its dialysis in a cellulose sack, which retains substances with molecular masses greater than 12 kDa. The residual antimutagenic activity of the dialyzed culture liquid was probably related to the interaction of the mutagen with thiols, rather than to the presence of organic acids (acetic or propionic). Thiols may also contribute to the antimutagenic activity of the Propionibacterium shermanii cells.     

The apeE Gene of Salmonella typhimurium Encodes an Outer Membrane Esterase Not Present in Escherichia coli

Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine β-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674–680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C₆ to C₁₆ fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli.     

沙门菌外膜蛋白D的原核表达及多克隆抗体制备

目的:在大肠杆菌中表达沙门菌外膜蛋白(OMP)D,纯化后制备兔抗OMPD抗体。方法:用PCR方法从鼠伤寒沙门菌中扩增出ompD基因,并插入融合表达载体pET-28a(+)的多克隆位点,构建重组表达质粒pET28a(+)-ompD;以重组质粒转化大肠杆菌BL21(DE3),筛选阳性重组菌株,经IPTG诱导目的蛋白表达,在变性条件下对目的蛋白进行亲和层析纯化;以表达的OMPD蛋白免疫家兔,制备抗OMPD的多克隆抗体并进行鉴定。结果:扩增了ompD基因,测序证实正确后亚克隆于表达载体pET-28a(+)中,经PCR筛选和酶切鉴定获得阳性克隆,经诱导在大肠杆菌中表达出相对分子质量为40×103的目的蛋白并进行纯化;纯化的OMPD免疫家兔后,能有效地刺激特异性抗体的产生,抗血清的效价达到1∶10000以上,且具有良好的特异性。结论:构建ompD基因的原核表达载体,并在大肠杆菌中获得高效表达;制备出兔抗OMPD抗体,效价及特异性均良好,为进一步制备肠黏膜高亲和力疫苗奠定了基础。     

Outer membrane of Salmonella typhimurium: Transmembrane diffusion of some hydrophobic substances

The outer membrane, which is composed of lipopolysaccharide, phospholipids, and proteins, is a layer of the cell wall of Gram-negative bacteria, and apparently acts as a penetration barrier for various substances. It had been shown by other workers that “deep rough” mutants of Salmonella typhimurium, whose lipopolysaccharides lack most of the saccharide chains, were much more sensitive than the wild type strain to certain antibiotics and dyes, but not to others. We found that the former group of agents are usually hydrophobic and the latter group mostly hydrophilic. All hydrophilic antibiotics had molecular weights lower than 650, and one of them was shown to diffuse through the outer membrane at 0 °C. In contrast, some hydrophobic antibiotics had molecular weights in excess of 1200, and the rate of diffusion of one of them was shown to be extremely dependent both on temperature and on the structure of lipopolysaccharide present. These data and results presented elsewhere suggest, but do not necessarily prove, that most hydrophilic antibiotics diffuse through aqueous pores, whereas hydrophobic antibiotics and dyes mainly penetrate by dissolving into the hydrocarbon interior of the outer membrane. In contrast to the outer membrane of deep rough mutants, that of the wild type strain and less defective rough mutants was unusual among biological membranes in that it was practically impermeable to hydrophobic agents. It is proposed that the difference in hydrophobic permeability between the two types of strain is due to radical differences in the organization of the outer membrane, more specifically to the presence or absence of exposed phospholipid bilayer regions.     

Effect of Energy Metabolism on Protein Motility in the Bacterial Outer Membrane

We demonstrate the energy dependence of the motion of a porin, the λ-receptor, in the outer membrane of living Escherichia coli by single molecule investigations. By poisoning the bacteria with arsenate and azide, the bacterial energy metabolism was stopped. The motility of individual λ-receptors significantly and rapidly decreased upon energy depletion. We suggest two different causes for the ceased motility upon comprised energy metabolism: One possible cause is that the cell uses energy to actively wiggle its proteins, this energy being one order-of-magnitude larger than thermal energy. Another possible cause is an induced change in the connection between the λ-receptor and the membrane structure, for instance by a stiffening of part of the membrane structure. Treatment of the cells with ampicillin, which directly targets the bacterial cell wall by inhibiting cross-linking of the peptidoglycan layer, had an effect similar to energy depletion and the motility of the λ-receptor significantly decreased. Since the λ-receptor is closely linked to the peptidoglycan layer, we propose that λ-receptor motility is directly coupled to the constant and dynamic energy-consuming reconstruction of the peptidoglycan layer. The result of this motion could be to facilitate transport of maltose-dextrins through the porin.     

Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi

A new immunogenic outer membrane protein, Omp-28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp-28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp-28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N-terminal amino acid sequence of Omp-28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c-DNA. ELISA and Western blotting identified Omp-28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp-28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp-28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp-28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.     

Outer membrane of Salmonella typhimurium. Electron spin resonance studies

The supramolecular structure of the outer membrane of Salmonella typhimurium that produces an Rc-type lipopolysaccharide was studied by adding spin-labeled fatty acid probes to membranes as well as model bilayers. Lipopolysaccharide of this organism apparently formed a bilayer structure in 0.2 M NaCl/0.01 M MgCl₂, and the electron spin resonance spectra suggested that the motion of the segments of hydrocarbon chains near the carboxyl end was quite restricted even at high temperature; this is presumably due to the anchoring of more than a dozen fatty acid residues to a single backbone structure. In the presence of Mg²⁺, we could produce lipopolysaccharide-phospholipid mixed bilayers containing up to 50% (by weight) lipopolysaccharide. Their spectra showed no sign of major heterogeneity, and the maximum hypertine splitting values were considerably larger than in phospholipid-only liposomes; these results suggest that the two components are finely interspersed and that the mobility of phospholipid hydrocarbons in severely restricted by the hydrocarbon chains of lipopolysaccharide. In spite of the presence of lipopolysaccharide in an amount equal to or exceeding that of phospholipids, the outer membrane produced spectra remarkably similar to those of the inner membrane, which does not contain lipopolysaccharide, and there was little sign of immobilization by lipopolysaccharides. Signals corresponding to the pure lipopolysaccharide phase were not detected, either. These results suggest that the phospholipids and lipopolysaccharides are segregated into separate domains in the outer membrane, and the fatty acid probes enter almost exclusively into the phospholipid domains. This conclusion was fully corroborated by determining, through the exchange broadening of line width, the total area of the domains that accommodated the spin label probes.     

Location of Sulfate-binding Protein in Salmonella typhimurium

A method is described for location of proteins in bacteria. It depends upon two techniques. One technique is the inactivation of the protein by a reagent which is incapable of penetrating the bacterial membrane (permeability barrier). Proteins inside this membrane cannot be inactivated unless the cells are disrupted; proteins on or outside the membrane can be inactivated. The second technique depends upon inactivation of the protein by specific antibody. Antibody should not penetrate the external bacterial wall, and therefore should only inactivate proteins that are on the wall surface. Thus, proteins can be localized inside the membrane, in the wall-membrane area, or outside the wall. One reagent developed for use with the first technique is diazo-7-amino-1,3-naphthalene-disulfonate. It inactivated beta-galactoside transport, but not beta-galactosidase of intact Escherichia coli. Similarly, it inactivated sulfate binding and transport but not uridine phosphorylase activity of Salmonella typhimurium. This indicates that the sulfate-binding protein is on or outside the cell membrane, and that uridine phosphorylase is inside the cell. The organic mercurial compounds used also showed that the sensitive parts of the sulfate and alpha-methylglucoside transport systems are less reactive than the sensitive part of the beta-galactoside system. Antibody to the sulfate-binding protein inactivated the purified protein but did not inactivate this protein when intact bacteria were employed. Thus, it appears that the sulfate-binding protein does not protrude outside the cell wall. The conclusion that the binding protein is located in the wall-membrane region is supported by its release upon spheroplast formation or osmotic shock, and also by its ability to combine with sulfate in bacteria which cannot transport sulfate into the cell.     

Mutations of putP that alter the lithium sensitivity of Salmonella typhimurium

The putP gene encodes the major proline permease in Salmonella typhimurium that couples transport of proline to the sodium electrochemical gradient. To identify residues involved in the cation binding site, we have isolated putP mutants that confer resistance to lithium during growth on proline. Wild-type S. typhimurium can grow well on proline as the sole carbon source in media supplemented with NaCl, but grows poorly when LiCl is substituted for NaCl. In contrast to the growth phenotype, proline permease is capable of transporting proline via Na+/proline or Li+/proline symport. Therefore, we selected mutants that grow well on media containing proline as the sole carbon source in the presence of lithium ions. All of the mutants assayed exhibit decreased rates of Li+/proline and Na+/proline cotransport relative to wild type. The location of each mutation was determined by deletion mapping: the mutations cluster in two small deletion intervals at the 5' and 3' termini of the putP gene. The map positions of these lithium resistance mutations are different from the locations of the previously isolated substrate specificity mutations. These results suggest that Lir mutations may define domains of the protein that fold to form the cation binding site of proline permease.     

Location of trpR Mutations in the serB-thr Region of Salmonella typhimurium

Tryptophan biosynthesis in Salmonella is controlled by at least one regulatory gene, trpR, which is cotransducible with thr genes and not with the trp operon. Mutations in trpR cause derepression of tryptophan enzyme synthesis and confer resistance to growth inhibition by 5-methyltryptophan. Nineteen trpR mutations were mapped with respect to thrA and serB markers by two-point (ratio) and three-point transduction tests. The results are all consistent with the site order serB80-trpR-thrA59 on the Salmonella chromosome. Very low or undetectable levels of recombination between different trpR mutations have so far prevented the determination of fine structure in the trpR gene. Thirteen other 5-methyltryptophan-resistant mutants previously found not to be cotransducible with either the trp operon or thrA, and designated trpT, were also used in these experiments. Lack of cotransducibility with thrA was confirmed, and no linkage with serB was detected. The nature and location of trpT mutations remain obscure.     

Mutations affecting glutamine synthetase activity in Salmonella typhimurium.

A positive selection procedure has been devised for isolating mutant strains of Salmonella typhimurium with altered glutamine synthetase activity. Mutants are derived from a histidine auxotroph by selecting for ability to grow on D-histidine as the sole histidine source. We hypothesize that the phenotype may be based on a regulatory increase in the activities of the D-histidine racemizing enzymes, but this has not been established. Spontaneous glutamine-requiring mutants isolated by the above selection procedure have two types of alterations in glutamine synthetase activity. Some have less than 10% of parent activity. Others have significant glutamine synthetase activity, but the enzyme have an altered response to divalent cations. Activity in mutants of the second type mimics that of highly adenylylated wild-type enzyme, which is believed to be in-active in vivo. Glutamine synthetase from one such mutant is more heat labile than wild-type enzyme, indicating that it is structurally altered. Mutations in all strains are probably in the glutamine synthetase structural gene (glnA). They are closely linked on the Salmonella chromosome and lie at about min 125. The mutants have normal glutamate dehydrogenase activity.     

Cold-sensitive Mutations in Salmonella typhimurium Which Affect Ribosome Synthesis

A number of mutations (45) expressed as cold-sensitive conditional lethal pheno-types were screened by transduction for their linkage to the streptomycin-resistance locus; 7 showed such linkage. Of these, two were studied in greater detail. The sedimentation profiles of ribosomes from cultures grown at low temperature differed from wild type and from one another. Both mutants lost ribonucleic acid control at low temperature. It is suggested that a high proportion of mutants expressing a cold-sensitive phenotype harbor mutations in genes affecting ribosome synthesis or regulation.     

Mutations that affect the regulation of phs in Salmonella typhimurium.

The regulation of phs [production of hydrogen sulphide (H2S)] in Salmonella typhimurium is complex. Previous studies have shown that expression is dependent upon the presence of reduced sulphur and anaerobiosis and is modulated by carbon source and growth stage. Transposon mutagenesis failed to find any potential trans-acting factors effective in the regulation of phs in relation to oxygen. Spontaneous mutants capable of expressing phs-lac aerobically were isolated and characterized. These mutations are closely linked to phs and affect not only oxygen regulation but also the requirement for cyclic AMP and reduced sulphur. Analysis of merodiploid strains indicates that these mutations cis-acting and that phs is not subject to autoregulation.     

Atomistic Scale Effects of Lipopolysaccharide Modifications on Bacterial Outer Membrane Defenses

Lipopolysaccharides (LPS) are a main constituent of the outer membrane of Gram-negative bacteria. Salmonella enterica, like many other bacterial species, are able to chemically modify the structure of their LPS molecules through the PhoPQ pathway as a defense mechanism against the host immune response. These modifications make the outer membrane more resistant to antimicrobial peptides (AMPs), large lipophilic drugs, and cation depletion, and are crucial for survival within a host organism. It is believed that these LPS modifications prevent the penetration of large molecules and AMPs through a strengthening of lateral interactions between neighboring LPS molecules. Here, we performed a series of long-timescale molecular dynamics simulations to study how each of three key S. enterica lipid A modifications affect bilayer properties, with a focus on membrane structural characteristics, lateral interactions, and the divalent cation bridging network. Our results discern the unique impact each modification has on strengthening the bacterial outer membrane through effects such as increased hydrogen bonding and tighter lipid packing. Additionally, one of the modifications studied shifts Ca²⁺ from the lipid A region, replacing it as a major cross-linking agent between adjacent lipids and potentially making bacteria less susceptible to AMPs that competitively displace cations from the membrane surface. These results further improve our understanding of outer membrane chemical properties and help elucidate how outer membrane modification systems, such as PhoPQ in S. enterica, are able to alter bacterial virulence.     

Outer membrane of Salmonella typhimurium: Identification of proteins exposed on cell surface

Proteins exposed on the outer surface of the outer membrane of Salmonella typhimurium were identified by reacting intact cells with a covalent labeling reagent. Since the outer membrane permitted the free diffusion of small hydrophilic molecules, we used a macromolecular reagent, CNBr-activated dextran, as the non-penetrating labeling agent. We also used a mutant producing a lipopolysaccharide with a very short (i.e. hexasaccharide) carbohydrate chain, in order to avoid steric hindrance by the carbohydrates on membrane surface. Results showed that out of the four “major” proteins of molecular weight around 35 000, three were exposed, and that at least six other proteins were also exposed on cell surface. Only two or three outer membrane proteins consistently did not react with the reagent in intact cells.