Ultrastructure of the outer membrane of Salmonella typhimurium bacteriocin-resistant mutants deficient in the 33K protein.

Outer membrane mutants of Salmonella typhimurium deficient in one, two, or three of the 33,000-dalton (33K), 34K, and 36K outer membrane proteins (7) were studied by using thin sectioning and freeze-fracturing electron microscopy techniques. The outer concave fracture face of all mutants deficient in the 33K protein had numerous particleless patches. In contrast to all previously examined 34K to 36K-deficient mutants, the 33K-deficient mutants showed marked heterogeneity in the size and distribution of such "empty" patches between cells of a culture. One mutant was deficient in both the 33K and the 34K to 36K "porin" protein complex; its outer membrane had very large particleless smooth areas. It is concluded that the 33K protein on one hand and the porin on the other are both able to form intramembraneous particles.     

New Salmonella typhimurium mutants with altered outer membrane permeability

We describe three new classes of Salmonella typhimurium mutants with increased sensitivity to hydrophobic agents. In contrast to many previously described mutants, the phage sensitivity pattern of these mutants did not give any indication of defective lipopolysaccharide. Furthermore, they had no detectable changes in their phospholipid or outer membrane protein composition, and their growth rate and cell morphology were normal. Class B mutants were nearly as sensitive to novobiocin, fusidic acid, erythromycin, rifampin, and clindamycin as are deep rough (heptoseless) mutants; in addition they were sensitive to methicillin, penicillin (to which heptoseless mutants are resistant), gentian violet, and anionic and cationic detergents. Class A and C mutants had less sensitive, but characteristic phenotypes. None of the three classes were sensitive to serum bactericidal action. The class B mutation mapped between map positions 7 and 11 on the S. typhimurium chromosome, and the class C mutation mapped between positions 5 and 7. The map position for the class A mutation remained undefined, but it was separate from the class B and C mutations and, like those, did not correspond to any gene loci known to participate in the synthesis of major outer membrane constituents.     

Investigations on the outer membrane proteins of Salmonella typhimurium.

The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.     

Excretion of unassembled flagellin by Salmonella typhimurium mutants deficient in hook-associated proteins.

Of the flagellar filamentless mutants of Salmonella typhimurium, the flaV, flaU, and flaW mutants, which are defective in hook-associated proteins, synthesized flagellin molecules, but flagella did not polymerize at the tips of the mutant hooks and were excreted into the culture medium as intact monomers.     

Heptose-deficient mutants ofEscherichia coli K12 deficient in up to three major outer membrane proteins

Molecular Genetics and Genomics - Mutants ofEscherichia coli K12, deficient in up to three major outer membrane proteinsb,c andd have been constructed. Mutants that lack the lipopolysaccharide...     

Isolation and characterization of catalase deficient mutants of Salmonella typhimurium.

Summary Catalase deficient mutants (kat) ofSalmonella typhimurium have been isolated. The mutationskatA1, katC6 andkatD9 appear to map at about minute 10 on theSalmonella chromosome. ThekatC6 andkatD9 lesions are complemented by theE. coli F128 (lac+ pro+) episome but thekatA1 lesion is not.KatB2 maps at about minute 100. None of the mutants are oxygen sensitive; they grow as well as wild type bacteria, even when aerated.     

Aspartate-specific peptidases in Salmonella typhimurium: mutants deficient in peptidase E.

The only dipeptide found to serve as a leucine source for a Salmonella strain lacking peptidases N, A, B, D, P, and Q was alpha-L-aspartyl-L-leucine. A peptidase (peptidase E) that specifically hydrolyzes Asp-X peptides was identified and partially purified from cell extracts. The enzyme (molecular weight, 35,000) is inactive toward dipeptides with N-terminal asparagine or glutamic acid. Mutants (pepE) lacking this enzyme were isolated by screening extracts for loss of the activity. Genetic mapping placed the pepE locus at 91.5 map units and established the gene order metA pepE zja-861::Tn5 malB. Duplications of the pepE locus showed a gene dosage effect on levels of peptidase E, suggesting that pepE is the structural gene for this enzyme. Mutations in pepE resulted in the loss of the ability to grow on Asp-Pro as a proline source but did not affect utilization of other dipeptides with N-terminal aspartic acid. Loss of peptidase E did not cause a detectable impairment in protein degradation. Two other peptidases present in cell extracts of mutants lacking peptidases N, A, B, D, P, Q, and E also hydrolyze many Asp-X dipeptides.     

Genetic and physiological regulation of intrinsic proteins of the outer membrane of Salmonella typhimurium.

Four major outer membrane polypeptides, accounting for approximately 20% of the total protein of the outer membrane of Salmonella typhimurium, were induced by growth in minimal medium. The polypeptides were tightly bound membrane components. Physiological and genetic evidence indicates that the four polypeptides fall in two separate regulation groups. Synthesis of one of these groups was coordinately regulated by the concentration of iron in the medium, and a mutant strain has been identified in which there is constitutive synthesis of this group of major outer membrane proteins.     

Comparison of outer membrane porin proteins produced by Escherichia coli and Salmonella typhimurium

The OmpC, OmpF, and Lc (NmpC) porin proteins of Escherichia coli K-12 have been shown to be similar to the OmpC (36K), OmpF (35K) and OmpD (34K) porin proteins of Salmnella typhimurium LT2 in terms of function, regulation of expression, and, in the case of OmpC and OmpF proteins, equivalence of the genetic loci determining their production. However, the corresponding pairs of proteins from these two species showed only limited similarity in peptide maps and no similarity in terms of migration on polyacrylamide gels.     

Degradation of abnormal proteins in peptidase-deficient mutants of Salmonella typhimurium.

The degradation of abnormal proteins produced as a result of incorporation of the arginine analog L-canavanine or generated by exposure to puromycin was studied in wild-type and multiply peptidase-deficient strains of Salmonella typhimurium. Both types of abnormal protein were rapidly degraded during growth of Pep+ strains of this organism. Peptidase--deficient mutants (lacking peptidases N, A, B, and D) could also degrade these abnormal proteins, although the rate of production of trichloroacetic acid-soluble degradation products was slower in the mutant strain than in a strain carrying a normal complement of peptidases. Analysis of these trichloroacetic acid-soluble degradation products of ion-exchange chromatography showed that free amino acid was the major breakdown product produced by the wild-type strain. The acid-soluble degradation product produced by the mutant strain, however, was a complex mixture that contained a variety of small peptides as well as free amino acids. These results indicate that the same group of peptidases shown previously to function in the degradation of exogenously supplied peptides and in protein turnover during carbon starvation also lie on the pathway by which abnormal proteins are degraded.     

Major outer membrane protein in Salmonella typhimurium induced by maltose.

A maltose-induced major outer membrane protein (the 44K protein) is demonstrated in Salmonella typhimurium. This protein resembles the lambda receptor of Escherichia coli in its location, induction properties, apparent molecular weight, and association with the peptidoglycan layer of the cell wall. The 44K protein is missing in certain Salmonella Mal- mutants, which are also missing a protein analogous to the maltose-binding protein of E. coli. Thus, these mutants may be defective in the control of maltose genese in Salmonella. The proteins appear to be closely related, as indicated by cross-reaction of the Salmonella protein with the antiserum raised against the lambda receptor; however, they are not identical, since the peptide patterns obtained after limited proteolysis are completely different. Bacteriophage lambda does not use the 44K protein as a receptor.     

Topology of the outer membrane phospholipase A of Salmonella typhimurium.

The outer membrane phospholipase A (OMPLA) of Enterobacteriaceae has been proposed to span the membrane 14 times as antiparallel amphipathic beta-strands, thereby exposing seven loops to the cell surface. We have employed the epitope insertion method to probe the topology of OMPLA of Salmonella typhimurium. First, missense mutations were introduced at various positions in the pldA gene, encoding OMPLA, to create unique BamHI sites. These BamHI sites were subsequently used to insert linkers, encoding a 16-amino-acid B-cell epitope. Proper assembly of all mutant proteins was revealed by their heat modifiability in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The accessibility of the inserted epitopes was assessed. Immunofluorescence analysis of intact cells with antibodies against the inserted epitope showed that three of seven predicted loops are indeed cell surface exposed. Trypsin accessibility experiments verified the cell surface exposure of two additional loops and provided support for the proposed periplasmic localization of three predicted turns. For two other predicted exposed loops, the results were not conclusive. These results support to a large extent the proposed topology model of OMPLA. Furthermore, the observation that the substitutions Glu66Pro and Glu247Gly virtually abolished enzymatic activity indicates that these residues might play a major role in catalysis.     

Genetic analysis of mutants of Salmonella typhimurium deficient in formate dehydrogenase activity

Summary A genetical study of mutants of Salmonella typhimurium deficient in formate dehydrogenase activity was performed. The affected gene was designated fdh A and mapped at 116 min, the order of genes in that region being xyl-fdh A-mtl-cys E.Abbreviations FHL formate hydrogenylase - FDH (PMS) formate dehydrogenase (phenazine methosulfate) - FDH (BV) formate dehydrogenase (benzyl viologen) - HYD hydrogenase - NR nitrate-reductase - TTR tetrathionate-reductase     

Oligopeptidase-deficient mutants of Salmonella typhimurium.

An oligopeptidase that hydrolyzes N-acetyl-L-alanyl-L-alanyl-L-alanyl-L-alanine (AcAla4) has been identified in extracts of Salmonella typhimurium. Mutants lacking this activity have been isolated in dcp mutant strains by screening extracts of mutagenized clones for failure to hydrolyze AcAla4 or by screening colonies for inability to use AcAla4 as a nitrogen source. Double mutants (dcp optA) lacking both oligopeptidase A and dipeptidyl carboxypeptidase cannot use AcAla4 as a nitrogen source, although dcp+ optA and dcp optA+ strains grow on this peptide. The mutations responsible for the loss of activity map at a locus (optA) between asd (75 map units) and xylA (78 map units). Oligopeptidase A hydrolyzes certain N-blocked tetrapeptides, unblocked pentapeptides, and unblocked hexapeptides, usually but not always liberating the C-terminal tripeptide. These two activities seem to be responsible for the production of a large fraction of the dipeptides that accumulate during protein breakdown in a pepN pepA pepB pepD strain.     

Asymmetric localization of lipopolysaccharides on the outer membrane of Salmonella typhimurium.

Treatment of intact cells of Salmonella typhimurium with galactose oxidase (EC 1.1.3.9) resulted in an extensive oxidation of the susceptible galactose residues in the lipopolysaccharides. Comparison with the extent of oxidation in isolated lipopolysaccharides indicated that most lipopolysaccharide molecules were located in the outer leaflet of the outer membrane in intact cells.