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.     

Method of isolation of cysteine constitutive mutants of the cysteine regulon in Salmonella typhimurium.

Summary Size and shape of mitochondrial DNA molecules of Schizosaccharomyces pombe were analyzed by electron microscopy. Besides numerous linear molecules, circular molecules ranging from 0.83 m to 12.81 m were found. Depending on the method of preparation, both closed and open circular molecules were found. Most of the circular molecules could be assigned to five major size classes of 0.83±0.05 m, 1.7±0.05 m, 4.74±0.04 m, 5.74±0.04 m, and 8.32±0.07 m. Possible explanations for the different size classes of mitochondrial DNA molecules are discussed.     

5-Fluoroorotate-resistant mutants of Salmonella typhimurium.

Spontaneously occurring mutants of Salmonella typhimurium resistant to 5-fluoroorotate (5-FOA) were isolated. One class of mutant showed marked derepression of pyrimidine biosynthetic enzymes and had the unusual property of being unable to grow on nutrient agar. However, when the osmotic strength of nutrient agar was increased, the mutants were able to grow. The genetic basis for the osmotic fragility and elevated pyr enzyme synthesis was the result of mutations affecting pyrH, encoding the enzyme uridine 5'-monophosphate kinase.     

Dipeptidyl carboxypeptidase-deficient mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium deficient in dipeptidyl carboxypeptidase have been isolated by screening for clones unable to use N-acetyl-L-alanyl-L-alanyl-L-alanine (AcAla3) as the sole nitrogen source. An insertion of the transposable element Tn10 near dcp (the locus coding for dipeptidyl carboxypeptidase) has been isolated and used to map the locus in the interval between purB and trp, an otherwise genetically silent region of the S. typhimurium map. All dcp mutants could still grow using N-acetyl-L-alanyl-L-alanyl-L-alanyl-L-alanine (AcAla4) as the sole nitrogen source. Crude extracts from the dcp mutants failed to hydrolyze AcAla3 but retained approximately 80% of the wild-type activity toward AcAla4. Several lines of evidence indicate that hydrolysis of AcAla4 in the dcp mutant results from the action of a new peptidase distinct from dipeptidyl carboxypeptidase. A mutant strain lacking dipeptidyl carboxypeptidase in addition to peptidases N, A, B, and D showed reduced protein breakdown during carbon starvation compared with a strain lacking only peptidases N, A, B, and D.     

Membrane potential independent binding of azidopyrene to LPS mutants of Salmonella typhimurium

Abstract The hydrophobic photoprobe azidopyrene has been used to probe the interaction of hydrophobic molecules with strains of Salmonella typhimurium carrying defined LPS mutations. We have found that whereas binding of this probe to cells having normal LPS is inversely related to the membrane potential (deenergized cells showed enhanced azidopyrene binding), mutants with defective LPS bind excess probe independent of the membrane potential. These results emphasize the role of LPS as a barrier to hydrophobic molecules.     

Salmonella typhimurium mutants lacking protease II.

Mutants of Salmonella typhimurium lacking protease II, an endoprotease with trypsin-like specificity, have been isolated. These mutants can be identified by using the chromogenic substrate N-methyl-N-p-toluenesulfonyl-L-lysine beta-naphthyl ester to screen colonies growing on agar for the presence of the enzyme. All of the mutations isolated map at locus tlp (typsin-like protease) which is cotransducible (approximately 1%) using phage P1 with tre (trehalose utilization) at approximately 58 min on the Salmonella map. Double mutants lacking both protease I and protease II have been constructed. These strains grew normally. They were able to degrade abnormal proteins and to carry out protein turnover during carbon starvation at the same rate as the wild type.     

Deoxynucleoside-sensitive mutants of Salmonella typhimurium

Summary Thymineless mutants ofSalmonella typhimurium which are able to grow with low added concentrations of thymine (20 M) fall into two classes on the basis of growth on deoxyribose as sole carbon source. Those which can grow are deoxyribomutase negative and those which cannot are deoxyriboaldolase negative. The former class are inhibited by deoxynucleosides and this provides a method for discriminating between different classes oftlr mutants ofEscherichia coli K12, which cannot utilize deoxyribose as a carbon source. It is suggested that the sensitivity of deoxyriboaldolase negative strains is due to the accumulation of deoxyribose-5-phosphate. The data also indicate that deoxyribose-5-phosphate is the inducer of thymidine phosphorylase. It seems that one or both of the deoxyribose phosphates is the toxic compound, and that reversal of inhibition by ribonucleosides is due to inhibition of the enzymes catalysing their formation from deoxynucleosides. We propose that the symbolsdrm anddra be used to denote the structural genes for deoxyribomutase and deoxyriboaldolase respectively.     

Salmonella typhimurium mutants lacking NAD pyrophosphatase.

NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome.     

Peptidase mutants of Salmonella typhimurium

Six peptidase activities have been distinguished electrophoretically in cell extracts of Salmonella typhimurium with the aid of a histochemical stain. The activities can also be partially separated by chromatography on diethylaminoethyl-cellulose. These peptidases show overlapping substrate specificities. Mutants (pepN) of the parent strain leu-485 lacking one of these enzymes (peptidase N) were obtained by screening for colonies that do not hydrolyze the chromogenic substrate l-alanyl-beta-naphthylamide. The absence of this broad-specificity peptidase in leu-485 pepN(-) mutants allowed the selection of mutants unable to use l-leucyl-l-alaninamide as a leucine source. These mutants (leu-485 pepN(-)pepA(-)) lack a broad-specificity peptidase (peptidase A) similar to aminopeptidase I previously described in Escherichia coli. Mutants (pepD) lacking a dipeptidase (peptidase D) have been isolated from a leu-485 pepN(-)pepA(-) parent by penicillin selection for mutants unable to use l-leucyl-l-glycine as a leucine source. Mutants (pepB) lacking a fourth peptidase (peptidase B) have been isolated from a leu-485 pepN(-)pepA(-)pepD(-) strain by penicillin selection for failure to utilize l-leucyl-l-leucine as a source of leucine. Single recombinants were obtained by transduction for each of the peptidases missing in a leu-485 pepN(-)pepA(-)pepD(-)pepB(-) strain. The growth response of these recombinants to leucine peptides shows that all of these peptidases can function in the catabolism of peptides and that they display overlapping substrate specificities in vivo.     

rfaP mutants of Salmonella typhimurium

Salmonella typhimurium rfaP mutants were isolated and characterised with respect to their sensitivity towards hydrophobic antibiotics and detergents, and their lipopolysaccharides were chemically analysed. The rfaP mutants were selected after diethylsulfate mutagenesis or as spontaneous mutants. The mutation in two independent mutants SH7770 (line LT2) and SH8551 (line TML) was mapped by cotransduction with cysE to the rfa locus. The mutants were sensitive to hydrophobic antibiotics (clindamycin, erythromycin and novobiocin) and detergents (benzalkoniumchloride and sodium dodecyl sulfate). Analysis of their lipopolysaccharides by chemical methods and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that their saccharide portion was, to a large extent, of chemotype Rc with small proportions of material containing a more complete core oligosaccharide and O-specific chains. Only 2.5 mol phosphate/mol lipopolysaccharide was found whereas the phosphate content of the lipopolysaccharide of a galE mutant strain was 4.8 mol. Thus the rfaP mutant lipopolysaccharides lacked more than two phosphate residues. Assessment of the location of phosphate groups in rfaP lipopolysaccharides revealed the presence of at least 2 mol phosphate in lipid A, indicating that the core oligosaccharide was almost devoid of phosphate. The chemical, physiological and genetic data obtained for these mutants are in full agreement with those reported earlier for rfaP mutants of Salmonella minnesota.     

Temperature-sensitive glutamate dehydrogenase mutants of Salmonella typhimurium.

Mutants of Salmonella typhimurium defective in glutamate dehydrogenase activity were isolated in parent strains lacking glutamate synthase activity by localizcd mutagenesis or by a general mutagenesis combined with a cycloserine enrichment for glutamate auxotrophs. Two mutants with temperature-sensitive phenotypes had glutamate dehydrogenase activities that were more thermolabile than that of an isogenic control strain. Eight other mutants had less than 10% of the wild-type glutamate dehydrogenase activity. All the mutations were cotransducible with a Tn10 element (zed-2:Tn10) located at approximately 23 U on the S. typhimurium linkage map. These data strongly indicate that this region contains the structural gene (gdhA) for glutamate dehydrogenase.     

Subunit-specific phenotypes of Salmonella typhimurium HU mutants.

Salmonella hupA and hupB mutants were studied to determine the reasons for the high degree of conservation in HU structure in bacteria. We found one HU-1-specific effect; the F'128 plasmid was 25-fold less stable in hupB compared with hupA or wild-type cells. F' plasmids were 120-fold more unstable in hupA hupB double mutants compared with wild-type cells, and the double mutant also had a significant alteration in plasmid DNA structure. pBR322 DNA isolated from hupA hupB strains was deficient in supercoiling by 10 to 15% compared with wild-type cells, and the topoisomer distribution was significantly more heterogeneous than in wild-type or single-mutant strains. Other systems altered by HU inactivation included flagellar phase variation and phage Mu transposition. However, Mu transposition rates were only about fourfold lower in Salmonella HU double mutants. One reason that Salmonella HU double mutants may be less defective for Mu transposition than E. coli is the synthesis in double mutants of a new, small, basic heat-stable protein, which might partially compensate for the loss of HU. The results indicate that although either HU-1 or HU-2 subunit alone may accommodate the cellular need for general chromosomal organization, the selective pressure to conserve HU-1 and HU-2 structure during evolution could involve specialized roles of the individual subunits.     

Reconstitution of the phosphoglycerate transport protein of Salmonella typhimurium

Operation of the phosphoglycerate transport protein (PgtP) of Salmonella typhimurium has been studied in proteoliposomes by using a technique in which membrane protein is solubilized and reconstituted directly from small volumes of cell cultures. When protein from induced cells was reconstituted into phosphate (Pi)-loaded proteoliposomes, it was possible to demonstrate a PgtP-mediated exchange of internal and external phosphate. For this homologous Pi:Pi antiport, kinetic analysis indicated a Michaelis constant (Kt) of 1 mM and a maximal velocity of 26 nmol/min mg of protein; arsenate inhibited with a Ki of 1.3 mM, suggesting that PgtP did not discriminate between these two inorganic substrates. Pi-loaded proteoliposomes also accumulated 3-phosphoglycerate and phosphoenolpyruvate, establishing for each of them a concentration gradient (in/out) of about 100-fold; phosphoenolpyruvate (Ki = 70 microM) rather than 3-phosphoglycerate (Kt = 700, Ki = 900 microM) was the preferred substrate for these conditions. We also concluded that such heterologous exchange was a neutral event, since its rate and extent were unaffected by the presence of a protonophore and unresponsive to the imposition of a membrane potential (positive or negative inside). In quantitative work, we found a stoichiometry of 1:1 for the exchange of Pi and 3-phosphoglycerate, and given an electroneutral exchange, this finding is most easily understood as the overall exchange of divalent Pi against divalent phosphoglycerate. These experiments establish that PgtP functions as an anion exchange protein and that it shares important mechanistic features with the Pi-linked antiporters, GlpT and UhpT, responsible for transport of glycerol 3-phosphate and hexose 6-phosphates into Escherichia coli.     

Salmonella typhimurium mutants generally defective in chemotaxis.

The mutations of eight chemotaxis-deficient strains of Salmonella typhimurium, including five new mutants in strain LT2, were mapped by P22 transduction in relation to various fla mot deletions in S. abortus-equi. Seven recessive che mutations mapped between motB and flaC: three, all nontumbling, the che region I, adjacent to motB, and four, including one ever-tumbling, in che region II, adjacent to flaC. Mutant che-107, never-tumbling and dominant to wild type, mapped at flaAII, other mutations of which cause either absence of flagella or lack of locomotor function. We surmise that gene flaAII specifies a protein that polymerizes to form an essential component of the basal apparatus (so that absence of gene product prevents formation of flagela); that a component built up from certain mutationally altered proteins cannot transmit (or generate) active rotation of the hook and flagellum, and so causes the Mot (paralysis) phenyotype; and that a component built up from protein with the che-107 alteration permits only counterclockwise rotation, so that the tumble, normally produced by transient clockwise rotation, cannot be effected.     

6-Aminonicotinamide-resistant mutants of Salmonella typhimurium

Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD.     

Temperature-sensitive,lipopolysaccharide-deficient mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium LT2, which were temperature-sensitive for lipopolysaccharide (LPS) synthesis, were isolated from a galE ^- strain based on their resistance to phage C21 and sensitivity to sodium deoxycholate at 42°C. They produced LPS of chemotype Rc at 30°C and deep-rough LPS at 42°C. P22-mediated transductional analysis showed that the mutations responsible for temperature sensitivity are located in the rfa cluster where several genes involved in the synthesis of the LPS core are mapped. A plasmid, carrying rfaC, D and F genes of Escherichia coli K-12, complemented these mutations. These genes are responsible for the synthesis of the inner-core region of the LPS molecule. This indicates that genetic defects in these temperature-sensitive mutants affect the inner-core region of LPS.