Aerobic excretion of 1,2-propanediol by Salmonella typhimurium.

Salmonella typhimurium excreted the rhamnose fermentation product 1,2-propanediol not only under anaerobic conditions, but also under aerobic conditions. The absence of an aldehyde dehydrogenase enzymatic activity that oxidizes to lactate the lactaldehyde formed in the dissimilation of rhamnose raised the intracellular concentration of the aldehyde which was alternatively reduced to the excretable 1,2-propanediol by a residual propanediol oxidoreductase activity.     

Utilization of d-amino acids by dadR mutants of Salmonella typhimurium

Utilization of d-amino acids being substrates of d-amino acid dehydrogenase of Salmonella typhimurium was examined. The experiments were done with wild type strains and the mutants dadA missing the enzyme activity and dadR in which its synthesis is released from catabolite repression. Growth on d-tryptophan, d-histidine and d-methionine used as precursors of the l-amino acids was faster when the respective auxotrophs carried dadR mutations. The dadR mutants grew faster when d-or l-alanine was present as a sole source of nitrogen. Experiments with d-amino acid dehydrogenase in vitro provided evidence that d-tryptophan is its substrate with a very low affinity to the dehydrogenase.     

Regions of Salmonella typhimurium flagellin essential for its polymerization and excretion.

Immunological methods were used to examine the flagellin production of Salmonella typhimurium strains that carried a mutation in one of the two possible genes for flagellin (H1 or H2) and also were incapable of expressing the other gene. Some mutants produced flagellin that was excreted into the culture medium; others accumulated flagellin intracellularly. These two phenotypes were detected in both H1 and H2 mutants. The mutation sites were mapped on the corresponding deletion map (consisting of 21 segments in the case of H1 and 31 segments in the case of H2). H1 and H2 mutations causing excretion of flagellin were clustered mainly in segment 12 and segment 6 from the proximal end, respectively, suggesting that the corresponding segments of the flagellins play a role in polymerization. Mutations causing accumulation in the cytoplasm were clustered in segments 19 to 21 of the H1 map and in segments 25 to 29 of the H2 map, suggesting that an essential region for flagellin transport exists toward the C terminus of flagellin.     

Chemotaxis of Salmonella typhimurium to amino acids and some sugars.

Patterns of chemotaxis by Salmonella typhimurium strain LT-2 to l-amino acids and to several sugars were quantitated by the Adler capillary procedure. Competition experiments indicated that LT-2 possesses three predominant receptors, or interacting sets of receptors, for amino acids. These were termed the aspartate, serine, and alanine classes, respectively. Studies with strains carrying point and deletion mutations affecting components of the phosphoenolpyruvate: glycose phosphotransferase system (PTS) made unlikely a role in primary reception of d-glucose by the three soluble PTS components, namely HPr, enzyme I, and factor III. A ptsG mutant defective in membrane-bound enzyme IIB' of the high-affinity glucose transport system was shown to exhibit normal chemotaxis providing pleiotropic effects of the mutation were eliminated by its genotypic combination with other pts mutations or, phenotypically, by addition of cyclic AMP and substrate. A correlation was demonstrated between chemotaxis to glucose and activity of the low-affinity glucose transport complex, membrane-bound enzymes IIB:IIA, and an enzyme IIB:IIA mutant was shown to have a preponderant defect in chemotaxis to glucose and mannose. Of four systems capable of galactose transport, only the beta-methylgalactoside transport system was implicated in chemotaxis to galactose. Some properties of a mutant possibly defective in processing of signals for chemotaxis to sugars is described.     

Reversal by aromatic amino acids of 2-thiazole-DL-alanine inhibition of Salmonella typhimurium

Of 21 l-amino acids tested (at 1.2 x 10(-4)m), only histidine and the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) protect Salmonella typhimurium strains from inhibition of growth and immediately reverse the growth inhibition by 5 x 10(-4)m 2-thiazole-dl-alanine.     

Spatio-temporal patterns generated by Salmonella typhimurium.

We present experimental results on the bacterium Salmonella typhimurium which show that cells of chemotactic strains aggregate in response to gradients of amino acids, attractants that they themselves excrete. Depending on the conditions under which cells are cultured, they form periodic arrays of continuous or perforated rings, which arise sequentially within a spreading bacterial lawn. Based on these experiments, we develop a biologically realistic cell-chemotaxis model to describe the self-organization of bacteria. Numerical and analytical investigations of the model mechanism show how the two types of observed geometric patterns can be generated by the interaction of the cells with chemoattractant they produce.     

Redox potential-dependent nitrite metabolism by Salmonella typhimurium.

Salmonellae are generally resistant to the inhibitory effects of NaNO2. Removal of the lipopolysaccharide of Salmonella typhimurium by ethylenediaminetetraacetic acid pretreatment did not result in subsequent inhibtion of growth by NaNO2, indicating that lipopolysaccharide does not function to exclude NaNO2 from the cell. NaNO2 disappeared from the medium while the cells were growing, but, after stationary phase was reached, no further losses were observed unless the pH was maintained above 7.0. Similar losses were observed in a cell-free system if the redox potential of the medium was between -250 and -175 mV. If the disrupted cell suspension was first heated in a boiling water bath for 15 to 18 min, no NaNO2 loss was observed regardless of the redox potential. S. typhimurium is capable of metabolizing NaNO2, possibly by means of a nitrite-reducing enzyme function which is redox controlled.     

Redox potential-dependent nitrite metabolism by Salmonella typhimurium.

Salmonellae are generally resistant to the inhibitory effects of NaNO2. Removal of the lipopolysaccharide of Salmonella typhimurium by ethylenediaminetetraacetic acid pretreatment did not result in subsequent inhibtion of growth by NaNO2, indicating that lipopolysaccharide does not function to exclude NaNO2 from the cell. NaNO2 disappeared from the medium while the cells were growing, but, after stationary phase was reached, no further losses were observed unless the pH was maintained above 7.0. Similar losses were observed in a cell-free system if the redox potential of the medium was between -250 and -175 mV. If the disrupted cell suspension was first heated in a boiling water bath for 15 to 18 min, no NaNO2 loss was observed regardless of the redox potential. S. typhimurium is capable of metabolizing NaNO2, possibly by means of a nitrite-reducing enzyme function which is redox controlled.     

Proline excretion and indirect suppression in Escherichia coli and Salmonella typhimurium

The last step in proline biosynthesis in Escherichia coli K-12, Salmonella typhimurium LT7, and a number of other enterobacterial isolates is regulated so that no proline is excreted, even if excess Delta(1)-pyrroline-5-carboxylate, the immediate precursor of proline, is added to a culture. In proline auxotrophs blocked at an early step in proline biosynthesis (proA or proB), reversion to prototrophy is often due to a mutation in the arginine pathway which diverts N-acetyl glutamate gamma-semialdehyde to proline synthesis, thus bypassing the proA or proB block. In such double mutants (proAB, argD), the last step in proline synthesis appears to be unregulated, since proline is excreted. Feedback inhibition and repression of the arginine pathway overcomes indirect suppression (restoring the Pro(-) phenotype), but proline regulation is not restored; double mutants still excrete proline when fed Delta(1)-pyrroline-5-carboxylate exogeneously. A new class of proline analogue-resistant mutant, due to mutation at argD, is also described.     

Proton magnetic resonance studies of alpha-keto acids.

Pulsed Fourier transform proton magnetic resonance was used to study alpha-ketoglutaramic, and several other alpha-keto acids in aqueous solutions as a function of pH. Most alpha-keto acids were found to exist in equilibrium with the hydrate (gem-doil). The equilibrium position favors the nonhydrated alphs-keto acid at neutral pH, but at low pH values (below the pKa of the alpha-carboxylic acid group) the hydrate predominates. We found evidence that alpha-ketoglutaric acid exists in a third equilibrium form which is assigned to the lactol. alpha-Ketoglutaramic acid (the alpha-keto acid analog of glutamine) which is known to exist predominantly in a cyclic form at pH 7.0 was shown to exist as a cyclic structure over a wide pH range. However, the cyclic form is an equilibrium mixture of 2-pyrrolidone-5-hydroxy-5-carboxylic and 1-pyrrolin-2-one-5-carboxylic acids.     

Excretion of unassembled hook-associated proteins by Salmonella typhimurium.

Hook-associated proteins (HAPs) were excreted into the culture medium of the Fla+ strain as well as into the growth medium of the filamentless mutants of Salmonella typhimurium. This indicates that the bacteria synthesize HAPs excessively, beyond the amount required for construction of flagella. The extra HAPs are shed into the culture medium after a definite amount of each HAP has been assembled into the flagellar structure.     

Inhibition of cathepsin B and papain by peptidyl alpha-keto esters, alpha-keto amides, alpha-diketones, and alpha-keto acids

A series of peptidyl alpha-keto esters, alpha-keto amides, alpha-keto acids, and alpha-diketones were synthesized which reversibly inhibit papain and cathepsin B. Methyl 3-(N-benzyloxycarbonyl-L-phenylalanyl)amino-2-oxopropionate (a dicarbonyl compound) inhibits papain with a Ki value of 1 microM, whereas the Ki of 3-(N-acetyl-L-phenylalanyl)aminopropanone (a monocarbonyl compound) is 1.5 mM (M. R. Bendall et al., 1979. Eur. J. Biochem. 79, 201-209). Both carbonyl groups are required for effective inhibition. Extension of these inhibitors by addition of P substituents (e.g., hexyl) does not affect the Ki for papain, but reduces Ki for cathepsin B 33-fold. For these two enzymes slow binding inhibition was observed with slow on rates (kappa on, 5.2 X 10(2) M-1 s-1 for papain, and 2.7 X 10(3) M- s-1 for cathepsin B). Addition of a P3 substituent (glycine) has no effect on Ki. We propose that the mechanism of inhibition involves the formation of a hemithioketal by addition of the active-site thiol to the carbonyl group of the inhibitor closer to the N-terminus. The hemithioketal intermediate is most likely stabilized by the electron withdrawing effect of the second carbonyl group.     

IlvHI locus of Salmonella typhimurium.

In Escherichia coli K-12, the ilvHI locus codes for one of two acetohydroxy acid synthase isoenzymes. A region of the Salmonella typhimurium genome adjacent to the leucine operon was cloned on plasmid pBR322, yielding plasmids pCV47 and pCV49 (a shortened version of pCV47). This region contains DNA homologous to the E. coli ilvHI locus, as judged by hybridization experiments. Plasmid pCV47 did not confer isoleucine-valine prototrophy upon either E. coli or S. typhimurium strains lacking acetohydroxy acid synthase activity, suggesting that S. typhimurium lacks a functional ilvHI locus. However, isoleucine-valine prototrophs were readily isolated from such strains after mutagenesis with nitrosoguanidine. In one case we found that the Ilv+ phenotype resulted from an alteration in bacterial DNA on the plasmid (new plasmid designated pCV50). Furthermore, a new acetohydroxy acid synthase activity was observed in Ilv+ revertants; this enzyme was similar to E. coli acetohydroxy acid synthase III in its lack of activity at low pH. This new activity was correlated with the appearance in minicells of a new polypeptide having an approximate molecular weight of 61,000. Strains carrying either pCV49 or pCV50 produced a substantial amount of ilvHI-specific mRNA. These results, together with results from other laboratories, suggest that S. typhimurium has functional ilvB and ilvG genes and a cryptic ilvHI locus. E. coli K-12, on the other hand, has functional ilvB and ilvHI genes and a cryptic ilvG locus.     

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.