Identification and characterization of a relA-dependent starvation-inducible locus (sin) in Salmonella typhimurium

By use of Mu cts d1(Ap lac) phage, a strain of Salmonella typhimurium was isolated containing a Mu d insertion in a locus (sinA) which is induced during nicotinate, thiamine, purine, amino acid, phosphate, and carbon starvation conditions. Depending on the starvation condition, a 2- to 10-fold increase in beta-galactosidase activity was demonstrated. The sinA locus, which mapped at 32 U, became induced after a decline in growth rate due to starvation. The introduction of relA into the sinA-lac strain prevented induction by nicotinate starvation and partially prevented induction by phosphate starvation. The data suggest that sinA responds to changes in growth rate due to various nutrient starvation conditions and probably responds in part to changes in guanosine tetraphosphate levels.     

Flow cytometric analysis of the cellular DNA content of Salmonella typhimurium and Alteromonas haloplanktis during starvation and recovery in seawater.

Flow cytometry was used to investigate the heterogeneity of the DNA content of Salmonella typhimurium and Alteromonas haloplanktis cells that were starved and allowed to recover in seawater. Hoechst 33342 (bisbenzimide) was used as a DNA-specific dye to discriminate between DNA subpopulations. The DNA contents of both strains were heterogeneous during starvation. S. typhimurium cells contained one or two genomes, and A. haloplanktis cells contained up to six genomes. S. typhimurium genomes were fully replicated at the onset of starvation. Each replication cycle was completed in the early stage of starvation for A. haloplanktis by stopping cells in the partition step of the cell cycle prior to division. Multigenomic marine cells can undergo rapid cell division without DNA synthesis upon recovery, resulting in large fluctuations in the DNA contents of individual cells. In contrast, the heterogeneity of the DNA distribution of S. typhimurium cells was preserved during recovery. The fluctuations in the DNA fluorescence of this strain seem to be due to topological changes in DNA. Flow cytometry may provide a new approach to understanding dynamic and physiological changes in bacteria by detecting cellular heterogeneity in response to different growth conditions.     

Growth of Coliphage T7 in Salmonella typhimurium

A mutant of Salmonella typhimurium was found to be sensitive to killing by coliphage T7 because of an alteration in its surface properties. However, the infections were abortive and studies with (32)P-labeled T7 grown in Escherichia coli B (T7.B) indicated that the phage DNA was restricted by S. typhimurium. When a mutant T7 which survived the restriction and produced plaques on Salmonella (T7.S) was passed through one cycle of growth in E. coli B, its ability to grow in Salmonella was lost, indicating that host-controlled restriction and modification are operative in this system. Restrictionless S. typhimurium mutants were isolated that permit the growth of not only T7.S but also T7.B and coliphage T3. The physiology of T7 production in the restrictionless host is nearly identical to that in Escherichia coli.     

Ultrastructural organization of Salmonella typhimurium cells during long-term starvation and transfer to an unculturable state

Electron microscopic and immunocytochemical studies of Salmonella typhimurium culture were carried out under conditions of cell transfer into an unculturable state induced by carbon, phosphorus, and nitrogen starvation. Morphological variants of bacterial cells were detected in the course of cell culturing under conditions of starvation. Electron microscopy showed that O-antigen was retained in salmonella after long starvation and transfer into an unculturable state.     

Role of protein degradation in the survival of carbon-starved Escherichia coli and Salmonella typhimurium.

When an Escherichia coli K-12 culture was starved for glucose, 50% of the cells lost viability in about 6 days. When a K-12 mutant lacking five distinct peptidase activities, CM89, was starved in the same manner, viability was lost much more rapidly; 50% of the cells lost viability in about 2 days, whereas a parent strain lacking only one peptidase activity lost 50% viability in about 4 days. Compared with the wild-type strain and with its parent strain CM17, CM89 was defective in both protein degradation and protein synthesis during carbon starvation. Similar results were obtained with glucose-starved Salmonella typhimurium LT2 and LT2-derived mutants lacking various peptidase activities. An S. typhimurium mutant lacking four peptidases, TN852, which was deficient in both protein degradation and synthesis during carbon starvation (Yen et al., J. Mol. Biol. 143:21-33, 1980), was roughly one-third as stable as the isogenic wild type. Isogenic S. typhimurium strains that lacked various combinations of three of four peptidases and that displayed protein degradation and synthesis rates intermediate between those of LT2 and TN852 (Yen et al., J. Mol. Biol. 143:21-33, 1980) displayed corresponding stabilities during carbon starvation. These results point to a role for protein degradation in the survival of bacteria during starvation for carbon.     

Enzyme Alterations in Tyrosine and Phenylalanine Auxotrophs of Salmonella typhimurium

The enzyme activities specified by the tyrA and pheA genes were studied in wildtype strain Salmonella typhimurium and in phenylalanine and tyrosine auxotrophs. As in Aerobacter aerogenes and Escherichia coli, the wild-type enzymes of Salmonella catalyze two consecutive reactions: chorismate --> prephenate --> 4-hydroxy-phenylpyruvate (tyrA), and chorismate --> prephenate --> phenylpyruvate (pheA). A group of tyrA mutants capable of interallelic complementation had altered enzymes which retained chorismate mutase T activity but lacked prephenate dehydrogenase. Similarly, pheA mutants (in which interallelic complementation does not occur) had one group with altered enzymes which retained chorismate mutase P but lacked prephenate dehydratase. Tyrosine and phenylalanine auxotrophs outside of these categories showed loss of both activities of their respective bifunctional enzyme. TyrA mutants which had mutase T were considerably derepressed in this activity by tyrosine starvation and consequently excreted prephenate. A new and specific procedure was developed for assaying prephenate dehydrogenase activity.     

Mutants of Salmonella typhimurium deficient in an endoprotease.

Three bands of hydrolytic activity toward the chromogenic protease substrate N-acetyl-DL-phenylalanine beta-naphthyl ester (NAPNE) can be observed after gel electrophoresis of crude extracts of Salmonella typhimurium or Escherichia coli. Mutants deficient in one of these three activities have been isolated using a staining procedure that identifies colonies that show reduced ability to hydrolyze NAPNE. These mutants lack the strongest of the three bands of activity. The Salmonella mutations (designated apeA) are all co-transducible with purE, and the order (pro)-apeA-Hfr K17 origin-purE has been established. Strains carrying apeA mutations have wild-type doubling times. None of the apeA mutants isolated gains an auxotrophic requirement as a result of loss of the apeA gene product. The rates and extents of protein degradation during starvation for a carbon source or during growth after exposure to the amino acid analogue canavanine do not seem to be affected by apeA mutations. Revertants of apeA mutations (selected by screening for clones that have regained the ability to hydrolyze NAPNE) frequently contain a new enzymatic activity not found in wild-type cells.     

Global control in Salmonella typhimurium: two-dimensional electrophoretic analysis of starvation-, anaerobiosis-, and heat shock-inducible proteins.

The response of Salmonella typhimurium to various forms of environmental stress was examined by using O'Farrell two-dimensional gel electrophoresis. Polypeptides (a total of 110) which quantitatively increased during various starvations, anaerobiosis, or heat shock were identified and cataloged in reference to a standard polypeptide map. Although significant overlap was noted during comparison of proteins induced by different starvations, only a few proteins produced during heat shock or anaerobiosis were also identified as starvation inducible.     

Stabilization of a HemA-LacZ hybrid protein against proteolysis during carbon starvation in atp mutants of Salmonella typhimurium.

Transposon insertions that stabilize the beta-galactosidase activity of a HemA-LacZ hybrid protein following carbon starvation were mapped to the atp operon of Salmonella typhimurium. This effect is similar to that seen with nuo mutants defective in the energy-conserving type I NADH dehydrogenase. Insertions in several other genes, including such highly pleiotropic mutants as rpoS, polA, and hfq, were isolated with the same phenotypic screen, but they do not affect the beta-galactosidase activity of HemA-LacZ. All of these mutants act indirectly to alter the colony color of many different fusion strains on indicator plates.     

Inhibition by antibiotics of the bacterial response to long-term starvation of Salmonella typhimurium and the colon microbiota of mice

The number of viable cells of two strains of Salmonella typhimurium and the number of viable cells and the cell size of the colon microbiota of mice were examined during non-growing conditions after exposure to antibiotics with known modes of action. Salmonella typhimurium starved for 1, 2, 4, 5, 12 and 20 d in a phosphate buffer saline solution and subsequently exposed for 2 and 6 h showed the following characteristics. The protein synthesis inhibitors gentamicin and tetracycline, the RNA synthesis inhibitor rifampicin and the membrane potential inhibitor polymyxin all impaired survival of starved cells. The reduction in the number of viable cells caused by the addition of gentamicin, rifampicin and polymyxin was generally more pronounced with extended exposure to energy and nutrient deprivation. Both 2- and 6-h exposure of tetracycline, however, had diminishing inhibitory effects after 20 d compared with 5 d of starvation. Control experiments to verify non-growing conditions in the starvation regime showed that DNA and cell wall synthesis inhibitors had no inhibitory effect after 24-h starvation. The rough mutant strain displayed a lower sensitivity to a hydrophobic rather than a hydrophilic inhibitor as compared to the smooth wild-type strain. The cell size reduction but not viability was partly prevented by protein synthesis inhibitors as seen for both in vivo and in vitro colon microbiota studies.     

Carbon starvation of Salmonella typhimurium does not cause a general increase of mutation rates.

Mutation rates in bacteria can vary depending on the genetic target studied and the specific growth conditions of the cells. Here, two different methods were used to determine how rates of mutation to antibiotic resistance, auxotrophy, and prototrophy were influenced by carbon starvation on agar plates. The rate of mutation to rifampin resistance was increased by starvation as measured by fluctuation tests, similar to what has been reported previously for Escherichia coli. In contrast, the rates of mutation to various types of auxotrophy were unaffected or decreased as measured by both fluctuation tests and a repeated-streaking procedure. Similarly, the rates of reversion to prototrophy of his and lac nonsense and missense mutations were unaffected by starvation. Thus, mutation rates of different genetic targets can be affected differently by starvation and we conclude that carbon starvation is not generally mutagenic in Salmonella typhimurium.     

Inhibition by antibiotics of the bacterial response to long-term starvation of Salmonella typhimurium and the colon microbiota of mice

The number of viable cells of two strains of Salmonella typhimurium and the number of viable cells and the cell size of the colon microbiota of mice were examined during non-growing conditions after exposure to antibiotics with known modes of action. Salmonella typhimurium starved for 1, 2, 4, 5, 12 and 20 d in a phosphate buffer saline solution and subsequently exposed for 2 and 6 h showed the following characteristics. The protein synthesis inhibitors gentamicin and tetracycline, the RNA synthesis inhibitor rifampicin and the membrane potential inhibitor polymyxin all impaired survival of starved cells. The reduction in the number of viable cells caused by the addition of gentamicin, rifampicin and polymyxin was generally more pronounced with extended exposure to energy and nutrient deprivation. Both 2- and 6-h exposure of tetracycline, however, had diminishing inhibitory effects after 20 d compared with 5 d of starvation. Control experiments to verify non-growing conditions in the starvation regime showed that DNA and cell wall synthesis inhibitors had no inhibitory effect after 24-h starvation. The rough mutant strain displayed a lower sensitivity to a hydrophobic rather than a hydrophilic inhibitor as compared to the smooth wild-type strain. The cell size reduction but not viability was partly prevented by protein synthesis inhibitors as seen for both in vivo and in vitro colon microbiota studies.     

Heterogeneity of the Stability of Messenger Ribonucleic Acid in Salmonella typhimurium

Cells of Salmonella typhimurium strain SL 282, deflagellated by mechanical shear, regenerated their flagella in the absence of tryptophan, an amino acid required for growth but not found in flagellin. Ribonucleic acid (RNA) synthesis was severely inhibited by tryptophan starvation. These findings suggested that the messenger RNA (mRNA) for flagellin might be stable. Actinomycin D was used to inhibit RNA synthesis in ethylenediaminetetraacetate-treated bacteria. The introduction of an F(lac) episome into strain SL 282 permitted the simultaneous study of the synthesis of flagellin, beta-galactosidase, and total protein. In the actinomycin-treated bacteria protein and beta-galactosidase syntheses were inhibited by 90%, whereas flagellin synthesis was unaffected. We conclude that the mRNA for flagellin synthesis is stable and that species of mRNA vary with respect to metabolic stability in S. typhimurium.     

Phosphate starvation regulon of Salmonella typhimurium.

Several phosphate-starvation-inducible (psi) genetic loci in Salmonella typhimurium were identified by fusing the lacZ gene to psi promoters by using the Mu d1 and Mu d1-8 bacteriophages. Although several different starvation conditions were examined, the psi loci responded solely to phosphate deprivation. A regulatory locus, psiR, was identified as controlling the psiC locus. The psiR locus did not affect the expression of the Escherichia coli phoA locus or any of the other psi loci described.     

Gene expression in response to multiple nutrient-starvation conditions in Salmonella typhimurium

Abstract In nature, bacteria encounter a variety of environmental conditions, among the most frequent of these is the limitation or starvation of one or more essential nutrients. It is reasonable to assume, therefore, that bacteria have evolved mechanisms to enhance their survival over prolonged periods of nutrient starvation. We have identified eight genetic loci in the enteropathogen Salmonella typhimurium , using Mu d -directed lacZ operon fusion technology, that were induced in response to two or more different starvation conditions (sti) . In simultaneous studies, we also identified genetic loci, using Mu d-lac fusions, which respond to only phosphate-starvation conditions (psi) . We further characterized these loci as to their induction-characteristics, kinetics of induction, expression during growth on different carbon sources, and approximate location on the S. typhimurium genetic map. In concurrent studies, we analyzed whole cell extracts of S. typhimurium grown under a variety of nutrients oxydation conditions as well as under nonlimiting conditions, using two-dimensional polyacrylamide gel electrophoresis. Results from these studies correlated well with our gene fusion studies. In more recent studies, we have demonstrated a complex genetic regulation of a number of these starvation-inducible loci, and have implicated at least four of these loci in the long term starvation-survival of S. typhimurium .     

Regulation and Mechanism of Phosphoribosylpyrophosphate Synthetase: Repression by End Products

Phosphoribosylpyrophosphate (PRPP) synthetase participates in the biosynthesis in bacteria of purine nucleotides, pyrimidine nucleotides, tryptophan, and histidine. The regulation of the synthesis of PRPP synthetase in Salmonella typhimurium was studied. Addition of end products to the growth medium, singly or in combination, resulted in small decreases in the specific activity of PRPP synthetase, but levels of the enzyme were never decreased to less than half of those found when the bacteria were grown on minimal medium. Growth of the bacteria on several different carbon sources or starvation for phosphate had little effect on the specific activity of PRPP synthetase. Over-production of histidine in a histidine regulatory mutant, which would be expected to result in a depletion of intracellular PRPP pools, did not alter PRPP synthetase specific activity. PRPP synthetase levels were examined in auxotrophic strains of S. typhimurium that had been starved for the end products of PRPP. In each case derepression of an enzyme in the biosynthetic pathway for the limiting end product was demonstrated. However, only alterations in the levels of pyrimidine bases in the culture medium brought about derepression and repression of PRPP synthetase. Excess pyrimidines do not completely repress the enzyme. Deprivation of exponentially growing cells for pyrimidines by growth of an auxotrophic mutant on media containing orotic acid, which enters the cells slowly, resulted in a 10-fold derepression of PRPP synthetase. Derepression of PRPP synthetase during uracil starvation was prevented by chloramphenicol. The PRPP synthetase activities of extracts from repressed and derepressed cells responded in identical fashion to heat inactivation, cellulose acetate electrophoresis at several pH values, and in kinetic experiments.     

Comparison of methods for specific depletion of ATP in Salmonella typhimurium.

Three methods of ATP depletion in Salmonella typhimurium were compared. ATP concentrations were lowest after arsenate treatment. Arsenate or alpha-methylglucoside-plus-azide treatment nonspecifically lowered all nucleotide triphosphate levels. Histidine starvation in a hisF mutant was relatively specific for ATP depletion and therefore has potential in distinguishing ATP-dependent processes from processes dependent on other nucleotides.     

Genetic activity of allyl chloride.

Allyl chloride (3-chloroprene) is mutagenic for Salmonella typhimurium and it induces gene conversions in Saccharomyces cerevisiae. It also displays DNA-modifying activity for E. coli. This is in contrast to a recent study which reported its lack of genetic activity for Salmonella typhimurium.