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.     

Nucleotide pool in pho regulon mutants and alkaline phosphatase synthesis in Escherichia coli.

The intracellular nucleotide pool of Escherichia coli W3110 reproducibly changes from conditions of growth in phosphate excess to phosphate starvation, with at least two nucleotides appearing under starvation conditions and two nucleotides appearing only under excess phosphate conditions. Strains bearing a deletion of the phoA gene show the same pattern, indicating that dephosphorylation by alkaline phosphatase is not responsible for the changes. Strains with mutations in the phoU gene, which result in constitutive expression of the pho regulon, show the nucleotide pattern of phosphate-starved cells even during phosphate excess growth. These changes in nucleotides are therefore due to phoU mutation but not to alkaline phosphatase constitutivity. In fact, a phoR (phoR68) mutant strain has the patterns of the wild type in spite of being constitutive for alkaline phosphatase. That these nucleotides might be specific signals for pho regulon expression was supported by the fact that the two nucleotides appearing under phosphate starvation induced the synthesis of alkaline phosphatase in repressed permeabilized wild-type cells under conditions of phosphate excess.     

Oxygen regulation in Salmonella typhimurium

Regulation by oxygen of the peptidase T (pepT) locus of Salmonella typhimurium was studied by measuring beta-galactosidase levels in strains containing a pepT::Mu d1(Apr lac) operon fusion. beta-Galactosidase was induced in anaerobic cultures and late-exponential and stationary-phase aerated cultures. Peptidase T activity also was induced under these growth conditions. pepT+ but not pepT strains will utilize as amino acid sources the tripeptides Leu-Leu-Leu and Leu-Gly-Gly only when grown anaerobically. Mutations at two loci, oxrA and oxrB (oxygen regulation) prevent induction of the pepT locus. The oxrA locus is homologous to the fnr locus of Escherichia coli. We have isolated 12 independent Mu d1 insertions (oxd::Mu d1, oxygen dependent) that show induction of beta-galactosidase in anaerobic cultures and stationary-phase aerated cultures. These insertions fall into nine classes based on map location. All of the oxd::Mu d1 insertions are regulated by oxrA and oxrB and therefore define a global regulon that responds to oxygen limitation.     

Thymineless bacteriophage induction in Staphylococcus aureus. I. High-frequency transduction with lysates containing a bacteriophage related to bacteriophage phi 11.

A thymine-requiring mutant of Staphylococcus aureus, strain 8325 (PI258)thy, undergoes prophage induction and lysis after thymine starvation. Four different phages were isolated from the lysate in low titers, among which was a phage designated phi 14, which differs from phage phi 11 in its immunity locus. The thymineless induced lysates of strain 8325(PI258)thy transduce the penicillinase plasmid at high frequency (10(-1), whereas transduction of chromosomal markers is inefficient. A plasmic-cured derivative of strain 8325(PI258)thy is also lysed by thymine starvation and be used for high-frequency transduction of other plasmids. Reconstitution of a strain of S. aureus that responds to thymine starvation was only partially successful, but this system can effectively be used to transduce plasmids or plasmid derivatives.     

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 .     

Response of Thiobacillus ferrooxidans to phosphate limitation

Abstract: Phosphate ion is an essential nutrient for all cells. Consequently, starvation for this component may constitute a stressing condition which affects the bioleaching capacity of the biomining microorganisms. Therefore, we have studied the manner in which the chemolithotroph Thiobacillus ferrooxidans responds to phosphate limitation. Under these circumstances the bacteria reduced its growth rate, capacity to oxidize ferrous iron and to fix CO,. Concomitant with these changes, the cells showed an increased synthesis of several proteins, some of which were exclusively synthesized during phosphate starvation. When intact cells grown in the absence of phosphate were labelled with ¹²⁵I, several proteins were iodinated in addition of those observed in control cells, suggesting that the lack of phosphate induces some proteins located in the membranes or the periplasmic space of the bacteria. It is expected that by measuring the levels of expression of some of the proteins induced by the shortage of phosphorus, it might be possible to estimate in situ the relative physiological condition of the bacteria in a given bioleaching operation.     

Responses to nutrient starvation in Pseudomonas putida KT2442: analysis of general cross-protection, cell shape, and macromolecular content.

The physiology of Pseudomonas putida KT2442 with respect to growth and carbon starvation was studied. During the transition from growth to nongrowth, the cell shape changes from cylindrical to spheric, a change which is accompanied by reductions in cell size, DNA and ribosome content, and the rate of total protein synthesis. In addition, a pattern of general cross-protection develops, which enables the cells to survive environmental stresses such as high and low temperatures, elevated osmolarity, solvents, and oxidative agents. Cultures are almost fully viable during 1 month of carbon, nitrogen, and multiple-nutrient starvation and are considered to be in an active nondormant state. In contrast, strain KT2442 does not survive well under conditions of sulfate and phosphate starvation.     

Complex regulation of two target genes encoding SPX-MFS proteins by rice miR827 in response to phosphate starvation

Here we report on the characterization of rice osa-miR827 and its two target genes, OsSPX-MFS1 and OsSPX-MFS2, which encode SPX-MFS proteins predicted to be implicated in phosphate (Pi) sensing or transport. We first show by Northern blot analysis that osa-miR827 is strongly induced by Pi starvation in both shoots and roots. Hybridization of osa-miR827 in situ confirms its strong induction by Pi starvation, with signals concentrated in mesophyll, epidermis and ground tissues of roots. In parallel, we analyzed the responses of the two OsSPX-MFS1 and OsSPX-MFS2 gene targets to Pi starvation. OsSPX-MFS1 mRNA is mainly expressed in shoots under sufficient Pi supply while its expression is reduced on Pi starvation, revealing a direct relationship between induction of osa-miR827 and down-regulation of OsSPX-MFS1. In contrast, OsSPX-MFS2 responds in a diametrically opposed manner to Pi starvation. The accumulation of OsSPX-MFS2 mRNA is dramatically enhanced under Pi starvation, suggesting the involvement of complex regulation of osa-miR827 and its two target genes. We further produced transgenic rice lines overexpressing osa-miR827 and T-DNA knockout mutant lines in which the expression of osa-miR827 is abolished. Compared with wild-type controls, both target mRNAs exhibit similar changes, their expression being reduced and increased in overexpressing and knockout lines, respectively. This suggests that OsSPX-MFS1 and OsSPX-MFS2 are both negatively regulated by osa-miR827 abundance although they respond differently to external Pi conditions. We propose that this is a complex mechanism comprising fine tuning of spatial or temporal regulation of both targets by osa-miR827.     

Cephamycin C production is regulated by relA and rsh genes in Streptomyces clavuligerus ATCC27064

The effects of growth rate and nutrient uptake rate on the production of cephamycin C were determined in the parental strain, deltarelA mutant, and deltarsh null mutant of S. clavuligerus. Production of cephamycin C was inversely related to mycelium growth and the phosphate feed rate was more critical for the production of cephamycin C. On the contrary, the production of cephamycin C was completely abolished in the deltarelA mutant, but not in deltarsh mutant. The changes in the cephamycin C production by disruption of the relA and rsh genes are presumably associated with the consequent ability of the mutants to accumulate (p)ppGpp under nutrient starvation. Therefore, it is concluded that the stringent response of S. clavuligerus to starvation for nutrients is governed mainly by RelA rather than Rsh and that the response is more apparently regulated by the limitation of phosphate.     

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.     

Guanosine pentaphosphate and guanosine tetraphosphate accumulation and induction of Myxococcus xanthus fruiting body development

Development of multicellular fruiting bodies of Myxococcus xanthus can be induced by limitation of any of a number of different classes of amino acids. Investigated were amino acids that wild-type strains of M. xanthus are unable to synthesize (isoleucine, leucine, and valine), can synthesize at a low rate (phenylalanine), or can normally synthesize at an adequate rate (tryptophan and serine). In general, gradual rather than abrupt starvation for an essential amino acid was required for the induction of fruiting. Perhaps gradual starvation in general minimizes antagonism between amino acids present in the medium, as was documented for valine starvation. The previously reported induction of fruiting by a high concentration of threonine was shown to be specifically reversed by lysine. Threonine addition may starve cells for lysine by feedback inhibition of aspartokinase activity. Starvation for carbon-energy sources or inorganic phosphate also induced fruiting. As in other bacteria, amino acid starvation of M. xanthus leads to increases in cellular guanosine polyphosphate, usually consisting of large increases in the amount of guanosine pentaphosphate with smaller increases in the level of guanosine tetraphosphate. Guanosine polyphosphate accumulation is thus shown to be correlated with nutritional conditions that induce fruiting, and therefore may serve as an intracellular signal to trigger cells to end vegetative growth and initiate fruiting body development.     

OsCYCP4s coordinate phosphate starvation signaling with cell cycle progression in rice

Phosphate starvation leads to a strong reduction in shoot growth and yield in crops. The reduced shoot growth is caused by extensive gene expression reprogramming triggered by phosphate deficiency, which is not itself a direct consequence of low levels of shoot phosphorus. However, how phosphate starvation inhibits shoot growth in rice is still unclear. In this study, we determined the role of OsCYCP4s in the regulation of shoot growth in response to phosphate starvation in rice. We demonstrate that the expression levels of OsCYCP4s, except OsCYCP4;3, were induced by phosphate starvation. Overexpression of the phosphate starvation induced OsCYCP4s could compete with the other cyclins for the binding with cyclin‐dependent kinases, therefore suppressing growth by reducing cell proliferation. The phosphate starvation induced growth inhibition in the loss‐of‐function mutants cycp4;1, cycp4;2, and cycp4;4 is partially compromised. Furthermore, the expression of some phosphate starvation inducible genes is negatively modulated by these cyclins, which indicates that these OsCYCP4s may also be involved in phosphate starvation signaling. We conclude that phosphate starvation induced OsCYCP4s might coordinate phosphate starvation signaling and cell cycle progression under phosphate starvation stress.     

Identification of a nodD-dependent locus in the Rhizobium strain NGR234 activated by phenolic factors secreted by soybeans and other legumes

Transfer of the strain NGR234nodD 1 gene into the narrow host range R. trifolii strain ANU843 on either a 6.7-kb HindIII or 17-kb XhoI fragment broadens the host range of this bacterium to include the tropical legumes Vigna unguiculata, Glycine ussuriensis, Leucaena leucocephala, and siratro (Macroptilium atropurpureum). Contrary to previous data (Bassam et al. 1986), mutagenesis of the 17-kb XhoI fragment with a mini-Mu lac transposon (Mu dII1734) showed that a functional nodD 1 gene was essential for extended host range. Gene expression studies using both Mu dII1734 fusions and a promoter-cloning vector indicated that several loci, including the nodD 1 gene, are constitutively expressed. No evidence was found for regulation of the strain NGR234 nodD 1 gene by its product. Another locus nod-81, was induced only in the presence of exudates from various plant species, including soybean (Glycine max). Whereas the expression of nod-81 was dependent on the presence of a functional nodD 1 gene product, a regulatory nod-box DNA sequence was not detected 5' to this gene by using available oligonucleotide hybridization probes. The nod-81 locus was induced by genistein, daidzein, naringenin, and coumestrol from both cotyledon and root tissue of freshly germinated soybean seedlings. A broad spectrum of commercially available phenolic compounds stimulated induction of the nod-81 locus, including some that antagonize nod gene induction in other Rhizobium species. The nodD 1 gene product from strain NGR234 was shown to determine the spectrum of compounds that induce nod-81 expression.     

Survival, stress resistance, and alterations in protein expression in the marine vibrio sp. strain S14 during starvation for different individual nutrients.

The response of the marine Vibrio sp. strain S14 to starvation for carbon, nitrogen, or phosphorus and to simultaneous depletion of all these nutrients (multiple-nutrient starvation) was examined with respect to survival, stress resistance, quantitative and qualitative alterations in protein and RNA synthesis, and the induction of the stringent control. Of the conditions tested, carbon starvation and multiple-nutrient starvation both promoted long-term starvation resistance and a rapid induction of the stringent control, as deduced from the kinetics of RNA synthesis. Carbon- and multiple-nutrient-starved cells were also found to become increasingly resistant to heat, UV, near-UV, and CdCl2 stress. Nitrogen- and phosphorus-starved cells demonstrated a poor ability to survive in the presence of carbon and did not develop a marked resistance to the stresses examined. The carbon, nitrogen, and phosphorus starvation stimulons consisted of about 20 proteins each, while simultaneous starvation for all the nutrients elicited an increased synthesis of 42 polypeptides. Nine common proteins were found to be induced regardless of the starvation condition used and were tentatively termed general starvation proteins. It was also demonstrated that the total number of proteins induced in response to multiple-nutrient starvation was not a predictable sum of the different individual starvation stimulons. Multiple-nutrient starvation induced 14 proteins which were not detected at increased levels of expression in response to individual starvation conditions. Furthermore, four out of five phosphorus starvation-specific polypeptides were not induced during simultaneous starvation for phosphorus, nitrogen, and carbon. The results are discussed in light of the physiological alterations previously described for Vibrio sp. strain S14 cells starved for carbon, nitrogen, and phosphorus simultaneously.