scholarly journals The yvyD Gene of Bacillus subtilis Is under Dual Control of ςB and ςH

1998 ◽  
Vol 180 (24) ◽  
pp. 6674-6680 ◽  
Author(s):  
Kathrin Drzewiecki ◽  
Christine Eymann ◽  
Gerhard Mittenhuber ◽  
Michael Hecker
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Gene Product ◽  
Stress Proteins ◽  
Stringent Response ◽  
Primer Extension ◽  
Wild Type ◽  
Computer Aided ◽  
Protein Gels ◽  
Amino Acid Depletion

ABSTRACT During a search by computer-aided inspection of two-dimensional (2D) protein gels for ςB-dependent general stress proteins exhibiting atypical induction profiles, a protein initially called Hst23 was identified as a product of the yvyD gene of Bacillus subtilis. In addition to the typical ςB-dependent, stress- and starvation-inducible pattern,yvyD is also induced in response to amino acid depletion. By primer extension of RNA isolated from the wild-type strain and appropriate mutants carrying mutations in the sigB and/orspo0H gene, two promoters were mapped upstream of theyvyD gene. The ςB-dependent promoter drives expression of yvyD under stress conditions and after glucose starvation, whereas a ςH-dependent promoter is responsible for yvyD transcription following amino acid limitation. Analysis of Northern blots revealed that yvyDis transcribed monocistronically and confirmed the conclusions drawn from the primer extension experiments. The analysis of the protein synthesis pattern in amino acid-starved wild-type and relAmutant cells showed that the YvyD protein is not synthesized in therelA mutant background. It was concluded that the stringent response plays a role in the activation of ςH. TheyvyD gene product is homologous to a protein which might modify the activity of ς54 in gram-negative bacteria. The expression of a ςL-dependent (ςL is the equivalent of ς54 in B. subtilis)levD-lacZ fusion is upregulated twofold in ayvyD mutant. This indicates that the yvyD gene product, being a member of both the ςB and ςH regulons, might negatively regulate the activity of the ςL regulon. We conclude that (i) systematic, computer-aided analysis of 2D protein gels is appropriate for the identification of genes regulated by multiple transcription factors and that (ii) YvyD might form a junction between the ςB and ςH regulons on one side and the ςL regulon on the other.

Synthese von Hitzeschockproteinen nach einer Aminosäure-und Sauerstofflimitation in Bacillus subtilis relA+-und relA-Stämmen / Synthesis of Heat Shock Proteins during Amino Acid or Oxygen Limitation Bacillus subtilis relA+ and relA

1987 ◽  
Vol 42 (7-8) ◽  
pp. 941-947 ◽  
Author(s):  
M. Hecker ◽  
A. Richter ◽  
A. Schroeter ◽  
L. Wölfel ◽  
F. Mach
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Environmental Conditions ◽  
Stress Proteins ◽  
Stringent Response ◽  
Oxygen Limitation ◽  
Adaptation Mechanism ◽  
Shock Proteins

Some of the presumable heat shock proteins will be produced in Bacillus subtilis in response to different environmental conditions, e.g. heat shock, amino acid limitation or oxygen limitation. During amino acid limitation or during oxygen limitation the relA+ strain is able of synthesizing this set of proteins but the relA strain is not. We suggest that the accelerated rate of the synthesis of some heat shock proteins depends on the induction of the stringent response because the (p)ppGpp production does not occur in the relA strain during amino acid or oxygen limitation. On the other hand the relA strain can produce heat shock proteins under heat stress. Therefore different mechanisms must be responsible for the expression of this set of genes during heat and other stress stimuli. It can be supposed that in B. subtilis the (p)ppGpp-dependent stringent control is a central defense reaction against different adverse environmental conditions and furthermore, that the synthesis of “stress” proteins as an essential component of the stringent response is part of a general adaptation mechanism under non-growing conditions.

scholarly journals Bacillus subtilis Tolerance of Moderate Concentrations of Rifampin Involves the σB-Dependent General and Multiple Stress Response

2002 ◽  
Vol 184 (2) ◽  
pp. 459-467 ◽  
Author(s):  
Julia Elisabeth Bandow ◽  
Heike Brötz ◽  
Michael Hecker
Keyword(s):  
Bacillus Subtilis ◽  
Stress Response ◽  
Rna Polymerase ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Growth Arrest ◽  
Wild Type ◽  
General Stress ◽  
Inhibition Of Growth ◽  
Increased Sensitivity

ABSTRACT Low concentrations of the RNA polymerase inhibitor rifampin added to an exponentially growing culture of Bacillus subtilis led to an instant inhibition of growth. Survival experiments revealed that during the growth arrest the cells became tolerant to the antibiotic and the culture was able to resume growth some time after rifampin treatment. l-[35S]methionine pulse-labeled protein extracts were separated by two-dimensional polyacrylamide gel electrophoresis to investigate the change in the protein synthesis pattern in response to rifampin. The σB-dependent general stress proteins were found to be induced after treatment with the antibiotic. Part of the oxidative stress signature was induced as indicated by the catalase KatA and MrgA. The target protein of rifampin, the β subunit (RpoB) of the DNA-dependent RNA polymerase, and the flagellin protein Hag belonging to the σD regulon were also induced. The rifampin-triggered growth arrest was extended in a sigB mutant in comparison to the wild-type strain, and the higher the concentration, the more pronounced this effect was. Activity of the RsbP energy-signaling phosphatase in the σB signal transduction network was also important for this protection against rifampin, but the RsbU environmental signaling phosphatase was not required. The sigB mutant strain was less capable of growing on rifampin-containing agar plates. When plated from a culture that had already reached stationary phase without previous exposure to the antibiotic during growth, the survival rate of the wild type exceeded that of the sigB mutant by a factor of 100. We conclude that the general stress response of B. subtilis is induced by rifampin depending on RsbP activity and that loss of SigB function causes increased sensitivity to the antibiotic.

scholarly journals Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis

2002 ◽  
Vol 184 (9) ◽  
pp. 2500-2520 ◽  
Author(s):  
Christine Eymann ◽  
Georg Homuth ◽  
Christian Scharf ◽  
Michael Hecker
Keyword(s):  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Profile Analysis ◽  
Bacterial Species ◽  
Stringent Response ◽  
Dependent Manner ◽  
Wild Type ◽  
Stringent Control ◽  
In The Wild ◽  
Growth And Reproduction

ABSTRACT The stringent response in Bacillus subtilis was characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type and relA mutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response to dl-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in the relA mutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknown y-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

scholarly journals The PAL1 gene product is a peroxisomal ATP-binding cassette transporter in the yeast Saccharomyces cerevisiae.

1996 ◽  
Vol 132 (4) ◽  
pp. 549-563 ◽  
Author(s):  
E E Swartzman ◽  
M N Viswanathan ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oleic Acid ◽  
Amino Acid ◽  
Carbon Source ◽  
Amino Acid Sequence ◽  
Gene Product ◽  
Atp Binding ◽  
Wild Type ◽  
Peroxisomal Membrane ◽  
Atp Binding Cassette

The PAL1 gene was isolated using PCR and degenerate oligonucleotide primers corresponding to highly conserved amino acid sequence motifs diagnostic of the ATP-binding cassette domain of the superfamily of membrane-bound transport proteins typified by mammalian multidrug resistance transporter 1 and Saccharomyces cerevisiae Ste6. The deduced PAL1 gene product is similar in length to, has the same predicted topology as, and shares the highest degree of amino acid sequence identity with two human proteins, adrenoleukodystrophy protein and peroxisomal membrane protein (70 kD), which are both presumptive ATP-binding cassette transporters thought to be constituents of the peroxisomal membrane. As judged by hybridization of a PAL1 probe to isolated RNA and by expression of a PAL1-lacZ fusion, a PAL1 transcript was only detectable when cells were grown on oleic acid, a carbon source which requires the biogenesis of functional peroxisomes for its metabolism. A pal1delta mutant grew normally on either glucose- or glycerol-containing media; however, unlike PAL1+ cells (or the pal1delta mutant carrying the PAL1 gene on a plasmid), pal1delta cells were unable to grow on either a solid medium or a liquid medium containing oleic acid as the sole carbon source. Antibodies raised against a chimeric protein in which the COOH-terminal domain of Pal1 was fused to glutathione S-transferase specifically recognized a protein in extracts from wild-type cells only when grown on oleic acid; this species represents the PAL1 gene product because it was missing in pal1delta cells and more abundant in pal1delta cells expressing PAL1 from a multicopy plasmid. The Pal1 polypeptide was highly enriched in the organellar pellet fraction prepared from wild-type cells by differential centrifugation and comigrated upon velocity sedimentation in a Nycodenz gradient with a known component of the peroxisomal matrix, e-oxoacyl-CoA thiolase. As judged by both subcellular fractionation and indirect immunofluorescence, localization of 3-oxoacyl-CoA thiolase to peroxisomes was unchanged whether Pal1 was present, absent, or overexpressed. These findings demonstrate that Pal1 is a peroxisome-specific protein, that it is required for peroxisome function, but that it is not necessary for the biogenesis of peroxisomes or for the import of 3-oxoacyl-CoA thiolase (and at least two other peroxisomal matrix proteins).

scholarly journals Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis

2011 ◽  
Vol 55 (9) ◽  
pp. 4326-4337 ◽  
Author(s):  
Anna-Barbara Hachmann ◽  
Elif Sevim ◽  
Ahmed Gaballa ◽  
David L. Popham ◽  
Haike Antelmann ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Resistance Mechanism ◽  
Point Mutations ◽  
Stringent Response ◽  
Resistant Isolate ◽  
Cross Resistance ◽  
Dependent Manner ◽  
Membrane Composition ◽  
Wild Type ◽  
Content Type

ABSTRACTDaptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca2+-dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, DapR1, from the model bacteriumBacillus subtilis168. DapR1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. DapR1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of DapR1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator genemreB, the stringent response generelA, and the phosphatidylglycerol synthase genepgsA. Genetic reconstruction studies indicated that thepgsA(A64V) allele is primarily responsible for DAP resistance. Allelic replacement with wild-typepgsArestored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca2+-DAP complex.

scholarly journals Inorganic Polyphosphate in Escherichia coli: the Phosphate Regulon and the Stringent Response

1998 ◽  
Vol 180 (8) ◽  
pp. 2186-2193 ◽  
Author(s):  
Narayana N. Rao ◽  
Shengjiang Liu ◽  
Arthur Kornberg
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Response Regulator ◽  
Stringent Response ◽  
Amino Acid Starvation ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Polyp Accumulation ◽  
Phosphate Regulon

ABSTRACT Escherichia coli transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (Pi), in media deficient in both Pi and amino acids. This transient accumulation is preceded by the appearance of nucleotides ppGpp and pppGpp, generated in response to nutritional stresses. Mutants which lack PhoB, the response regulator of the phosphate regulon, do not accumulate polyP even though they develop wild-type levels of (p)ppGpp when subjected to amino acid starvation. When complemented with a phoB-containing plasmid,phoB mutants regain the ability to accumulate polyP. PolyP accumulation requires high levels of (p)ppGpp independent of whether they are generated by RelA (active during the stringent response) or SpoT (expressed during Pi starvation). Hence, accumulation of polyP requires a functional phoB gene and elevated levels of (p)ppGpp. A rapid assay of polyP depends on its adsorption to an anion-exchange disk on which it is hydrolyzed by a yeast exopolyphosphatase.

scholarly journals Accelerated breakdown of reticulocyte protein formed under conditions of amino acid depletion

1978 ◽  
Vol 176 (1) ◽  
pp. 151-158 ◽  
Author(s):  
C S Chandler ◽  
F J Ballard
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Stringent Response ◽  
Molecular Weights ◽  
Protein Inhibition ◽  
Deficient Medium ◽  
Amino Acid Depletion ◽  
Rabbit Reticulocytes

1. Labile protein is formed when rat or rabbit reticulocytes are incubated in medium deficient in individual amino acids, especially histidine, valine or alanine. The fraction of unstable protein is increased to about 35% of the total protein synthesized when the histidinyl-tRNA-charging inhibitor, histidinol, is added to histidine-deficient media. 2. The molecular weights of the labile proteins measured by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of urea are less than haemoglobin and probably represent prematurely terminated haemoglobin chains. 3. Although protein synthesis is always lower under conditions that produce labile protein, inhibition of protein synthesis by fluoride or cycloheximide does not give an effect similar to amino acid depletion. 4. The synthesis of protein in deficient medium does not alter the degradation rate of pre-existing protein in reticulocytes and is thus unrelated to the stringent response in bacteria. 5. We propose that amino acid-deficient medium leads to a decreased charging of the appropriate tRNA, a concomitant decrease in protein synthesis and the degradation of nascent peptides.

scholarly journals Role of the (p)ppGpp Synthase RSH, a RelA/SpoT Homolog, in Stringent Response and Virulence of Staphylococcus aureus

2010 ◽  
Vol 78 (5) ◽  
pp. 1873-1883 ◽  
Author(s):  
Tobias Geiger ◽  
Christiane Goerke ◽  
Michaela Fritz ◽  
Tina Schäfer ◽  
Knut Ohlsen ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Acid Metabolism ◽  
Stringent Response ◽  
Essential Amino Acids ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Stringent Control ◽  
Nutrient Limitations

ABSTRACT In most bacteria, nutrient limitations provoke the stringent control through the rapid synthesis of the alarmones pppGpp and ppGpp. Little is known about the stringent control in the human pathogen Staphylococcus aureus, partly due to the essentiality of the major (p)ppGpp synthase/hydrolase enzyme RSH (RelA/SpoT homolog). Here, we show that mutants defective only in the synthase domain of RSH (rsh syn) are not impaired in growth under nutrient-rich conditions. However, these mutants were more sensitive toward mupirocin and were impaired in survival when essential amino acids were depleted from the medium. RSH is the major enzyme responsible for (p)ppGpp synthesis in response to amino acid deprivation (lack of Leu/Val) or mupirocin treatment. Transcriptional analysis showed that the RSH-dependent stringent control in S. aureus is characterized by repression of genes whose products are predicted to be involved in the translation machinery and by upregulation of genes coding for enzymes involved in amino acid metabolism and transport which are controlled by the repressor CodY. Amino acid starvation also provoked stabilization of the RNAs coding for major virulence regulators, such as SaeRS and SarA, independently of RSH. In an animal model, the rsh syn mutant was shown to be less virulent than the wild type. Virulence could be restored by the introduction of a codY mutation into the rsh syn mutant. These results indicate that stringent conditions are present during infection and that RSH-dependent derepression of CodY-regulated genes is essential for virulence in S. aureus.

scholarly journals Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium, Thermus thermophilus

2006 ◽  
Vol 188 (20) ◽  
pp. 7111-7122 ◽  
Author(s):  
Koji Kasai ◽  
Tomoyasu Nishizawa ◽  
Kosaku Takahashi ◽  
Takeshi Hosaka ◽  
Hiroyuki Aoki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Polymerase ◽  
Thermus Thermophilus ◽  
Stringent Response ◽  
Polymerase Activity ◽  
Thermophilic Bacterium ◽  
Rrna Gene ◽  
Wild Type ◽  
Rna Polymerase Activity

ABSTRACT Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel +; wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNASer aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

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