Cloning and characterization of a relA/spoT homologue from Bacillus subtilis

Wendrich, Thomas M.; Marahiel, Mohamed A.

doi:10.1046/j.1365-2958.1997.5511919.x

Cited by 148 publications

(140 citation statements)

References 62 publications

(65 reference statements)

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“…1F). These requirements are far more stringent than those of ⌬relA cells, which have only a strong requirement for valine and modest requirements for leucine, isoleucine, and methionine (9).…”

Section: Resultsmentioning

confidence: 99%

“…In other bacteria, (p)ppGpp is also required for viability in the absence of amino acids. In the Gram-positive bacterium Bacillus subtilis, (p)ppGpp is synthesized by three enzymes: RelA, YjbM, and YwaC (9)(10)(11). Deleting all three (p)ppGpp synthetase genes results in cells that do not form colonies on minimal medium containing glucose but form colonies efficiently when all 20 amino acids are added, confirming that these cells are auxotrophic for certain amino acids (12).…”

mentioning

confidence: 90%

“…Deleting all three (p)ppGpp synthetase genes results in cells that do not form colonies on minimal medium containing glucose but form colonies efficiently when all 20 amino acids are added, confirming that these cells are auxotrophic for certain amino acids (12). While deletion of relA alone was shown to render B. subtilis cell growth strongly dependent on externally supplemented valine and weakly dependent on leucine, isoleucine, and methionine (9), the precise auxotrophic requirements of (p)ppGpp 0 cells have not been characterized. (p)ppGpp 0 cells have high levels of GTP, and the inability of (p)ppGpp 0 cells to form colonies on minimal medium can be suppressed by mutations in guaA, guaB, and gmk, which encode enzymes in the GTP biosynthesis pathway, and by mutations in codY, which encodes the global transcriptional regulator CodY (12).…”

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confidence: 95%

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GTP Dysregulation in Bacillus subtilis Cells Lacking (p)ppGpp Results in Phenotypic Amino Acid Auxotrophy and Failure To Adapt to Nutrient Downshift and Regulate Biosynthesis Genes

Kriel

Brinsmade

Tse

et al. 2013

Journal of Bacteriology

125

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The nucleotide (p)ppGpp inhibits GTP biosynthesis in the Gram-positive bacterium Bacillus subtilis. Here we examined how this regulation allows cells to grow in the absence of amino acids. We showed that B. subtilis cells lacking (p)ppGpp, due to either deletions or point mutations in all three (p)ppGpp synthetase genes, yjbM, ywaC, and relA, strongly require supplementation of leucine, isoleucine, valine, methionine, and threonine and modestly require three additional amino acids. This polyauxotrophy is rescued by reducing GTP levels. Reduction of GTP levels activates transcription of genes responsible for the biosynthesis of the five strongly required amino acids by inactivating the transcription factor CodY, which represses the ybgE, ilvD, ilvBHC-leuABCD, ilvA, ywaA, and hom-thrCB operons, and by a CodY-independent activation of transcription of the ilvA, ywaA, hom-thrCB, and metE operons. Interestingly, providing the eight required amino acids does not allow for colony formation of (p)ppGpp 0 cells when transitioning from amino acid-replete medium to amino acid-limiting medium, and we found that this is due to an additional role that (p)ppGpp plays in protecting cells during nutrient downshifts. We conclude that (p)ppGpp allows adaptation to amino acid limitation by a combined effect of preventing death during metabolic transitions and sustaining growth by activating amino acid biosynthesis. This ability of (p)ppGpp to integrate a general stress response with a targeted reprogramming of gene regulation allows appropriate adaptation and is likely conserved among diverse bacteria.

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Section: Resultsmentioning

confidence: 99%

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confidence: 90%

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confidence: 95%

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GTP Dysregulation in Bacillus subtilis Cells Lacking (p)ppGpp Results in Phenotypic Amino Acid Auxotrophy and Failure To Adapt to Nutrient Downshift and Regulate Biosynthesis Genes

Kriel

Brinsmade

Tse

et al. 2013

Journal of Bacteriology

125

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“…Activation of the SigmaB regulon results in the induction of general stress proteins and a variety of other proteins essential to the stress response. The SigmaB regulon has been well-characterized in many bacteria, but is incompletely understood in Streptococcus (33,34). We postulate that increased expression of RsbU represents an attempt by S. pyogenes to activate the SigmaB stress response to cope with the effects of fluoride.…”

Section: Effect Of Fluoride On Protein Expression In S Pyogenes Cultmentioning

confidence: 99%

Fluoride Exposure Attenuates Expression of Streptococcus pyogenes Virulence Factors

Thongboonkerd¹,

Luengpailin²,

Cao³

et al. 2002

Journal of Biological Chemistry

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Fluoridation causes an obvious reduction of dental caries by interference with cariogenic streptococci. However, the effect of fluoride on group A streptococci that causes rheumatic fever and acute poststreptococcal glomerulonephritis is not known. We have used proteomic analysis to create a reference proteome map for Streptococcus pyogenes and to determine fluoride-induced protein changes in the streptococci. Cellular and extracellular proteins were resolved by two-dimensional polyacrylamide gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. 183 protein spots were visualized, and 74 spots representing 60 unique proteins were identified. A 16-h exposure to sodium fluoride caused decreased expression of proteins required to respond to cellular stress, including anti-oxidants, glycolytic enzymes, transcriptional and translational regulators, and protein folding. Fluoride caused decreased cellular expression of two well-characterized S. pyogenes virulence factors. Fluoride decreased expression of glyceraldehyde-3-phosphate dehydrogenase, which acts to bind fibronectin and promote bacterial adherence. We also performed proteomic analysis of protein released by S. pyogenes into the culture supernatant and observed decreased expression of M proteins following fluoride exposure. These data provide evidence that fluoride causes decreased expression by S. pyogenes proteins used to respond to stress, virulence factors, and implicated in nonsuppurative complications of S. pyogenes, including glomerulonephritis and rheumatic fever.The incidence of some sequelae of group A streptococcal infection such as rheumatic fever and acute post-streptococcal glomerulonephritis (APSGN) 1 has decreased over the last five decades in the United States and western countries (1-3). However, these disorders continue unabated and are important public health problems in developing countries (1,3,4). The effects of fluoride on cariogenic Streptococcus mutans and other bacteria have been extensively studied. Fluoride inhibits enolase, a glycolytic enzyme (5, 6), inhibits F-ATPase activity resulting in less acidurance (7,8), reduces glucan-binding lectin activity (9), and decreases glucose incorporation (10). However, the effects of fluoride on group A Streptococci have not been examined. To examine simultaneous changes in multiple virulence factors, we performed a proteomic analysis of S. pyogenes exposed to fluoride. Western blotting and other immunological methods have been successfully used to study protein expression of various microorganisms, cells, and tissues. However, these techniques are constrained by the limited number of proteins that can be studied in each experiment and the availability of specific antibodies. Proteomic techniques date to 1975, when two-dimensional PAGE was simultaneously described by O'Farrell and Klose (11, 12) and applied to the study of a large number of proteins simultaneously. In twodimensional PAGE proteins are separated by differential isoelectric point (pI)...

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“…Spontaneous Sm-resistant mutants were obtained as colonies that grew within 3 days after cells were spread on solid L medium (10 g tryptone, 5 g yeast extract, and 5 g NaCl per liter) containing Sm at a concentration of 10 to 15 g/ml. We used a relA1 mutation with a slightly leaky phenotype, accumulating 1/10 of the amount of ppGpp accumulated by the wild type upon nutritional downshift (31), rather than the relA-null mutation, because the latter results in a severe growth defect (32).…”

Section: Methodsmentioning

confidence: 99%

The mthA Mutation Conferring Low-Level Resistance to Streptomycin Enhances Antibiotic Production in Bacillus subtilis by Increasing the S -Adenosylmethionine Pool Size

et al. 2014

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bCertain Str r mutations that confer low-level streptomycin resistance result in the overproduction of antibiotics by Bacillus subtilis. Using comparative genome-sequencing analysis, we successfully identified this novel mutation in B. subtilis as being located in the mthA gene, which encodes S-adenosylhomocysteine/methylthioadenosine nucleosidase, an enzyme involved in the S-adenosylmethionine (SAM)-recycling pathways. Transformation experiments showed that this mthA mutation was responsible for the acquisition of low-level streptomycin resistance and overproduction of bacilysin. The mthA mutant had an elevated level of intracellular SAM, apparently acquired by arresting SAM-recycling pathways. This increase in the SAM level was directly responsible for bacilysin overproduction, as confirmed by forced expression of the metK gene encoding SAM synthetase. The mthA mutation fully exerted its effect on antibiotic overproduction in the genetic background of rel ؉ but not the rel mutant, as demonstrated using an mthA relA double mutant. Strikingly, the mthA mutation activated, at the transcription level, even the dormant ability to produce another antibiotic, neotrehalosadiamine, at concentrations of 150 to 200 g/ml, an antibiotic not produced (<1 g/ml) by the wild-type strain. These findings establish the significance of SAM in initiating bacterial secondary metabolism. They also suggest a feasible methodology to enhance or activate antibiotic production, by introducing either the rsmG mutation to Streptomyces or the mthA mutation to eubacteria, since many eubacteria have mthA homologues.

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Cloning and characterization of a relA/spoT homologue from Bacillus subtilis

Cited by 148 publications

References 62 publications

GTP Dysregulation in Bacillus subtilis Cells Lacking (p)ppGpp Results in Phenotypic Amino Acid Auxotrophy and Failure To Adapt to Nutrient Downshift and Regulate Biosynthesis Genes

GTP Dysregulation in Bacillus subtilis Cells Lacking (p)ppGpp Results in Phenotypic Amino Acid Auxotrophy and Failure To Adapt to Nutrient Downshift and Regulate Biosynthesis Genes

Fluoride Exposure Attenuates Expression of Streptococcus pyogenes Virulence Factors

The mthA Mutation Conferring Low-Level Resistance to Streptomycin Enhances Antibiotic Production in Bacillus subtilis by Increasing the S -Adenosylmethionine Pool Size

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