Initiation of antibiotic production by the stringent response of Bacillus subtilis Marburg

Bacillus subtilis Marburg was found to produce an appreciable amount of an antibiotic in a synthetic medium. Antibiotic activity was produced in parallel with cell growth, and production stopped at the end of exponential growth. When the synthetic medium was supplemented with a small amount of Casamino acids, however, antibiotic was made only at the end of growth and in lesser amounts. The ability of cells to produce the antibiotic increased when stringent (rel+ = wild-type) cells underwent a partial stringent response. These conditions also initiated extensive sporulation. An isogenic relaxed (rel) strain produced little antibiotic activity, which decreased under partial amino acid deprivation. In rel+ cells, the addition of a low concentration of chloramphenicol, which reduces ppGpp synthesis, also reduced antibiotic synthesis in both normal and amino acid-starved bacteria, without appreciably affecting their growth rate. Guanosine starvation of a gua mutant initiated sporulation, but decreased antibiotic production. The results show that the stringent response initiates both sporulation (differentiation) and antibiotic production (secondary metabolism), but by different mechanisms. It appears that sporulation results from a decrease of GTP, whereas antibiotic synthesis results from a different effect of the stringent response.     

Involvement of the stringent response in degradation of glutamine phosphoribosylpyrophosphate amidotransferase in Bacillus subtilis

Glutamine phosphoribosylpyrophosphate amidotransferase, the first enzyme of purine biosynthesis, has previously been shown to be rapidly inactivated and degraded in Bacillus subtilis cells at the end of growth. The loss of enzyme activity appears to involve the oxidation of an iron-sulfur cluster in the enzyme. The degradation of the inactive enzyme involves some elements of the stringent response because it is inhibited in relA and relC mutants. Intracellular pools of guanosine tetra- and pentaphosphate were measured by an improved extraction procedure in cells that had been manipulated in various ways to induce or inhibit amidotransferase degradation. The results are consistent with the hypothesis that one or both of these nucleotides stimulates the synthesis of a protein involved in degradation. An elevated level of these nucleotides was not required for the continued degradation of amidotransferase once it had begun.     

Initiation of Bacillus subtilis sporulation by the stringent response to partial amino acid deprivation

We have controlled the rates at which three different amino acids were available to auxotrophs of Bacillus subtilis by avoiding active transport of the respective substrate. The active transport of oxomethylvalerate, a precursor of isoleucine, was prevented by a kauA mutation, the uptake of L-aspartate was competed by 20 mM L-glutamate, and D-methionine was used instead of L-methionine. When in this way conditions of partial amino acid deprivation were achieved, a partial "stringent response" occurred which included the increase of ppGpp and pppGpp, and the decrease of GTP; such conditions initiated sporulation. In the corresponding relaxed (relA) mutants, the changes of guanine nucleotides were greatly reduced and no sporulation was observed at any substrate concentration; but addition of decoyinine produced a further decrease of GTP and caused sporulation.     

Iron starvation triggers the stringent response and induces amino acid biosynthesis for bacillibactin production in Bacillus subtilis

Iron deprivation in bacteria causes the derepression of genes controlled by the ferric uptake regulator (Fur). The present microarray analysis of iron-starved Bacillus subtilis cells grown in minimal medium unveils additional physiological effects on a large number of genes linked to stringent-response regulation and to genes involved in amino acid biosynthesis associated with pathways essential for bacillibactin production.     

The stringent response blocks DNA replication outside the ori region in Bacillus subtilis and at the origin in Escherichia coli

When the Bacillus subtilis dnaB37 mutant, defective in initiation, is returned to permissive temperature after growth at 45 degrees C, DNA replication is synchronized. Under these conditions, we have shown previously that DNA replication is inhibited when the Stringent Response is induced by the amino acid analogue, arginine hydroxamate. We have now shown, using DNA-DNA hybridization analysis, that substantial replication of the oriC region nevertheless occurs during the Stringent Response, and that replication inhibition is therefore implemented downstream from the origin. On the left arm, replication continues for at least 190 x 10(3) base-pairs to the gnt gene and for a similar distance on the right arm to the gerD gene. When the Stringent Response is lifted, DNA replication resumed downstream from oriC on both arms, confirming that DNA replication is regulated at a post-initiation level during the Stringent Response in B. subtilis. Resumption of DNA synthesis following the lifting of the Stringent Response did not require protein or RNA synthesis or the initiation protein DnaB. We suggest, therefore, that a specific control region, involving Stringent Control sites, facilitate reversible inhibition of fork movement downstream from the origin via modifications of a replisome component during the Stringent Response. In contrast, in Escherichia coli, induction of the Stringent Response appears to block initiation of DNA replication at oriC itself. No DNA synthesis was detected in the oriC region and, upon lifting the Stringent Response, replication occurred from oriC. Post-initiation control in B. subtilis therefore results in duplication of many key genes involved in growth and sporulation. We discuss the possibility that such a control might be linked to differentiation in this organism.     

Formation of extracellular neutral proteinase and the stringent response inBacillus subtilis

The kinetics of extracellular neutral proteinase synthesis by an isogenic stringent (IS58) and a relaxed (IS56) strain ofB. subtilis were compared. The specific enzyme formation rate by the stringent strain was higher than that of the relaxed one. Norvaline addition (1 mg/mL) induced the formation of pppGpp and ppGpp, respectively, as well as the appearance of extracellular neutral proteinase activities in cultures of the stringent strain IS58 and a strain with high proteinase production (ZF-178) only. These correlations support the suggestion that (p)ppGpp are involved in the regulation processes responsible for production of extracellular neutral proteinases byB. subtilis. Dedicated to Prof. Dr. F. Mach on the occasion of his 60th birthday.     

The oxidative stress response in Bacillus subtilis

Abstract Bacillus subtilis undergoes a typical bacterial stress response when exposed to low concentrations (0.1 mM) of hydrogen peroxide. Protection is thereby induced against otherwise lethal, challenge concentrations (10 mM) of this oxidant and a number of proteins are induced including the scavenging enzymes, catalase and alkyl hydroperoxide reductase, and a putative DNA binding and protecting protein. Induced protection against higher concentrations (10–30 mM) of hydrogen peroxide is eliminated in a catalase-deficient mutant. Both RecA and Spo0A influence the basal but not the induced resistance to hydrogen peroxide. A regulatory mutation has been characterized that affects the inducible phenotype and is constitutively resistant to high concentrations of hydrogen peroxide. This mutant constitutively overexpresses the proteins induced by hydrogen peroxide in the wild-type. The resistance of spores to hydrogen peroxide is partly attributable to binding of small acid soluble proteins by the spore DNA and partly to a second step which coincides with the depletion of the NADH pool, which may inhibit the generation of hydroxyl radicals from hydrogen peroxide.     

Heat-shock and general stress response in Bacillus subtilis

The induction of stress proteins is an important component of the adaptional network of a non-growing cell of Bacillus subtilis . A diverse range of stresses such as heat shock, salt stress, ethanol, starvation for oxygen or nutrients etc. induce the same set of proteins, called general stress proteins. Although the adaptive functions of these proteins are largely unknown, they are proposed to provide general and rather non-specific protection of the cell under these adverse conditions. In addition to these non-specific general stress proteins, all extracellular signals induce a set of specific stress proteins that may confer specific protection against a particular stress factor. In B. subtilis at least three different classes of heat-inducible genes can be defined by their common regulatory characteristics: Class I genes, as exemplified by the dnaK and groE operons, are most efficiently induced by heat stress. Their expression involves a σ^A-dependent promoter, an inverted repeat (called the CIRCE element) highly conserved among eubacteria, and probably a repressor interacting with the CIRCE element. The majority of general stress genes (class II, more than 40) are induced at σ^B-dependent promoters by different growth-inhibiting conditions. The activation of σ^B by stress or starvation is the crucial event in the induction of this large stress regulon. Only a few genes, including lon clpC clpP , and ftsH, can respond to different stress factors independently of σ^B or CIRCE (class III). Stress induction of these genes occurs at promoters presumably recognized by σ^A and probably involves additional regulatory elements which remain to be defined.     

The stringent response to unacylated tRNA,energy- and temperature-downshift in Bacillus stearothermophilus

The response of the thermophile Bacillus stearothermophilus to inhibition of tRNA acylation, energy starvation and temperature downshift was characterized. We found that B. stearothermophilus, like other prokaryotic organisms, reacts with the so-called stringent response, which includes the accumulation of the unusual nucleotides guanosine 3′,5′ bis (dipphosphate) [ppGpp] and guanosine 3′-diphosphate, 5′-triphosphate [pppGpp] and concomitantly the reduction of RNA synthesis and growth rate. The amount of (p)ppGpp formed depended on the cause of the stringent response: when tRNA acylation was inhibited (p)ppGpp synthesis was much higher than after energy starvation or temperature downshift whereas RNA synthesis was totally blocked in each case.     

The replication checkpoint control in Bacillus subtilis: identification of a novel RTP-binding sequence essential for the replication fork arrest after induction of the stringent response

We have shown previously that induction of the stringent response in Bacillus subtilis resulted in the arrest of chromosomal replication between 100 and 200 kb either side of oriC at distinct stop sites, designated LSTer and RSTer, left and right stringent terminators respectively. This replication checkpoint was also shown to involve the RTP protein, normally active at the chromosomal terminus. In this study, we show that the replication block is absolutely dependent upon RelA, correlated with high levels of ppGpp, but that efficient arrest at STer sites also requires RTP. DNA-DNA hybridization data indicated that one or more such LSTer sites mapped to gene yxcC (-128 kb from oriC). A 7.75 kb fragment containing this gene was cloned into a theta replicating plasmid, and plasmid replication arrest, requiring both RelA and RTP, was demonstrated. This effect was polar, with plasmid arrest only detected when the fragment was orientated in the same direction with respect to replication, as in the chromosome. This LSTer2 site was further mapped to a 3.65 kb fragment overlapping the next40 probe. Remarkably, this fragment contains a 17 bp sequence (B'-1) showing 76% identity with an RTP binding site (B sequence) present at the chromosomal terminus. This B'-1 sequence, located in the gene yxcC, efficiently binds RTP in vitro, as shown by DNA gel retardation studies and DNase I footprinting. Importantly, precise deletion of this sequence abolished the replication arrest. We propose that this modified B site is an essential constituent of the LSTer2 site. The differences between arrest at the normal chromosomal terminus and arrest at LSTer site are discussed.     

Cloning of the Bacillus subtilis sulfanilamide resistance gene in Bacillus subtilis

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A single colony was isolated containing the recombinant plasmid pKO101. This 6.3-megadalton plasmid simultaneously conferred resistance to neomycin and sulfanilamide when transferred into sensitive Rec+ or Rec- cells by either transduction or transformation.     

Adaptive response of Bacillus subtilis to N-methyl-N'-nitro-N-nitrosoguanidine.

Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated from an aceEF (pyruvate dehydrogenase-deficient) strain by selection for a complete absence of growth on medium lacking acetate. Extracts of two of the mutants were shown to contain normal levels of pyruvate oxidase antigen, although the enzymatic activities of the extracts were reduced or absent. The poxB locus was mapped by using closely linked transposon insertions to min 18.7 of the E. coli linkage map between the cmlA and aroA loci, a location far removed from that of the regulatory gene, poxA.