Cloning and characterization of a bifunctional RelA/SpoT homologue from Mycobacterium tuberculosis.

A 2.2kb relA/spoT homologue was isolated from Mycobacterium tuberculosis (Mtb) genomic DNA by PCR-amplification. The Mtb gene encodes a protein of 738 amino acid residues, and is flanked upstream by an ORF that is highly similar to the apt gene, and downstream by an ORF that is highly similar to the cypH gene. This dual function Mtb homologue belongs to the relA/spoT family of genes that mediate the stringent response by regulating the synthesis and degradation of guanosine 3',5'-bis(diphosphate) (ppGpp) and pppGpp. In vitro biochemical data indicate that purified RelMtb is a ribosome- and tRNA-independent ATP:GTP/GDP/ITP 3'-pyrophosphoryltransferase. Additionally, purified RelMtb is an Mn2+-dependent, ribosome and tRNA-independent, (p)ppGpp 3'-pyrophosphohydrolase. These reactions were also assessed in vivo in E. coli deleted in both the relA and spoT genes, which generates a (p)ppGpp0 phenotype. RelMtb can suppress this phenotype and can generate more (p)ppGpp than relA in the wild type E. coli control.     

Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene

RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli , V. cholerae Δ relA Δ spoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae . By repeated subculturing of a V. cholerae Δ relA Δ spoT mutant, a suppressor strain with (p)ppGpp⁰ phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene ( VC1224 ), which codes for a small protein (∼29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV . Using E. coli Δ relA or Δ relA Δ spoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (Δ relA Δ spoT relV676 ), which seems to be responsible for the (p)ppGpp⁰ phenotype. Analysis of a V. cholerae Δ relA Δ spoT Δ relV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.     

Protein sequences encoded by the relA and the spoT genes of Escherichia coli are interrelated

relA and spoT are designations for two unlinked Escherichia coli genes whose products function in the synthesis and degradation of guanosine 3',5'-bispyrophosphate during the stringent regulatory response to amino acid deprivation. The RelA protein catalyzes an ATP:GTP 3'-pyrophosphoryl group transfer reaction, and the SpoT protein has a guanosine 3',5'-bispyrophosphate 3'-pyrophosphohydrolyase activity. Both genes have been sequenced recently; the relA gene produces an 84-kDa protein, and the spoT gene is deduced to encode a 79-kDa protein. We report here that the protein sequences of the relA and spoT genes are extensively interrelated.     

SpoT governs Legionella pneumophila differentiation in host macrophages

During its life cycle, Legionella pneumophila alternates between a replicative and a transmissive state. To determine their contributions to L. pneumophila differentiation, the two ppGpp synthetases, RelA and SpoT, were disrupted. Synthesis of ppGpp was required for transmission, as relA spoT mutants were killed during entry to and exit from macrophages. RelA, which senses amino acid starvation induced by serine hydroxamate, is dispensable in macrophages, as relA mutants spread efficiently. SpoT monitors fatty acid biosynthesis (FAB), since following cerulenin treatment, wild-type and relA strains expressed the flaA transmissive gene, but relA spoT mutants did not. As in Escherichia coli , the SpoT response to FAB perturbation likely required an interaction with acyl-carrier protein (ACP), as judged by the failure of the spoT-A413E allele to rescue transmissive trait expression of relA spoT bacteria. Furthermore, SpoT was essential for transmission between macrophages, since secondary infections by relA spoT mutants were restored by induction of spoT , but not relA . To resume replication, ppGpp must be degraded, as mutants lacking spoT hydrolase activity failed to convert from the transmissive to the replicative phase in either bacteriological medium or macrophages. Thus, L. pneumophila requires SpoT to monitor FAB and to alternate between replication and transmission in macrophages.     

The stringent response genes relA and spoT are important for Escherichia coil biofilms under slow-growth conditions

In order to see whether the stringent response was involved in biofilm formation, Escherichia coli DS291 (MG1655), and its isogenic relA spoT derivative were grown for 48 h in a chemostat at dilution rates of 0.025 and 0.25 h(-1) under serine limitation. The absence of the stringent response genes relA and spoT had little effect on the planktonic cell concentrations. However, a significant (P < 0.001) reduction in biofilm cell density of the relA spoT mutants was seen at a doubling time of 40 h. At a doubling time of 4 h, differences in biofilm cell density were not significant. Scanning confocal laser microscopy demonstrated the cell densities of microcolonies in the relA spoT mutant to be lower than those in the wild type. Using a microtiter plate assay, we found biofilm formation in relA spoT mutants to be similarly reduced in minimal media but to be enhanced in rich media (Luria-Bertani broth). No significant differences in biofilm formation were observed between wild type and isogenic relA mutants under any growth conditions. Overall, these results suggest that both stringent response genes relA and spoT are important in nutrient-limited biofilms.     

The role of the omega subunit of RNA polymerase in expression of the relA gene in Escherichia coli

The rpoZ gene for the omega subunit of Escherichia coli RNA polymerase constitutes single operon with the spoT gene, which is responsible for the maintenance of stringent response under nutrient starvation conditions. To identify the physiological role of the omega subunit, we compared the gene expression profile of wild-type Escherichia coli with that of an rpoZ deleted strain by microarray analysis using an E. coli DNA chip. Here we report on a set of genes which show changes in expression profile following the removal of rpoZ. We have seen that relA, which is responsible for the synthesis of the stringent factor ppGpp and many ribosomal proteins, exhibited noticeable changes in mRNA levels and were therefore further analyzed for their expression using a GFP/RFP two-fluorescent protein promoter assay vector. In the absence of rpoZ, the promoter for the relA gene was severely impaired, but the promoters from the ribosomal protein genes were not affected as much. Taking these results together we propose that the omega subunit is involved in regulation of the relA gene, but induction of the stringently controlled genes in the absence of rpoZ is, at least in part, attributable to a decrease in ppGpp level.     

Temperature-sensitive growth and decreased thermotolerance associated with relA mutations in Escherichia coli

The relA gene of Escherichia coli encodes guanosine 3',5'-bispyrophosphate (ppGpp) synthetase I, a ribosome-associated enzyme that is activated during amino acid starvation. The stringent response is thought to be mediated by ppGpp. Mutations in relA are known to result in pleiotropic phenotypes. We now report that three different relA mutant alleles, relA1, relA2, and relA251::kan, conferred temperature-sensitive phenotypes, as demonstrated by reduced plating efficiencies on nutrient agar (Difco) or on Davis minimal agar (Difco) at temperatures above 41 degrees C. The relA-mediated temperature sensitivity was osmoremedial and could be completely suppressed, for example, by the addition of NaCl to the medium at a concentration of 0.3 M. The temperature sensitivities of the relA mutants were associated with decreased thermotolerance; e.g., relA mutants lost viability at 42 degrees C, a temperature that is normally nonlethal. The spoT gene encodes a bifunctional enzyme possessing ppGpp synthetase and ppGpp pyrophosphohydrolase activities. The introduction of the spoT207::cat allele into a strain bearing the relA251::kan mutation completely abolished ppGpp synthesis. This ppGpp null mutant was even more temperature sensitive than the strain carrying the relA251::kan mutation alone. The relA-mediated thermosensitivity was suppressed by certain mutant alleles of rpoB (encoding the beta subunit of RNA polymerase) and spoT that have been previously reported to suppress other phenotypic characteristics conferred by relA mutations. Collectively, these results suggest that ppGpp may be required in some way for the expression of genes involved in thermotolerance.     

Suppressive effect of l-phenylalanine on lignin peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract Guanosine-5'-diphosphate-3'-diphosphate (ppGpp), an effector for many metabolic pathways, is synthesized by the relA gene product after amino acid limitation. Studies of stringent controlled Escherichia coli CP78 (relA⁺) and relaxed controlled E. coli CP79 (relA⁻) were carried out to test whether these strains differ in the appearance of their cytoplasmic membranes after induction of stringent and relaxed response. Cytoplasmic membrane structures of the cells were investigated by freeze-fracture electron microscopy after cooling the cells. The obtained micrographs showed a net-like distribution of the particles in the cytoplasmic membranes of relaxed controlled cells whereas such a pattern was not detectable in the stringent controlled counterparts.     

Residual guanosine 3',5'-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations

It was known previously that 1) the relA gene of Escherichia coli encodes an enzyme capable of guanosine 3',5'-bispyrophosphate (ppGpp) synthesis, 2) an uncharacterized source of ppGpp synthesis exists in relA null strains, and 3) cellular degradation of ppGpp is mainly due to a manganese-dependent ppGpp 3'-pyrophosphohydrolase encoded by the spoT gene. Here, the effects of spoT gene insertions and deletions are compared with analogous alterations in neighboring genes in the spo operon and found to be lethal in relA+ strains as well as slower growing in relAl backgrounds than delta relA hosts. Cells with null alleles in both the relA and spoT genes are found no longer to accumulate ppGpp after glucose exhaustion or after chelation of manganese ions by picolinic acid addition; the inability to form ppGpp is reversed by a minimal spoT gene on a multicopy plasmid. Strains apparently lacking ppGpp show a complex phenotype including auxotrophy for several amino acids and morphological alterations. We propose that the SpoT protein can either catalyze or control the alternative pathway of ppGpp synthesis in addition to its known role as a (p)ppGpp 3'-pyrophosphohydrolase. We favor the possibility that the SpoT protein is a bifunctional enzyme capable of catalyzing either ppGpp synthesis or degradation.     

The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes

Campylobacter jejuni is a highly prevalent food-borne pathogen that causes diarrhoeal disease in humans. A natural zoonotic, it must overcome significant stresses both in vivo and during transmission despite the absence of several traditional stress response genes. Although relatively little is understood about its mechanisms of pathogenesis, its ability to interact with and invade human intestinal epithelial cells closely correlates with virulence. A C. jejuni microarray-based screen revealed that several known virulence genes and several uncharacterized genes, including spoT, were rapidly upregulated during infection of human epithelial cells. spoT and its homologue relA have been shown in other bacteria to regulate the stringent response, an important stress response that to date had not been demonstrated for C. jejuni or any other epsilon-proteobacteria. We have found that C. jejuni mounts a stringent response that is regulated by spoT. Detailed analyses of a C. jejuni delta spoT mutant revealed that the stringent response is required for several specific stress, transmission and antibiotic resistance-related phenotypes. These include stationary phase survival, growth and survival under low CO2/high O2 conditions, and rifampicin resistance. A secondary suppressor strain that specifically rescues the low CO2 growth defect of the delta spoT mutant was also isolated. The stringent response additionally proved to be required for the virulence-related phenotypes of adherence, invasion, and intracellular survival in two human epithelial cell culture models of infection; spoT is the first C. jejuni gene shown to participate in longer term survival in epithelial cells. Microarray analyses comparing wild-type to the delta spoT mutant also revealed a strong correlation between gene expression profiles and phenotype differences observed. Together, these data demonstrate a critical role for the C. jejuni stringent response in multiple aspects of C. jejuni biology and pathogenesis and, further, may lend novel insight into unexplored features of the stringent response in other prokaryotic organisms.     

Guanosine 3',5'-bispyrophosphate (ppGpp) synthesis in cells of Escherichia coli starved for Pi.

Cells of Escherichia coli which enter a phase of starvation for Pi induce the synthesis of the nucleotide guanosine 3',5'-bispyrophosphate (ppGpp). This induction is relA independent but depends on the spoT gene product. A mutant unable to produce ppGpp is impaired in the expression of two genes which belong to the pho regulon, a defect which is dependent on the product of spoT. We suggest that ppGpp is essential for the proper induction of the genes which belong to the pho regulon.     

Role of the stringent response in the expression and mechanism of near-ultraviolet induced growth delay

The near-ultraviolet (300-400 nm) induced growth delay of Escherichia coli cells was compared in isogenic relA+ and relA- cells illuminated either in the stationary or the exponential phase. In the latter case: (a) the relA- strains of K12 and B/r exhibited similar maximal growth lags (65 min and 55 min respectively); (b) the maximal lags were 1.5-fold and 4-fold longer, respectively, in the isogenic relA+ strains; (c) the rate of the relA- -dependent guanosine 3',5'-bis(diphosphate) (ppGpp) accumulation was three-times lower in the K12 relA+ strain as compared to the B/r relA- strain: (d) a K12 spoT mutant having an impaired rate of ppGpp degradation had a 2-fold longer lag. On the other hand, when illumination is performed in the stationary phase, isogenic relA+ and relA- cells (B/r or K12) exhibited similar growth lags at any fluences, indicating little if any involvement of the stringent response. These data extend previous observations of T.V. Ramabhadran an J. Jagger [(1976) Proc. Natl Acad. Sci. USA, 73, 59-63] but do not support their conclusion that the stringent response is the main factor responsible for growth delay. By monitoring the intracellular level of ppGpp in relA+ spoT- and relA+ spoT+ growing cells during illumination and the subsequent growth lag we observed that the initial burst of ppGpp decreases slowly all along the lag; in all relA+ strains checked the return of ppGpp to its basal level coincides with the recovery of normal growth. We conclude that it is the accumulation of ppGpp over the basal level due either to the stringent response or to prevention of ppGpp degradation that is responsible for an amplification of the growth lag.     

Functional analysis of a relA/spoT gene homolog from Streptococcus equisimilis.

We examined the functional attributes of a gene encountered by sequencing the streptokinase gene region of Streptococcus equisimilis H46A. This gene, originally called rel, here termed relS. equisimilis, is homologous to two related Escherichia coli genes, spoT and relA, that function in the metabolism of guanosine 5',3'-polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the highly expressed rel S. equisimilis gene encodes a strong (p)ppGppase and a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absence of the relA gene. We verified that the Rel S. equisimilis protein, purified from an E. coli relA spoT double mutant, catalyzed a manganese-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel S. equisimilis protein preparation also weakly catalyzed a ribosome-independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was restricted by the absence of manganese ions. An analogous activity has been deduced for the SpoT protein from genetic evidence. In addition, the Rel S. equisimilis protein displays immunological cross-reactivity with polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel S. equisimilis gene function in E. coli as being similar to that of the native spoT gene, disruptions of rel S. equisimilis in S. equisimilis abolish the parental (p)ppGpp accumulation response to amino acid starvation in a manner expected for relA mutants rather than spoT mutants.