Role of the all1549 (ana-rsh) gene, a relA/spoT homolog, of the Cyanobacterium Anabaena sp. PCC7120

The role of a single relA/spoT homolog all1549 (designated hereafter as ana-rsh) of the cyanobacterium Anabaena sp. PCC7120 was investigated. The complementation test in Escherichia coli showed that the protein encoded by ana-rsh possesses guanosine tetraphosphate (p)ppGpp-synthase/hydrolase activity. Under laboratory growth conditions, a low level of ppGpp was detected in Anabaena sp. PCC7120 and the loss of ana-rsh was lethal. Amino acid starvation induced ppGpp accumulation to an appropriate level, and nitrogen deficiency did not alter the ppGpp concentration in Anabaena cells. These data suggest that ana-rsh is required for cell viability under normal growth conditions and involved in the (p)ppGpp-related stringent response to amino acid deprivation, but not related to heterocyst formation and nitrogen fixation of Anabaena sp. PCC7120.     

Co-Inactivation of GlnR and CodY Regulators Impacts Pneumococcal Cell Wall Physiology

CodY, a nutritional regulator highly conserved in low G+C Gram-positive bacteria, is essential in Streptococcus pneumoniae (the pneumococcus). A published codY mutant possessed suppressing mutations inactivating the fatC and amiC genes, respectively belonging to iron (Fat/Fec) and oligopeptide (Ami) ABC permease operons, which are directly repressed by CodY. Here we analyzed two additional published codY mutants to further explore the essentiality of CodY. We show that one, in which the regulator of glutamine/glutamate metabolism glnR had been inactivated by design, had only a suppressor in fecE (a gene in the fat/fec operon), while the other possessed both fecE and amiC mutations. Independent isolation of three different fat/fec suppressors thus establishes that reduction of iron import is crucial for survival without CodY. We refer to these as primary suppressors, while inactivation of ami, which is not essential for survival of codY mutants and acquired after initial fat/fec inactivation, can be regarded as a secondary suppressor. The availability of codY ^- ami ⁺ cells allowed us to establish that CodY activates competence for genetic transformation indirectly, presumably by repressing ami which is known to antagonize competence. The glnR codY fecE mutant was then found to be only partially viable on solid medium and hypersensitive to peptidoglycan (PG) targeting agents such as the antibiotic cefotaxime and the muramidase lysozyme. While analysis of PG and teichoic acid composition uncovered no alteration in the glnR codY fecE mutant compared to wildtype, electron microscopy revealed altered ultrastructure of the cell wall in the mutant, establishing that co-inactivation of GlnR and CodY regulators impacts pneumococcal cell wall physiology. In light of rising levels of resistance to PG-targeting antibiotics of natural pneumococcal isolates, GlnR and CodY constitute potential alternative therapeutic targets to combat this debilitating pathogen, as co-inactivation of these regulators renders pneumococci sensitive to iron and PG-targeting agents.     

Phylogenetic analysis of proteins involved in the stringent response in plant cells

The nucleotide (p)ppGpp is a second messenger that controls the stringent response in bacteria. The stringent response modifies expression of a large number of genes and metabolic processes and allows bacteria to survive under fluctuating environmental conditions. Recent genome sequencing analyses have revealed that genes responsible for the stringent response are also found in plants. These include (p)ppGpp synthases and hydrolases, RelA/SpoT homologs (RSHs), and the pppGpp-specific phosphatase GppA/Ppx. However, phylogenetic relationship between enzymes involved in bacterial and plant stringent responses is as yet generally unclear. Here, we investigated the origin and evolution of genes involved in the stringent response in plants. Phylogenetic analysis and primary structures of RSH homologs from different plant phyla (including Embryophyta, Charophyta, Chlorophyta, Rhodophyta and Glaucophyta) indicate that RSH gene families were introduced into plant cells by at least two independent lateral gene transfers from the bacterial Deinococcus-Thermus phylum and an unidentified bacterial phylum; alternatively, they were introduced into a proto-plant cell by a lateral gene transfer from the endosymbiotic cyanobacterium followed by gene loss of an ancestral RSH gene in the cyanobacterial linage. Phylogenetic analysis of gppA/ppx families indicated that plant gppA/ppx homologs form an individual cluster in the phylogenetic tree, and show a sister relationship with some bacterial gppA/ppx homologs. Although RSHs contain a plastidial transit peptide at the N terminus, GppA/Ppx homologs do not, suggesting that plant GppA/Ppx homologs function in the cytosol. These results reveal that a proto-plant cell obtained genes for the stringent response by lateral gene transfer events from different bacterial phyla and have utilized them to control metabolism in plastids and the cytosol.     

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

Escherichia coli transiently accumulates large amounts of inorganic polyphosphate (polyP), up to 20 mM in phosphate residues (P_i), in media deficient in both P_i 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 P_i 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.     

CodY of Streptococcus pneumoniae: link between nutritional gene regulation and colonization

CodY is a nutritional regulator mainly involved in amino acid metabolism. It has been extensively studied in Bacillus subtilis and Lactococcus lactis. We investigated the role of CodY in gene regulation and virulence of the human pathogen Streptococcus pneumoniae. We constructed a codY mutant and examined the effect on gene and protein expression by microarray and two-dimensional differential gel electrophoresis analysis. The pneumococcal CodY regulon was found to consist predominantly of genes involved in amino acid metabolism but also several other cellular processes, such as carbon metabolism and iron uptake. By means of electrophoretic mobility shift assays and DNA footprinting, we showed that most of the targets identified are under the direct control of CodY. By mutating DNA predicted to represent the CodY box based on the L. lactis consensus, we demonstrated that this sequence is indeed required for in vitro DNA binding to target promoters. Similar to L. lactis, DNA binding of CodY was enhanced in the presence of branched-chain amino acids, but not by GTP. We observed in experimental mouse models that codY is transcribed in the murine nasopharynx and lungs and is specifically required for colonization. This finding was underscored by the diminished ability of the codY mutant to adhere to nasopharyngeal cells in vitro. Furthermore, we found that pcpA, activated by CodY, is required for adherence to nasopharyngeal cells, suggesting a direct link between nutritional regulation and adherence. In conclusion, pneumococcal CodY predominantly regulates genes involved in amino acid metabolism and contributes to the early stages of infection, i.e., colonization of the nasopharynx.     

(p)ppGpp——“魔斑”核苷酸在细菌中的研究进展

鸟苷四磷酸（guanosine tetraphosphate,ppGpp）/鸟苷五磷酸（guanosine pentaphosphate,pppGpp）是细菌严谨反应的信号分子,其合成和水解由Rel/SpoT同系物（RelA/SpoT homologue,RSH）家族的蛋白质合成和水解活性控制。（p）ppGpp介导的严谨反应能够提高细菌对营养匮乏的适应能力和抗生素抗性。近年来发现（p）ppGpp与细菌生长和细胞分裂、抗生素合成等都密切相关,是细胞内重要的全局调控因子。（p）ppGpp在细菌细胞中有许多靶点,使其可以调节DNA复制、转录、细胞周期、核糖体生物合成以及抗生素合成基因簇的表达。然而,（p）ppGpp如何控制转录和其他代谢过程取决于细菌种类,并在不同的微生物中通过不同的机制调节相同的过程。因此,本文通过综述（p）ppGpp的合成/水解酶的种类和调节机制,（p）ppGpp对微生物代谢调控机制、对细胞周期的影响机制,以及（p）ppGpp对抗生素合成和耐受性的调控机制,为细菌耐药性研究和细胞生理学研究奠定基础。     

Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

In Mycobacterium tuberculosis, the stringent response to amino acid starvation is mediated by the M. tuberculosis Rel (Rel_Mtb) enzyme, which transfers a pyrophosphate from ATP to GDP or GTP to synthesize ppGpp and pppGpp, respectively. (p)ppGpp then influences numerous metabolic processes. Rel_Mtb also encodes a second, distinct catalytic domain that hydrolyzes (p)ppGpp into pyrophosphate and GDP or GTP. Rel_Mtb is required for chronic M. tuberculosis infection in mice; however, it is unknown which catalytic activity of Rel_Mtb mediates pathogenesis and whether (p)ppGpp itself is necessary. In order to individually investigate the roles of (p)ppGpp synthesis and hydrolysis during M. tuberculosis pathogenesis, we generated Rel_Mtb point mutants that were either synthetase dead (Rel_Mtb^H344Y) or hydrolase dead (Rel_Mtb^H80A). M. tuberculosis strains expressing the synthetase-dead Rel_Mtb^H344Y mutant did not persist in mice, demonstrating that the Rel_Mtb (p)ppGpp synthetase activity is required for maintaining bacterial titers during chronic infection. Deletion of a second predicted (p)ppGpp synthetase had no effect on pathogenesis, demonstrating that Rel_Mtb was the major contributor to (p)ppGpp production during infection. Interestingly, expression of an allele encoding the hydrolase-dead Rel_Mtb mutant, Rel_Mtb^H80A, that is incapable of hydrolyzing (p)ppGpp but still able to synthesize (p)ppGpp decreased the growth rate of M. tuberculosis and changed the colony morphology of the bacteria. In addition, Rel_Mtb^H80A expression during acute or chronic M. tuberculosis infection in mice was lethal to the infecting bacteria. These findings highlight a distinct role for Rel_Mtb-mediated (p)ppGpp hydrolysis that is essential for M. tuberculosis pathogenesis.