Enzymes of the Rel/Spo family enable bacteria to survive prolonged periods of nutrient limitation by producing an intracellular signaling alarmone, (p)ppGpp, which triggers the so-called stringent response. Both the synthesis of (p)ppGpp from ATP and GDP(GTP), and its hydrolysis to GDP(GTP) and pyrophosphate, are catalyzed by Rel/Spo proteins. The 2.1 A crystal structure of the bifunctional catalytic fragment of the Rel/Spo homolog from Streptococcus dysgalactiae subsp. equisimilis, Rel(Seq), reveals two conformations of the enzyme corresponding to known reciprocal activity states: (p)ppGpp-hydrolase-OFF/(p)ppGpp-synthetase-ON and hydrolase-ON/synthetase-OFF. The hydrolase and synthetase domains bear remarkable similarities to the catalytic domains of the cyclic phosphodiesterase and nucleotidyltransferase superfamilies, respectively. The active sites, separated by more than 30 A, contain bound nucleotides including an unusual (p)ppGpp derivative, GDP-2':3'-cyclic monophosphate. Reciprocal regulation of the antagonistic catalytic activities, suggested by the structure, is supported by mutagenesis experiments and appears to involve ligand-induced signal transmission between the two active sites.

2We 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 rel S. 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.As typified by extensive studies with members of the family Enterobacteriacae, bacterial cells subjected to nutrient exhaustion respond with rapid and complex adjustments that involve the metabolism of GTP and GDP analogs bearing pyrophosphate derivatives at the ribose 3Ј-hydroxyl position, collectively abbreviated (p)ppGpp (8). Restriction of accumulation of most stable RNA species, stimulation of certain anabolic activities, and induction of stationary-phase-specific gene expression generally accompany these responses, presumably to facilitate adaptation to the nutritional stress or to ensure cell viability if adaptation is not possible (21).In Escherichia coli, the products of the relA and spoT genes can regulate the accumulation of (p)ppGpp by two apparently independent mechanisms. Synthesis is regulated by the RelA protein, which catalyzes the pyrophosphorylation of GTP (or GDP) using an ATP donor and is activated by codon-specific uncharged tRNA binding to ribosomes engaged in protein synthesis elongation (13,25,26,49). Increased ratios of uncharged to charged tRNA seem to comprise the sole signal for relA-dependent induction of (p)ppGpp synthesis (22). Degradation of (p)ppGpp is inhibited when a primary energy source becomes limiting (17) and leads to (p)ppGpp accumulation without necessarily increasing synthesis; this occurs by inhibiting the activity of a manganese-dependent (p)ppGpp 3Ј-pyrophosphohydrolase...

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Genomic DNA from Streptococcus equisimilis strain H46A was cloned in Escherichia coli by using the bacteriophage X replacement vector L47 and an in vitro packaging system. A casein/plasminogen overlay technique was used to screen the phage bank for recombinants carrying the streptokinase gene (skc). The gene was present with a frequency of 1 in 836 recombinants, and 10 independent clones containing skc were isolated and physically characterized. One recombinant clone was used to subclone skc in E. coli plasmid vectors. Plasmid pMF2 [10.4 kilobases (kb)] consisting of pACYC184 with a 6.4-kb H46A DNA fragment in the EcoRI site and pMF5 (6.9 kb) carrying a 2.5-kb fragment in the Pst I site of pBR322 were among the recombinant plasmids determining streptokinase production in three different E. coli host strains. Expression of skc was independent of its orientation in either vector, indicating that its own promoter was present and functional in E. coli. However, expression in pBR322 was more efficient in one orientation than in the other, suggesting that one or both of the bla gene promoters contributed to skc expression. Several lines of evidence, including proof obtained by the immunodiffusion technique, established the identity of E. coli streptokinase. Testing cell-free culture supernatant fluids, osmotic shock fluids, and sonicates of osmotically shocked cells for streptokinase activity revealed the substance to be present in all three principal locations, indicating that E. coli cells were capable of releasing substantial amounts of streptokinase into the culture medium.Streptokinases are a well-defined group of proteins exported by many strains of hemolytic streptococci to the growth medium. They interact stoichiometrically with the enzymatically inert plasma plasminogen to yield the active enzyme plasmin. The plasmin so formed then degrades, by limited proteolysis, the fibrin network to form soluble products (1, 2). Although, unlike other plasminogen activators, streptokinases are not proteases, the recently determined amino acid sequence of one streptokinase species revealed homology to the sequences of bovine trypsin and Streptomyces griseus proteases, suggesting that it evolved from a seine protease (3).The role of streptokinases in the pathogenicity of streptococci is unclear. Potentially, these substances may be determinants of virulence that contribute to the invasiveness of the organisms by preventing the formation of fibrin barriers around infectious lesions. Physical and immunological differences, paralleled by differences in substrate specificity, testify to the molecular heterogeneity of streptokinases from different sources (4, 5). Although these proteins are closely related in function, the genetic basis of their heterogeneity is unknown. To achieve a better understanding of the genetic aspects of this important streptococcal product, we have undertaken to clone a streptokinase gene from a group C Streptococcus and report here its expression in Escherichia coli. Besides providing approach...

Control over mRNA stability is an essential part of gene regulation that involves both endo-and exoribonucleases. RNase Y is a recently identified endoribonuclease in Gram-positive bacteria, and an RNase Y ortholog has been identified in Streptococcus pyogenes (group A streptococcus [GAS]). In this study, we used microarray and Northern blot analyses to determine the S. pyogenes mRNA half-life of the transcriptome and to understand the role of RNase Y in global mRNA degradation and processing. We demonstrated that S. pyogenes has an unusually high mRNA turnover rate, with median and mean half-lives of 0.88 min and 1.26 min, respectively. A mutation of the RNase Y-encoding gene (rny) led to a 2-fold increase in overall mRNA stability. RNase Y was also found to play a significant role in the mRNA processing of virulence-associated genes as well as in the rapid degradation of rnpB read-through transcripts. From these results, we conclude that RNase Y is a pleiotropic regulator required for mRNA stability, mRNA processing, and removal of read-through transcripts in S. pyogenes. RNA degradation is a strictly regulated process that involves both endo-and exoribonucleases (1). In prokaryotes, mRNA degradation is initiated by endonucleolytic cleavage and followed by exonuclease digestion (2-4). The first step is relatively slow and rate limiting, while the second step proceeds rapidly (1). In Escherichia coli, RNase E functions as the major endoribonuclease that initiates the bulk of mRNA degradation (5). Although RNase E is absent in Bacillus subtilis, the recently identified RNase Y is considered its functional analog (6). RNase E and RNase Y do not share sequence homology but are strikingly similar in function (7,8). Both RNases are membrane-bound proteins that interact with other components to form a complex called the RNA degradosome (7, 9). These components include other RNases, an RNA helicase, and two glycolytic enzymes (10, 11). Both RNase Y and RNase E prefer 5= monophosphorylated RNA substrates with downstream secondary structures (6, 12). The depletion of B. subtilis RNase Y results in the accumulation of about 550 mRNAs, including important transcriptional regulators for stress response and biofilm formation and metabolic operons for tryptophan biosynthesis and glycolytic enzymes (8). B. subtilis RNase Y also interacts with RNases J1 and III to control the abundance of total mRNAs (13). RNase Y of Staphylococcus aureus plays a major role in virulence gene regulation and is involved in the processing and stabilization of a global regulator system, SaePQRS (14). These observations suggest that RNase Y is the major endoribonuclease in mRNA degradation in B. subtilis and perhaps also in other Gram-positive pathogens, such as Streptococcus pyogenes.S. pyogenes (group A streptococcus [GAS]) causes a variety of human diseases ranging from mild local infections such as pharyngitis and impetigo to life-threatening systemic diseases such as toxic shock syndrome and necrotizing fasciitis (15). GAS infections often cau...