Characterization of the stringent and relaxed responses of Streptococcus equisimilis.

The 739-codon rel(Seq) gene of Streptococcus equisimilis H46A is bifunctional, encoding a strong guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase (ppGppase) and a weaker ribosome-independent ATP:GTP 3'-pyrophosphoryltransferase [(p)ppGpp synthetase]. To analyze the function of this gene, (p)ppGpp accumulation patterns as well as protein and RNA synthesis were compared during amino acid deprivation and glucose exhaustion between the wild type and an insertion mutant carrying a rel(Seq) gene disrupted at codon 216. We found that under normal conditions, both strains contained basal levels of (p)ppGpp. Amino acid deprivation imposed by pseudomonic acid or isoleucine hydroxamate triggered a rel(Seq)-dependent stringent response characterized by rapid (p)ppGpp accumulation at the expense of GTP and abrupt cessation of net RNA accumulation in the wild type but not in the mutant. Tetracycline added to block (p)ppGpp synthesis caused the accumulated (p)ppGpp to degrade rapidly, with a concomitant increase of the GTP pool (decay constant of ppGpp, approximately 0.7 min(-1)). Simultaneous addition of pseudomonic acid and tetracycline to mimic a relaxed response caused wild-type RNA synthesis to proceed at rates approximating those seen under either condition in the mutant. Glucose exhaustion provoked the (p)ppGpp accumulation response in both the wild type and the rel(Seq) insertion mutant, consistent with the block of net RNA accumulation in both strains. Although the source of (p)ppGpp synthesis during glucose exhaustion remains to be determined, these findings reinforce the idea entertained previously that rel(Seq) fulfils functions that reside separately in the paralogous reL4 and spoT genes of Escherichia coli. Analysis of (p)ppGpp accumulation patterns was complicated by finding an unknown phosphorylated compound that comigrated with ppGpp under two standard thin-layer chromatography conditions. Unlike ppGpp, this compound did not adsorb to charcoal and did not accumulate appreciably during isoleucine deprivation. Like ppGpp, the unknown compound did accumulate during energy source starvation.

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.

The streptokinase gene: allelic variation, genomic environment and expression control.

The genes for streptokinase, the most important prokaryotic plasmingoen activator, exhibit allelic variation predominantly due to the polymorphism of an internal 220-base pair fragment that divides the phylogenetic tree of their products into two primary branches. Current molecular genetic research seeks functional correlates of the allelic variation, aims at analyzing the genomic environment of the streptokinase gene, skc, and focuses on understanding its expression. Of the six genes cloned and sequenced in the skc region of Streptococcus equisimilis H46A, skc is expressed most abundantly in a fashion that involves two overlapping core promoters and upstream sequences rich of AT tracts. Transcription of skc is terminated at a hypersymmetrical site that functions bidirectionally and prevents convergent transcription of the oppositely oriented skc and rel-orf1 genes whose mRNA abundance differs by a factor of at least three orders of magnitude.

Inactivation of the streptokinase gene prevents Streptococcus equisimilis H46A from acquiring cell-associated plasmin activity in the presence of plasminogen.

The streptokinase gene of Streptococcus equisimilis H46 was inactivated by plasmid insertion mutagenesis to study the relationship between elaboration of streptokinase and acquisition of cell-associated plasmin activity after incubation of wild-type and mutant cells in media containing plasminogen or plasmin. The results showed that H46A binds both the zymogen and active enzyme, generates surface-associated plasmin activity in the presence of plasminogen when producing streptokinase, and expresses its plasmin(ogen) receptor(s) independently of a functional streptokinase gene. At least part of the plasmin(ogen) binding capacity may be due to the glyceraldehyde-3-phosphate dehydrogenase type of receptor molecule, as judged by the detection of the corresponding gene.

Genetic organization of the streptokinase region of the Streptococcus equisimilis H46A chromosome.

The complete nucleotide sequences of four genes and one open reading frame (ORF1) adjacent to the streptokinase gene, skc, from Streptococcus equisimilis H46A were determined. These genes are encoded on the opposite DNA strand to skc and are arranged as follows: dexB-abc-lrp-skc-ORF1-rel. The dexB gene, coding for an alpha-glucosidase (M(r) 61,733), and abc, encoding an ABC transporter (M(r) 42,080), are similar to the dexB and msmK genes, respectively, from the multiple sugar metabolism operon of S. mutans. The lrp gene specifies a leucine-rich protein (M(r) 32,302) that has a leucine-zipper motif at its C-terminus. The function of the Lrp protein is not known but appeared to be detrimental when overexpressed in Escherichia coli. Although lrp appears not to be an essential gene, as judged by plasmid insertion mutagenesis, it is conserved in all streptococcal strains carrying a streptokinase gene. The rel gene showed significant homology to the E. coli relA and spoT genes involved in the stringent response to amino acid deprivation. Multiple alignment of the amino acid sequences of Rel (M(r) 83,913), RelA and SpoT revealed 59.4% homology of the primary structures. Northern hybridization analyses of the genes in the skc region showed skc to be transcribed most abundantly. In addition to transcripts for skc, monocistronic mRNAs were detected for all three genes divergently transcribed from skc. Although there was also some read-through transcription from lrp into abc, and from abc into dexB, the transcription pattern suggests a high degree of transcriptional and functional independence not only of skc but also abc and dexB. Prominent structural features in intergenic regions included a static DNA bending locus located upstream and a putative bidirectional transcription terminator downstream of skc.