Characterization of the purified hyaluronan synthase from Streptococcus equisimilis

Hyaluronan synthase (HAS) utilizes UDP-GlcUA and UDP-GlcNAc in the presence of Mg(2+) to form the GAG hyaluronan (HA). The purified HAS from Streptococcus equisimilis (seHAS) shows high fidelity in that it only polymerizes the native substrates, UDP-GlcNAc and UDP-GlcUA. However, other uridinyl nucleotides and UDP-sugars inhibited enzyme activity, including UDP-GalNAc, UDP-Glc, UDP-Gal, UDP-GalUA, UMP, UDP, and UTP. Purified seHAS was approximately 40% more active in 25 mM, compared to 50 mM, PO(4) in the presence of either 50 mM NaCl or KCl, and displayed a slight preference for KCl over NaCl. The pH profile was surprisingly broad, with an effective range of pH 6.5-11.5 and the optimum between pH 9 and 10. SeHAS displayed two apparent pK(a) values at pH 6.6 and 11.8. As the pH was increased from approximately 6.5, both K(m) and V(max) increased until pH approximately 10.5, above which the kinetic constants gradually declined. Nonetheless, the overall catalytic constant (120/s) was essentially unchanged from pH 6.5 to 10.5. The enzyme is temperature labile, but more stable in the presence of substrate and cardiolipin. Purified seHAS requires exogenous cardiolipin for activity and is very sensitive to the fatty acyl composition of the phospholipid. The enzyme was inactive or highly activated by synthetic cardiolipins containing, respectively, C14:0 or C18:1(Delta9) fatty acids. The apparent E(act) for HA synthesis is 40 kJ (9.5 kcal/mol) disaccharide. Increasing the viscosity by increasing concentrations of PEG, ethylene glycol, glycerol, or sucrose inhibited seHAS activity. For PEGs, the extent of inhibition was proportional to their molecular mass. PEGs with average masses of 2.7, 11.7, and 20 kg/mol caused 50% inhibition of V(max) at 21, 6.5, and 3.5 mM, respectively. The apparent K(i) values for ethylene glycol, glycerol, and sucrose were, respectively, 4.5, 3.3, and 1.2 mM.     

Maturation of ribosomal RNA in stringent and relaxed bacteria

Experiments with relaxed and stringent strains of E. coli confirm that rRNA synthesis continues in both during amino acid starvation. rRNA species produced are exclusively in their precursor configurations and vulnerable to nuclease attack.     

Severe invasive streptococcal infection by Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

Streptococcus pyogenes, a group A Streptococcus (GAS), has been recognized as the causative pathogen in patients with severe invasive streptococcal infection with or without necrotizing fasciitis. In recent epidemiological studies, Streptococcus dysgalactiae subsp. equisimilis (SDSE) has been isolated from severe invasive streptococcal infection. Complete genome sequence showed that SDSE is the closest bacterial species to GAS, with approximately 70% of genome coverage. SDSE, however, lacks several key virulence factors present in GAS, such as SPE‐B, the hyaluronan synthesis operon and active superantigen against human immune cells. A key event in the ability of GAS to cause severe invasive streptococcal infection was shown to be the acquisition of novel genetic traits such as phages. Strikingly, however, during severe invasive infection, GAS destroys its own covRS two‐component system, which negatively regulates many virulence factor genes, resulting in a hyper‐virulent phenotype. In contrast, this phenomenon has not been observed in SDSE. The present review describes the epidemiology of severe invasive streptococcal infection and the detailed pathogenic mechanisms of GAS and SDSE, emphasizing findings from their genome sequences and analyses of gene expression.     

Regulation of polyamine and streptomycin transport during stringent and relaxed control in Escherichia coli.

Inhibition of polyamine uptake was observed during amino acid depletion in a stringent strain of Escherichia coli CP78 but not in a relaxed strain (CP79). Chloramphenicol was shown partially to relieve the inhibition of uptake. Stringent cells which were induced for a transport system common to both polyamines and streptomycin were found to restrict the uptake of spermidine as well as streptomycin.     

Regulation of intracellular protein breakdown in stringent and relaxed strains of E. coli,

Regulation of protein breakdown by amino acids is abolished in relaxed strains of E. coli, but these mutants do respond to the deprivation of inorganic phosphate. Protein synthesis is directly required for the control of protein degradation. We suggest that the failure of amino acid-deprived rel- strains to regulate protein breakdown may be due to defective protein synthesis.     

The Histidyl-tRNA Synthetase from Streptococcus equisimilis: Overexpression in Escherichia coli,Purification, and Characterization

ABSTRACT

We describe the high-level expression of the Streptococcus equisimilis histidyl-tRNA synthetase gene hisS) in Escherichia coli and the purification and characterization of the gene product. Due to a lack of an efficient E. coli ribosome binding sequence in the hisS gene, the coding region was fused in-frame to the expression vector pT7-7, thereby creating a fusion gene construct (pT7-7recIII), which is under the control of a strong bacteriophage T7 promoter. Another construct (pT-7recII) was used for low level expression of the native histidyl-tRNA synthetase (HisRS). The plasmids were electroporated into E. coli HB101, which already contained pGP1-2. After temperature induction, the fusion HisRS, which has an extra 15 amino acids between the initiator Met and the second amino acid, Lys, was expressed at a level of —18% of total cell protein (～50 mg'liter of bacterial culture). The fusion HisRS was purified to >99% by a combination of anion exchange and cation exchange chromatography of the S100 fraction. The predicted MWs of the native and fusion proteins are 47,932 and 49,717, respectively. The mass of the active fusion HisRS was estimated to be 94,000 Da by Sephacryl S-200 gel filtration chromatography and 108,200 Da by nondenaturing PAGE. Both methods show that the funtional enzyme is a dimer of two identical subunits. SDS-PAGE analysis of purified fusion HisRS with or without reduction showed a single band of M_r = 53.7 kDa.     

Mixed culture kinetics of stringent and relaxed Escherichia coli cells in glucose-limited chemostat

The mixed culture kinetics of stringent and relaxed Escherichia coli cells were investigated in a glucose-limited chemostat at different dilution rates. Independent of the dilution rate the stringent cells competed out the relaxed cells. But the number of generations necessary for displaying the relaxed cells by the stringent ones increased with increasing dilution rate. The results are discussed as a consequence of the regulatory role of guanosine-5'-diphosphate-3'-diphosphate (ppGpp) which is known to be present at different concentrations in stringent and relaxed cells under conditions of nutrient limitation. In addition, it is postulated that the coefficient of the maintenance metabolism according to PIRT (1965) is slower in stringent cells than in relaxed cells of E. coli.     

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.     

Molecular mechanisms of Streptococcus dysgalactiae subsp equisimilis enabling intravascular persistence

Streptococcus dysgalactiae subsp. equisimilis (SDSE) can cause recurrent bacteremic infection. We have characterized novel virulence properties of an SDSE isolate of type stG485.0 that caused severe sepsis three times in a patient despite that he had opsonizing antibodies to the isolate. An infected aortic aneurysm was suspected to be the focus for the persisting bacteria. For the first time we show that this SDSE isolate, as well as other invasive SDSE isolates, aggregate human platelets and efficiently internalize into human endothelial cells. These properties may aid SDSE to persist and could explain the tendency of SDSE to cause recurrent bacteremia.     

Polysome stability in relaxed and stringent strain of Escherichia coli during amino acid starvation

The influence of amino acid starvation on polysome content was examined in relaxed and stringent strains of Escherichia coli which were isogenic for the RC locus. No difference was observed between the polysome profiles obtained from two different sets of stringent and relaxed strains starved for the same amino acid. In both relaxed and stringent strains, starvation for amino acids other than methionine resulted in only a slight breakdown of polysomes with a concomitant increase of 70S ribosomes. However, starvation for methionine in both RC stringent and relaxed strains of E. coli resulted in a more extensive degradation of polysomes and accumulation of 70S ribosomes. The 70S ribosomes obtained as a result of methionine starvation were more sensitive to degradation to 50 and 30S subunits in 10(-3)m Mg(2+) than 70S monomers obtained either by degradation of polysomes with ribonuclease or by starvation of cells for amino acids other than methionine. The 70S ribosomes from methionine starvation were similar (sensitivity to 10(-3)m Mg(2+)) to 70S ribosomes obtained from cells in which initiation of protein synthesis had been prevented by trimethoprim, an inhibitor of formylation. Since N-formyl-methionyl-transfer ribonucleic acid is required for initiation, the 70S ribosomes obtained in both methionine-starved and trimethoprim-treated cells must result from association of 50 and 30S subunits for reasons other than reinitiation. These results suggest that the level of ribonucleic acid synthesis does not influence the distribution of ribosomes in the polysome profile and vice versa.