The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis. (9/7066)

MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem. In the present study, we have demonstrated complementation of defined cysG mutants of Sal. typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal. typhimurium and E. coli cysG). MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag. After purification, the functions of the His-tagged Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD+ and Co2+. The results demonstrated that Met8p acts as a dehydrogenase and chelatase in the biosynthesis of sirohaem. Moreover, despite the fact that S. cerevisiae does not make cobalamins de novo, we have shown also that MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference in the early stages of the anaerobic cobalamin biosynthetic pathways between Sal. typhimurium and Bacillus megaterium.  (+info)

An Escherichia coli strain with all chromosomal rRNA operons inactivated: complete exchange of rRNA genes between bacteria. (10/7066)

Current global phylogenies are built predominantly on rRNA sequences. However, an experimental system for studying the evolution of rRNA is not readily available, mainly because the rRNA genes are highly repeated in most experimental organisms. We have constructed an Escherichia coli strain in which all seven chromosomal rRNA operons are inactivated by deletions spanning the 16S and 23S coding regions. A single E. coli rRNA operon carried by a multicopy plasmid supplies 16S and 23S rRNA to the cell. By using this strain we have succeeded in creating microorganisms that contain only a foreign rRNA operon derived from either Salmonella typhimurium or Proteus vulgaris, microorganisms that have diverged from E. coli about 120-350 million years ago. We also were able to replace the E. coli rRNA operon with an E. coli/yeast hybrid one in which the GTPase center of E. coli 23S rRNA had been substituted by the corresponding domain from Saccharomyces cerevisiae. These results suggest that, contrary to common belief, coevolution of rRNA with many other components in the translational machinery may not completely preclude the horizontal transfer of rRNA genes.  (+info)

The Salmonella invasin SipB induces macrophage apoptosis by binding to caspase-1. (11/7066)

Recently, Salmonella spp. were shown to induce apoptosis in infected macrophages. The mechanism responsible for this process is unknown. In this report, we establish that the Inv-Spa type III secretion apparatus target invasin SipB is necessary and sufficient for the induction of apoptosis. Purified SipB microinjected into macrophages led to cell death. Binding studies show that SipB associates with the proapoptotic protease caspase-1. This interaction results in the activation of caspase-1, as seen in its proteolytic maturation and the processing of its substrate interleukin-1beta. Caspase-1 activity is essential for the cytotoxicity. Functional inhibition of caspase-1 activity by acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone blocks macrophage cytotoxicity, and macrophages lacking caspase-1 are not susceptible to Salmonella-induced apoptosis. Taken together, the data demonstrate that SipB functions as an analog of the Shigella invasin IpaB.  (+info)

Domain organization of flagellar hook protein from Salmonella typhimurium. (12/7066)

Hook forms a universal joint, which mediates the torque of the flagellar motor to the outer helical filaments. Domain organization of hook protein from Salmonella typhimurium was investigated by exploring thermal denaturation properties of its proteolytic fragments. The most stable part of hook protein involves residues 148 to 355 and consists of two domains, as revealed by deconvolution analysis of the calorimetric melting profiles. Residues 72-147 and 356-370 form another domain, while the terminal regions of the molecule, residues 1-71 and 371-403, avoid a compact tertiary structure in the monomeric state. These folding domains were assigned to the morphological domains of hook subunits known from EM image reconstructions, revealing the overall folding of hook protein in its filamentous state.  (+info)

Physiological states of individual Salmonella typhimurium cells monitored by in situ reverse transcription-PCR. (13/7066)

The possibility of using levels of specific mRNAs in individual bacteria as indicators of single-cell physiology was investigated. Estimates of the numbers of groEL and tsf mRNAs per cell in Salmonella typhimurium cells in different physiological states were obtained by Northern analysis. The average number of groEL mRNAs per cell was estimated to be 22 in fast-growing cultures and 197 in heat-shocked cultures. The average number of tsf mRNAs per cell was estimated to be 37 in fast-growing cultures, 4 in slow-growing cultures, and 0 in nongrowing cultures. The potential of mRNA-targeted in situ reverse transcription (RT)-PCR to monitor quantitatively different levels of groEL and tsf mRNA in individual cells and thus monitor both specific gene induction and general growth activity was assessed. Neither groEL nor tsf mRNA was present in stationary-phase cells, but it was shown that stationary-phase cells contain other RNA species at high levels, which may provide a possibility for monitoring directly stationary-phase individual cells by the use of in situ RT-PCR. The outcome of the in situ RT-PCR analyses indicated that a population of fast-growing cells is heterogeneous with respect to groEL mRNA single-cell contents, suggesting a cell-cycle-controlled expression of groEL in S. typhimurium, whereas a fast-growing culture is homogeneous with respect to tsf mRNA single-cell contents, suggesting that the level of tsf mRNA is relatively constant during the cell cycle.  (+info)

Tyrosine aminotransferase catalyzes the final step of methionine recycling in Klebsiella pneumoniae. (14/7066)

An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of alpha-ketomethiobutyrate to methionine, has been purified from Klebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, and Rhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with alpha-ketomethiobutyrate and of glutamate with alpha-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with alpha-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.  (+info)

Role of ArgR in activation of the ast operon, encoding enzymes of the arginine succinyltransferase pathway in Salmonella typhimurium. (15/7066)

The ast operon, encoding enzymes of the arginine succinyltransferase (AST) pathway, was cloned from Salmonella typhimurium, and the nucleotide sequence for the upstream flanking region was determined. The control region contains several regulatory consensus sequences, including binding sites for NtrC, cyclic AMP receptor protein (CRP), and ArgR. The results of DNase I footprintings and gel retardation experiments confirm binding of these regulatory proteins to the identified sites. Exogenous arginine induced AST under nitrogen-limiting conditions, and this induction was abolished in an argR derivative. AST was also induced under carbon starvation conditions; this induction required functional CRP as well as functional ArgR. The combined data are consistent with the hypothesis that binding of one or more ArgR molecules to a region between the upstream binding sites for NtrC and CRP and two putative promoters plays a pivotal role in modulating expression of the ast operon in response to nitrogen or carbon limitation.  (+info)

The Salmonella typhi melittin resistance gene pqaB affects intracellular growth in PMA-differentiated U937 cells, polymyxin B resistance and lipopolysaccharide. (16/7066)

Salmonella typhi is the causative agent of typhoid fever in humans. A cell-culture based assay involving the human monocyte macrophage cell line U937 has been developed to examine S. typhi invasion and survival. An S. typhi PhoP- (null) mutant was shown to be restricted in net growth in phorbol myristate acetate (PMA) differentiated U937 (PMA-U937) cells, and an S. typhi PhoPc (constitutive) mutant showed a defect in invasion. Neither of the phoP/Q mutants were growth impaired in HeLa cells, however the PhoPc mutant was impaired in invasion. As opposed to what was found for S. typhi, Salmonella typhimurium wild-type, PhoP- and PhoPc mutants grew equally well in PMA-U937 cells, indicating that the PhoP(-)-mediated net growth restriction in the PMA-U937 cells was S. typhi specific. An S. typhi mutation, pqaB::MudJ, recently shown to be a PhoP-activated locus, was shown to have a net growth defect in PMA-U937 cells. Sequencing of the S. typhipqaB gene revealed it had 98% identity to the fifth gene in a S. typhimurium PmrA/B regulated operon necessary for 4-aminoarabinose lipid A modification and polymyxin B resistance. The pqaB locus was regulated by PmrA/B (whose activity is modulated by PhoP-PhoQ) and the pqaB transposon mutant was sensitive to polymyxin B. The lipopolysaccharides (LPS) of S. typhi and S. typhimurium wild-type, PhoP- and PhoPc mutants, were compared by SDS-PAGE and silver staining. Differences in the LPS profile between the two Salmonella species were observed, and shown to be affected differently by the PhoPc mutation. Additionally, the pqaB::MudJ mutation affected S. typhi LPS. The effects on LPS may have ramifications for the difference between S. typhi and S. typhimurium infection of hosts.  (+info)