SUMMARY The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK) revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A survey of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria where (p)ppGpp regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.

In Salmonella typhimurium, thiamine pyrophosphate (TPP) is a required cofactor for several enzymes in central metabolism. Herein we identify a new thi operon, thiBPQ (designated sfuABC in Escherichia coli), required for the transport of thiamine and TPP into the cell. Insertions in the operon result in strains that are phenotypically and biochemically defective in thiamine and TPP transport. Data presented herein show that this operon is transcriptionally repressed in the presence of exogenous thiamine, with TPP the likely regulatory molecule. This work represents the first identification of thiamine transport genes in bacteria and demonstrates the function of a proposed ABC transporter in E. coli.

Thiamine pyrophosphate (TPP) is a required cofactor in Salmonella typhimurium that is generated de novo by the condensation of 4-amino-5-hydroxymethyl pyrimidine (HMP) pyrophosphate and 4-methyl-5-(␤-hydroxyethyl)-thiazole (THZ) monophosphate. The THZ and HMP moieties are independently synthesized, and labeling studies have demonstrated probable metabolic precursors to both. We present herein the initial characterization of thiI, a gene required for THZ synthesis. We show that thiI is a 1,449-bp open reading frame located at minute 9.65 on the S. typhimurium chromosome and that it encodes a 483-amino-acid protein with a predicted molecular mass of 55 kDa. Unlike genes in the thiamine biosynthetic operon at minute 90, thiI is not transcriptionally regulated by TPP.Thiamine pyrophosphate (TPP) is a required cofactor for a number of well-characterized enzymes (e.g., ␣-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and transketolase). Although several recent studies of Saccharomyces cerevisiae (20-23), Escherichia coli (2, 28), and Salmonella typhimurium (9, 25, 29) have furthered our understanding of the synthesis of this vitamin, the genetic and biochemical characterization of the biosynthetic pathway is still in the early stages. In S. typhimurium and E. coli, thiamine monophosphate (TMP) is generated by the condensation of 4-amino-5-hydroxymethyl pyrimidine pyrophosphate (HMP-PP) and 4-methyl-5-(␤-hydroxyethyl)-thiazole monophosphate (THZ-P) and then phosphorylated to TPP, the physiologically relevant form of the cofactor (Fig.

We report results of studies of the conversion of adenosylcobyric acid (AdoCby) to adenosylcobinamidephosphate, the last step of the de novo corrin ring biosynthetic branch of the adenosylcobalamin (coenzyme B 12 ) pathway of Salmonella enterica serovar Typhimurium LT2. Previous reports have implicated the CbiB protein in this step of the pathway. Hydropathy analysis predicted that CbiB would be an integral membrane protein. We used a computer-generated topology model of the primary sequence of CbiB to guide the construction of CbiB-LacZ and CbiB-PhoA protein fusions, which were used to explore the general topology of CbiB in the cell membrane. A refined model of CbiB as an integral membrane protein is presented. In vivo analyses of the effect of single-amino-acid changes showed that periplasm-and cytosol-exposed residues are critical for CbiB function. Results of in vivo studies also show that ethanolamine-phosphate (EA-P) is a substrate of CbiB, but L-Thr-P is not, and that CbiB likely activates AdoCby by phosphorylation. The latter observation leads us to suggest that CbiB is a synthetase not a synthase enzyme. Results from mass spectrometry and bioassay experiments indicate that serovar Typhimurium synthesizes norcobalamin (cobalamin lacking the methyl group at C176) when EA-P is the substrate of CbiB.

In Salmonella enterica, the last step of the synthesis of adenosylcobamide is catalysed by the cobalamin synthase enzyme encoded by the cobS gene of this bacterium. Overexpression of the S. enterica cobS gene in Escherichia coli elicited the accumulation of the phage shock protein PspA, a protein whose expression has been linked to membrane stress. Resolution of inner and outer membranes of S. enterica by isopycnic density ultracentrifugation showed CobS activity associated with the inner membrane, a result that was confirmed using antibodies against CobS. Computer analysis of the predicted amino acid sequence of CobS suggested it was an integral membrane protein.Results of experiments performed with strains carrying plasmids encoding CobS-alkaline phosphatase or CobS-b-galactosidase protein fusions were consistent with the membrane localization of the CobS protein. Modifications to the predicted model were made based on data obtained from experiments using protein fusions. The function encoded by the cobS orthologue in the methanogenic archaeon Methanobacterium thermoautotrophicum strain DH compensated for the lack of CobS during cobalamin synthesis in cobS strains of S. enterica. Cobalamin synthase activity was also detected in a membrane preparation of M. thermoautotrophicum. It was concluded that the assembly of the nucleotide loop of adenosylcobamides in archaea and bacteria is a membrane-associated process. Possible reasons for the association of adenosylcobamide biosynthetic enzymes with the cell membrane are discussed.