Mutations in the tryptophan operon allow PurF-independent thiamine synthesis by altering flux in vivo.

Phosphoribosyl amine (PRA) is an intermediate in purine biosynthesis and also required for thiamine biosynthesis in Salmonella enterica. PRA is normally synthesized by phosphoribosyl pyrophosphate amidotransferase, a high-turnover enzyme of the purine biosynthetic pathway encoded by purF. However, PurF-independent PRA synthesis has been observed in strains having different genetic backgrounds and growing under diverse conditions. Genetic analysis has shown that the anthranilate synthase-phosphoribosyltransferase (AS-PRT) enzyme complex, involved in the synthesis of tryptophan, can play a role in the synthesis of PRA. This work describes the in vitro synthesis of PRA in the presence of the purified components of the AS-PRT complex. Results from in vitro assays and in vivo studies indicate that the cellular accumulation of phosphoribosyl anthranilate can result in nonenzymatic PRA formation sufficient for thiamine synthesis. These studies have uncovered a mechanism used by cells to redistribute metabolites to ensure thiamine synthesis and may define a general paradigm of metabolic robustness.

Xenorhabdus nematophilus as a model for host-bacterium interactions: rpoS is necessary for mutualism with nematodes.

Xenorhabdus nematophilus, a gram-negative bacterium, is a mutualist of Steinernema carpocapsae nematodes and a pathogen of larval-stage insects. We use this organism as a model of host-microbe interactions to identify the functions bacteria require for mutualism, pathogenesis, or both. In many gram-negative bacteria, the transcription factor sigma(S) controls regulons that can mediate stress resistance, survival, or host interactions. Therefore, we examined the role of sigma(S) in the ability of X. nematophilus to interact with its hosts. We cloned, sequenced, and disrupted the X. nematophilus rpoS gene that encodes sigma(S). The X. nematophilus rpoS mutant pathogenized insects as well as its wild-type parent. However, the rpoS mutant could not mutualistically colonize nematode intestines. To our knowledge, this is the first report of a specific allele that affects the ability of X. nematophilus to exist within nematode intestines, an important step in understanding the molecular mechanisms of this association.

Isolation and characterization of the thermoresistant gluconokinase from Escherichia coli.

It is known that two gluconokinases are inducibly expressed during the utilization of gluconate by E. coli. One is thermoresistant (activity stable for 3 h at 30 degrees C) and the other thermosensitive (losses 75% or more of its activity under the above conditions). The thermoresistant gluconokinase (EC 2.7.1.12) was isolated, purified and characterized for the first time from the E. coli mutant Ca26, a K12 derivative which lacks the thermosensitive activity. The enzyme was purified 43 fold with a recovery of 11%. The M(r) of the enzyme was 100 kDa with three equal subunits of approximately 29.5 kDa. The enzyme exhibited Michaelis-Menten kinetics and the Km values for gluconate and ATP were 0.02 mM and 0.045 mM respectively.

The leader peptide is essential for the post-translational modification of the DNA-gyrase inhibitor microcin B17.

Microcin B17 (MccB17) is a ribosomally encoded DNA-gyrase inhibitor. Ribosomally encoded antibiotics are derived from precursors containing an N-terminal leader, which is removed during maturation, and a C-terminal structural peptide. PreMccB17, the translational product of mcbA, is modified into proMccB17 by the action of three enzymes, McbB, McbC, and McbD. A chromosomally encoded peptidase then converts proMccB17 into MccB17. The role of McbB, McbC, and McbD is to convert glycine, cysteine, and serine residues present in preMccB17 into four thiazole and four oxazole rings. Using a modification-specific antibody rather than antimicrobial activity, we show that the 26-amino-acid N-terminal leader of preMccB17 is essential for the conversion of preMccB17 into proMccB17. Neither a preMccB17 peptide lacking the leader nor a preMccB17-beta-galactosidase fusion lacking the leader are post-translationally modified.

MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli.

The Escherichia coli gene murZ was recently shown to encode UDP-N-acetylglucosamine enolpyruvyl transferase, which catalyzes the first committed step of peptidoglycan biosynthesis (J. L. Marquardt, D. A. Siegele, R. Kolter, and C. T. Walsh, J. Bacteriol. 174:5748-5752, 1992). The map position of murZ (69.3 min) differed from that determined for murA (90 min), a gene which had been previously proposed to encode the same activity (P.S. Venkateswaran and H. C. Wu, J. Bacteriol. 110:935-944, 1972). Here we describe the construction of a chromosomal deletion of murZ and a plasmid containing murZ under arabinose control. Growth of cells containing the murZ deletion was dependent on the expression of murZ from the plasmid. We conclude that murZ is an essential gene and encodes the sole UDP-N-acetylglucosamine enolpyruvyl transferase of E. coli. To simplify the nomenclature, we recommend that murA be used to designate the gene at 69.3 min that encodes this activity and that the designation murZ be abandoned.