We used the technique of Mu d-directed lac operon fusion formation in an effort to identify loci in Salmonella typhimurium which are transcriptionally regulated by nutrient starvation conditions. We identified lacZ operon fusions in eight genetic loci, all of which exhibited increased transcription when starved for two or more of the following nutrients: nicotinate, phosphate, ammonium, glucose, and sulfqte. The loci have been designated stiA to stiH for starvation-inducible loci. Mutations in two sti loci (stiC and stiD) significantly decreased cell viability during prolonged periods of nicotinate starvation. sti4 and stiD are linked and map at 30 min. The stiC, stiE, stiG, and stiH loci mapped at approximately 77, 43, 88, and 56 min, respectively, on the S. typhimurium linkage map.In nature, Salmonella typhimurium can encounter a variety of environmental conditions. Some of these conditions permit optimal or nearly optimal growth, whereas others can cause mild-to-severe metabolic physiological stress, creating feast-or-famine-like situations. It is reasonable to assume that physiological mechanisms have evolved to enable bacteria to survive periods of suboptimal growth conditions or physiological stress. Several different genetic systems which respond to various environmental stresses have been studied in Escherichia coli, including the heat shock regulon (16), the SOS regulon (27), and the phosphate starvation stimulon (28-30).The phosphate starvation stimulon of E. coli appears to be analogous to the system described in this communication for S. typhimurium. Wanner and McSharry (30) identified several genes regulated under phosphate starvation conditions, which were designated psi for phosphate starvation inducible. The psi loci were classified into different genetic and physiological types on the basis of the extent of their regulation by known pho regulatory genes (phoM, phoR, and phoB) (29,30).Since glucose, ammonium, and phosphate are important for NAD biosynthesis and because NAD(P) participates in hundreds of enzymatic reactions (both anabolic and catabolic), it was reasoned that nicotinate (NA) starvation might result in numerous stress signals which, in turn, would trigger the induction of many genes associated with the maintenance or restoration of balanced growth. We report the identification of a system in S. typhimurium which appears to be analogous to the psi system of E. coli (30) but in which the initial selection was for NA starvation-inducible genetic loci. We identified and characterized, using Mu ddirected lac operon fusion construction (1, 2), eight loci which respond to starvation of two or more of the following: NA, phosphate, ammonium, glucose, and sulfate. We refer to the genes which make up this stimulon as sti for starvation inducible. The locus stiA was originally reported as sinA (6). The genetic designation has been changed to avoid potential confusion with a previously used mnemonic. MATERIALS AND METHODSBacterial strains, phage, and transductions. The strains used in this study...
Transformation frequencies greater than 1 % for some single markers were obtained in Micrococcus radiodurans when bacteria in the exponential phase of growth were resuspended in fresh growth medium containing 0.03 M-Ca2+ before being incubated with transforming DNA. Mg2+, Sr2+ or Zn2+ could not replace Ca2+ in giving high frequencies of transformation. The time required for the maximum expression of transformed markers was 2 to 3 h for resistance to rifampicin and acriflavin and 6 to 8 h for resistance to erythromycin, kanamycin and streptomycin. The comparative frequency of transformants at maximum expression for each resistance marker was: kanamycin, 1 ; streptomycin, 1 ; acriflavin, 4; erythromycin, 25 ; rifampicin, 64. Cultures were competent during all stages of exponential growth, the frequency of transformants only falling during stationary phase. The minimum time for DNA to be taken up by M. radiodurans into a DNAase-resistant form was between 3 and 6 s. From 6 s to 10 min exposure to DNA, the number of transformants increased non-linearly with time as though the process was inducible. The transformation frequency was directly proportional to the DNA concentration up to 1 pg ml-l, although even at 88 pg ml-l the bacteria were not saturated. Attempts to measure the fraction of cells which were competent, using the unlinked marker technique, gave values well in excess of one. These were interpreted in terms of multiple genome copies and approximate values of between 0-25 and 0.72 were derived for the competent fractions.
The nadA and pnuC loci of S. typhimurium were cloned and found to reside within a 2.2-kilobase region. Two-dimensional O'Farrell gel electrophoresis of the proteins produced after chloramphenicol amplificaton and subsequent release from chloramphenicol inhibition revealed NadA and PnuC to be 43,000-and 25,000-molecular-weight proteins, respectively. The data indicated that nadA and pnuC represent two distinct genes.The initial reactions in the biosynthesis of NAD in Salmonella typhimurium are catalyzed by the products of the nadA and nadB loci. These proteins form a loosely associated complex referred to as quinolinic acid synthetase. Both nadA (17 min) and nadB (55 min) are regulated coordinately at the transcriptional level by the product of the nadR locus (99 min), as determined through the construction of operon fusions (3). The pnuC locus is situated adjacent to nadA and is involved in the utilization of exogenous nicotinamide mononucleotide as a pyridine source (9). Operon fusion studies with this locus also indicate its regulation by the nadR product. Both nadA and pnuC are induced under anaerobic conditions, and at least nadA appears to be controlled in part by cyclic AMP. A study of these genes at the molecular level will provide important information about their genetic structure and also will identify regulatory sequences responsible for controlling their expression.
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