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A gene encoding a polypeptide of 25 kDa is located immediately upstream of the gene for ribosomal protein S1, rpsA. In high gene copy number, this gene, mssA, was previously found to suppress defects in smbA, which is now known to be identical to pyrH, encoding UMP kinase. We show here that the 25-kDa polypeptide comprises CMP kinase and propose that the gene be designated cmk. In a strain deleted for cmk, the pools of CMP and dCMP were elevated approximately 30-fold. We constructed a plasmid from which synthesis of CMP kinase was regulated by the lac promoter-operator and measured the synthesis rates for RNA and DNA after induction in the ⌬cmk/lacPO-cmk ؉ strain. A specific increase in the rate of DNA synthesis was observed. Further analyses showed that the replication elongation rate was halved in the ⌬cmk strain, most likely caused by the reductions of the dCTP and dTTP pools to 30 and 70%, respectively, of the levels in the parental strain, but that this was compensated for by a doubling in the frequency of initiation. The ⌬cmk strain is viable at 37؇C but cold sensitive. The cold sensitivity may be related to defects in the synthesis of phospholipids or lipopolysaccharides. In addition to the physiological studies, the region upstream of cmk was sequenced, and 120 codons with strong homology to an uncharacterized protein of the speB operon were identified.The rpsA gene, encoding ribosomal protein S1, is located at 20.5 min on the Escherichia coli chromosome. Upstream of rpsA, and separated from it by a 110-bp intercistronic region, a gene specifying a 25-kDa polypeptide of unknown function was identified, and the protein was provisionally referred to as the P25 protein (22).The rpsA gene is expressed from three promoters, of which one, P 0 , is located 5Ј of the P25 gene; the two others, P 1 and P 3 , are localized downstream within the coding region of the gene. Since several conditionally lethal mutations have been described for the rpsA region (15), and ribosomal protein operons often encode proteins involved in the biosynthesis of other types of macromolecules (reviewed in reference 14), we considered the possibility that the P25 gene has an important, if not essential, role.Recently, it was shown that the gene for the P25 protein, when present on a multicopy plasmid, suppressed the conditional lethal phenotype of certain smbA mutants (suppressor mutants of mukB, a gene that is involved in partitioning of chromosomes prior to cell division [33]). The P25 gene was therefore designated mssA (multicopy suppressor of smbA). The deduced amino acid sequence of mssA indicated that it was related to a nucleoside monophosphate kinase (32).The nucleotide sequence of smbA and its chromosomal location between the tsf and frr genes (33) establish smbA as being identical to the pyrH gene, encoding the essential enzyme UMP kinase (25). In addition to the highly specific UMP kinase, enteric bacteria contain two other pyrimidine nucleoside monophosphate kinases: a dTMP kinase and a CMP (dCMP) kinase (20). Conditional le...

Mutants deficient in orotate utilization (initially termed out mutants) were isolated by selection for resistance to 5-fluoroorotate (FOA), and the mutations of 12 independently obtained isolates were found to map at 79 to 80 min on the Salmonella typhimurium chromosome. A gene complementing the mutations was cloned and sequenced and found to possess extensive sequence identity to characterized genes for C4-dicarboxylate transport (dctA) in Rhizobium species and to the sequence inferred to be the dctA gene of Escherichia coli. The mutants were unable to utilize succinate, malate, or fumarate as sole carbon source, an expected phenotype of dctA mutants, and introduction of the cloned DNA resulted in restoration of both C4-dicarboxylate and orotate utilization. Further, succinate was found to compete with orotate for entry into the cell. The S. typhimurium dctA gene encodes a highly hydrophobic polypeptide of 45.4 kDa, and the polypeptide was found to be enriched in the membrane fraction of minicells harboring a dctA ؉ plasmid. The DNA immediately upstream of the deduced ؊35 region contains a putative cyclic AMP-cyclic AMP receptor protein complex binding site, thus affording an explanation for the more effective utilization of orotate with glycerol than with glucose as carbon source. The E. coli dctA gene was cloned from a lambda vector and shown to complement C4-dicarboxylate and orotate utilization in FOA-resistant mutants of both E. coli and S. typhimurium. The accumulated results demonstrate that the dctA gene product, in addition to transporting C4-dicarboxylates, mediates the transport of orotate, a cyclic monocarboxylate.In Salmonella typhimurium and Escherichia coli, six enzymic reactions are involved in de novo pyrimidine nucleotide biosynthesis to form UMP, the ordered sequence of the unlinked genes for the enzymes being carAB and pyrBI, -C, -D, -E, and -F. Wild-type cells are not readily permeable to intermediates of the pyrimidine biosynthetic pathway, with the exception of orotate, which is formed from dihydroorotate by the action of dihydroorotate dehydrogenase (pyrD). Orotate satisfies the pyrimidine requirement of carAB, pyrBI, pyrC, or pyrD mutants and is effective at low to moderate concentrations, provided glycerol is used as the carbon source (29,39,40). Accordingly, 5-fluoroorotate (FOA) is a potent growth inhibitor in glycerol minimal medium and has been employed for the selection of pyrH (UMP kinase) mutants of both S. typhimurium (40) and E. coli (15). It has been observed that orotate is growth rate limiting for carAB and pyrBI mutants in a concentration-dependent manner, and culturing cells at varying orotate concentrations has been used as a means to establish partial pyrimidine starvation for purposes of studying the impact of pyrimidine nucleotide limitation on specific gene expression and general aspects of macromolecular synthesis (3,5,27,36,38). In contrast, reducing the concentration of uracil serves to lower only the growth yield, not the growth rate (36), and indicates that the ...

The repressive effects of exogenous cytidine on growing cells was examined in a specially constructed strain in which the pool sizes of endogenous uridine 5'diphosphate and uridine 5'-triphosphate cannot be varied by the addition of uracil and/or uridine to the medium. Five enzymes of the pyrimidine biosynthetic pathway and one enzyme of the arginine biosynthetic pathway were assayed from cells grown under a variety of conditions. Cytidine repressed the synthesis of dihydroorotase (encoded by pyrC), dihydroorotate dehydrogenase (encoded by pyrD), and ornithine transcarbamylase (encoded by argI). Moreover, aspartate transcarbamylase (encoded bypyrB) became further derepressed upon cytidine addition, whereas no change occurred in the levels of the last two enzymes (encoded by pyrE and pyrF) of the pyrimidine pathway. Quantitative nucleotide pool determinations have provided evidence that any individual riboor deoxyribonucleoside mono-, di-, or triphosphate of cytosine or uracil is not a repressing metabolite for the pyrimidine biosynthetic enzymes. Other nucleotide derivatives or ratios must be considered.

Expression of the Salmonella typhimurium pyrC and pyrD genes is regulated in response to fluctuations in the intracellular CTP/GTP pool ratio. The repressive mechanism involves the formation of a stable secondary structure (hairpin) at the 5' ends of the transcripts that precludes translational initiation by sequestering sequences required for ribosomal binding. The potential for hairpin formation is controlled through CTP/GTP-modulated selection of the transcriptional start site. Substitution of nucleotides in the region of transcriptional initiation has revealed that selection of the transcriptional start point in vivo depends on the nucleotide context within the initiation region and the nucleoside triphosphate pool ratios. For maximal control in response to CTP/GTP pool ratios, the wild-type CCGG start site motif appears to be optimal. Changing the -35 region in the pyrC promoter to the consensus sequence, or replacement of the pyrC promoter with the lac promoter from Escherichia coli, has served to illustrate that the ability of the RNA polymerase to select the initiation site in response to the intracellular nucleoside triphosphate pools is not promoter specific but is determined by the kinetic properties of the initiating RNA polymerase during the formation of the first phosphodiester bond of the transcript.The Salmonella typhimuriumpyrC andpyrD genes encode the third and the fourth enzymes of the UMP biosynthetic pathway, namely, dihydroorotase and dihydroorotate dehydrogenase. Expression of these unlinked genes is regulated in response to fluctuations in the intracellular CTP/GTP pool ratio through modulation of the leader region of the transcript (4,8,25). High CTP/GTP pool ratios repress expression as a consequence of the production of an mRNA initiated with a CTP, 6 or 7 bp downstream of the -10 region (C at position -1 or + 1 [ Fig. 1]); this transcript is inefficiently translated because of its capacity to form a stable secondary structure (hairpin) at the 5' terminus, thereby sequestering sequences necessary for ribosomal binding. Under a low CTP/GTP pool ratio, the predominant start site is not C at position -1 or + 1, but occurs with GTP, 2 or 3 bp further downstream (G at position +3). This shorter transcript has reduced potential to form a stable secondary structure at its 5' end, and translation occurs unhindered. Evidence that a similar regulatory mechanism controls expression of the Escherichia coli pyrC gene has recently been presented (26).The effect of the relative nucleoside triphosphate concentrations on the in vivo selection of the transcriptional start point is dependent on the nucleotide sequence in the initiation region. Relocating the site for the first G nucleotide within this region 1 bp closer to the -10 region of the S. typhimunium pyrC and pyrD promoters established it as the predominant start point even when the CTP/GTP pool ratio was high. However, when the G at position +3 was changed to a C, thereby effectively moving the first G 1 bp further downstream, C at position +1 ...

The Salmonella typhimurium pyrD gene encoding dihydroorotate dehydrogenase was cloned and sequenced. In total, a sequence of 1286 nucleotide pairs was determined wherein a single open-reading-frame of 1011 bp, encoding a polypeptide of 336 amino acids having 95% similarity with the Escherichia coli pyrD gene product, was identified. A region of hyphenated-dyad symmetry exists within the leader region affording the potential for the formation of a stable secondary structure in the 5' end of the transcript. Mutations from several regulatory mutants were located within the region of dyad symmetry which would impart changes in the transcript within the putative secondary structure, implicating the secondary structure in regulation. Primer extension analysis revealed multiple transcriptional start sites located six to nine nucleotides downstream from the Pribnow box, with the primary initiation site differing in repressing and derepressing growth conditions. The results are discussed in terms of a translational attenuation model for regulation of pyrD expression.In Salmonella typhimurium and Escherichia coli, the pyrD gene encodes dihydroorotate dehydrogenase, the fourth enzyme of the pyrimidine biosynthetic pathway. The enzyme is membrane-bound [l] and linked to the electron transport system of the cell [2]. The E. coli enzyme has been purified and shown to consist of two identical flavin-containing subunits [3, 41. The synthesis of dihydroorotate dehydrogenase is regulated in a complex manner by the intracellular nucleotide pools, being negatively correlated with the concentration of CTP [5, 61 and Each mutation would decrease the stability of the putative secondary structure in the transcript, suggesting that pyrC expression is controlled at the level of translational initiation. A similar involvement of the symmetry regions of the E. coli pyrD and pyrC genes has yet to be demonstrated.To gain further insight into the regulation of pyrD expression in S. typhimuriurn, the gene has been cloned and sequenced. Regulatory mutants overexpressing pyrD have been isolated and the mutations identified. Additionally, the transcriptional start sites in repressing and derepressing conditions have been determined. MATERIALS AND METHODS Bacterial strains, bacteriophages and plasmid vectorsplasmid vectors employed, are listed in Table 1.The S. typhimuriurn LT2 and E. coli K12 strains, and the Media and growth conditionsThe composition of the minimal medium [5] and LB medium [20] has been reported. Glucose and casamino acids were added to minimal medium at 0.2%. Other supplements were added at the following final concentrations: ampicillin (50 pg/ ml) ; tetracycline (10 pg/ml) ; 5-bromo-4-chloro-3-indolyl-~-~-galactopyranoside (40 pg/ml); cytidine (50 pg/ml) ; uracil Regulation was studied in KP1725 which has a mutant pyrH gene encoding a partially defective UMP kinase, and when grown in the absence of exogenous pyrimidines is partially starved for pyrimidine nucleotides. Addition of cytidine and uracil results in swelling of ...

The pyrC gene of Salmonella typhimurium, encoding the third enzyme of pyrimidine nucleotide biosynthesis, dihydroorotase, has been cloned into the multicopy plasmid pBR322. The recombinant plasmid, pJRCl , promoted the synthesis of 20 -30-fold elevated levels of dihydroorotase. The expression of pyrC was regulated to the same extent by pyrimidines whether present on the multicopy plasmid or in single copy on the chromosome. A comparison of the polypeptides encoded by pyrC-complementing and non-complementing plasmids showed the gene product to have a molecular mass of approximately 37 kDa. The nucleotide sequence of the gene and 400 base pairs upstream from the coding region was determined. An open-reading frame, encoding a protein with a calculated molecular mass of 38500 Da, was deduced to be the coding region for pyrC. S1 nuclease mapping indicated that transcription of pyrC is initiated 40 base pairs upstream from the translational start. Subcloning of a 184-base-pair DNA fragment, which included 118 base pairs upstream from the transcriptional start, and the first eight codons of the pyrC structural gene, into a galK expression vector, established that thepyrC promoter and regulatory region are harbored on this fragment. The leader region does not show any features resembling the attenuators found in front of the coding regions of pyrB and pyrE; however, it contains a region of dyad symmetry, which may allow the leader transcript to form a stable hairpin. The possible significance of this putative hairpin formation in the regulation of pyrC expression is discussed.Six consecutive enzymic reactions provide de novo synthesis of uridine 5'-monophosphate (UMP) in Escherichia coli and Salmonella typhimurium. The genes and operons encoding these enzymes are found at scattered positions on the chromosomes of these bacteria. Expression of the pyr genes/ operons is regulated by the intracellular nucleotide pools in a complex manner. Thus, expression of carAB (pyrA in S. typhimurium; encoding carbamoylphosphate synthase) is controlled through cumulative repression by arginine and pyrimidines [l -41, expression of pyrBZ, E and F (encoding aspartate carbamoyltransferase, orotate phosphoribosyltransferase and OMP decarboxylase respectively) is negatively controlled by UTP [3, 5, 61 and by a guanine nucleotide [7], and expression of pyrC and D (encoding dihydroorotase and dihydroorotate dehydrogenase) is repressed by a cytosine nucleotide [3,5,6] and stimulated by a guanine nucleotide [7]. Gaining an understanding of the molecular basis of these control mechanisms has been hampered by the complexities associated with the interconversions of nucleotides in vivo, and the past difficulties in acquiring genuine regulatory mutants (for a review see [S]). However, recent studies utilizing molecular approaches have established that pyrBZ and pyrE expression is controlled through UTP-mediated modulation of transcription termination at an attenuator located Correspondence to J. Neuhard, Enzymafdeling, Institut for Biologisk Kemi B...

Restriction fragment analysis of mitochondrial DNA (mtDNA) was used to examine genetic variation and population structure of the migratory grasshopper, Melanoplus sanguinipes. The total DNA of 89 individuals was digested with 10 restriction enzymes and probed with three cloned EcoRT fragments representing the entire mitochondrial genome (total length 16.1 kb). Five endonucleases revealed polymorphism, yielding 12 haplotypes in combination. Nucleotide diversity indices (6) between haplotypes ranged from 0.18 to 0.92 per cent with an average value (pr) of 0.27 per cent, a comparatively low figure that possibly reflects a history of chronically small population numbers prior to the species' colonization of a suitable habitat engendered by agricultural settlement. Little, if any, differentiation was evident among locations or between prairie and parkland regions, areas spanned by a colour trait dine. Selective forces with respect to the latter must, therefore, be sufficiently powerful to withstand the attenuating effects of widespread gene flow, implied by the mtDNA data.