The cmk gene encoding cytidine monophosphate kinase is located in the rpsA operon and is required for normal replication rate in Escherichia coli

Abstract

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...

Maximization of transcription of the serC (pdxF)-aroA multifunctional operon by antagonistic effects of the cyclic AMP (cAMP) receptor protein-cAMP complex and Lrp global regulators of Escherichia coli K-12

“…Sizes of protected bands were determined from standard curves of mobility plotted against the calculated sizes of the full-length probes and other RNA molecular markers (not shown) as described elsewhere (64). speB operon (17). Control hybridization reactions containing tRNA instead of mRNA showed that the apparent antisense transcription into the distal part of aroA (280-nt band in Fig.…”

The Deoxycytidine Pathway for Thymidylate Synthesis in Escherichia coli

“…CMP kinase, encoded by cmk, catalyzes the reversible interconversion of (d)CMP and (d)CDP. It is responsible for 94% of the ATPdependent CMP kinase activity in a cell extract of E. coli (9). Because the cytosine residue is synthesized in the cell from uracil at the triphosphate level, dCMP is not an intermediate in the biosynthesis of dCTP.…”

“…The slow growth produced by a cmk mutation (Fig. 3) was previously attributed (3,9) to the kinase's role in helping to regenerate the (d)CTP that is consumed during membrane Table 1. Saturated cultures were diluted in 10 mM MgSO 4 and plated on a minimal agar containing 0.4% glucose and 1% Norit-treated Difco Casamino Acids (28), both with and without Thd at 125 g/ml.…”

Escherichia coli thymidylate kinase: molecular cloning, nucleotide sequence, and genetic organization of the corresponding tmk locus

“…An amino acid sequence comparison of E. coli TMK with AMP, CMP, and GMP kinases (data not shown) has revealed significant regions of homology spanning a and c motifs as well as the invariant glycine residue (G-93 of the TMK enzyme) close to the b motif. No significant similarity of E. coli UMP kinase with E. coli TMK was found as previously described for other E. coli nucleoside monophosphate kinase (15). In fact, this enzyme appears more related to the aspartokinase family (43).…”

“…A possible involvement of this domain in protein oligomerization or allosteric regulation by nucleotides has yet to be demonstrated. The genes encoding other E. coli nucleoside monophosphate kinases specific for AMP, CMP, GMP, and UMP have been isolated and sequenced (5,15,17,43). Unlike UMP kinase which is specific for UMP, the other enzymes are active with the corresponding 2Ј deoxyribonucleotides.…”