Multicopy suppresors, mssA and mssB, of an smbA mutation of Escherichia coli

Yamanaka, Kunitoshi; Ogura, Teru; Koonin, Eugene V.; Niki, Hironori; Hiraga, Sota

doi:10.1007/bf00283870

Cited by 34 publications

(31 citation statements)

References 29 publications

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“…The kanamycin-resistant gene (1.3-kb HincII fragment) from pUC7Km(Pst) was inserted into the middle of the coding region of the csdA gene (alternatively, deaD and mssB) at the EcoRV site in pKX164 (65), yielding pKNJ9026. The linearized pKNJ9026 DNA fragment containing the disrupted csdA gene (csdA::kan) was then introduced into the chromosome of a recD mutant FS1576 (recD1009 thi-1 thr-1 leuB6 lacY1 tonA21 supE44).…”

Section: Methodsmentioning

confidence: 99%

“…5, the pnp mutant was extremely sensitive to low temperature and was unable to form colonies below 25°C, which agrees well with the results reported by Luttinger et al (41); the colony-forming ability dropped to 10 Ϫ7 . The cold-sensitive growth phenotype of the pnp mutant was fully complemented by transforming cells with plasmid pKX150 carrying the pnp ϩ gene (65), indicating that PNPase function is essential for growth at low temperature but not at high temperature.…”

Section: Figmentioning

confidence: 99%

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Selective mRNA Degradation by Polynucleotide Phosphorylase in Cold Shock Adaptation in Escherichia coli

2001

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Upon cold shock, Escherichia coli cell growth transiently stops. During this acclimation phase, specific cold shock proteins (CSPs) are highly induced. At the end of the acclimation phase, their synthesis is reduced to new basal levels, while the non-cold shock protein synthesis is resumed, resulting in cell growth reinitiation. Here, we report that polynucleotide phosphorylase (PNPase) is required to repress CSP production at the end of the acclimation phase. A pnp mutant, upon cold shock, maintained a high level of CSPs even after 24 h. PNPase was found to be essential for selective degradation of CSP mRNAs at 15°C. In a poly(A) polymerase mutant and a CsdA RNA helicase mutant, CSP expression upon cold shock was significantly prolonged, indicating that PNPase in concert with poly(A) polymerase and CsdA RNA helicase plays a critical role in cold shock adaptation.

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Section: Methodsmentioning

confidence: 99%

Section: Figmentioning

confidence: 99%

Selective mRNA Degradation by Polynucleotide Phosphorylase in Cold Shock Adaptation in Escherichia coli

2001

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“…For nucleotide pool determinations, the Tris-buffered medium of Edlin and Maaløe (12) with 0.3 mM phosphate was used. Cells were labeled for at least one generation of exponential growth in this medium containing 32 P i with a specific activity of 33 Ci/mol. A 0.5-ml aliquot of the labeled culture was filtered onto a 13-mm membrane filter (0.45-m pore size), and the filter was extracted with 0.2 ml of ice-cold 0.3 M formic acid.…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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

et al. 1995

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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...

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“…The N-terminal globular domain of MukB binds to filaments of the FtsZ protein polymer (12) and to eukaryotic microtubules in vitro (13). To analyze the in vivo function of MukB, various suppressor mutations and syntheticlethal mutations have been identified (10,(25)(26)(27). Mutations of the topA gene, encoding topoisomerase I, suppress the temperature-sensitive growth and anucleate cell production caused by null mutation of each muk gene.…”

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Null Mutation of thedamorseqAGene Suppresses Temperature-Sensitive Lethality but Not Hypersensitivity to Novobiocin ofmukNull Mutants

et al. 2000

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Escherichia coli mukF, mukE, and mukB null mutants have common phenotypes such as temperaturedependent colony formation, anucleate cell production, chromosome cutting by septum closure, and abnormal localization of SeqA-DNA clusters. We show here that the associated muk null mutations cause hypersensitivity to novobiocin. Null mutation of either dam or seqA suppressed partially the temperature-sensitive lethality but failed to suppress the anucleate cell production and the hypersensitivity to novobiocin caused by muk null mutations.The mukF, mukE, and mukB genes are essential for faithful partitioning of sister chromosomes into both daughter cells in Escherichia coli (15,17,28). Null mutation of each muk gene causes the medium-dependent, temperature-sensitive, lethal phenotype and produces a significant number of anucleate cells of normal size during growth at permissive low temperature (16, 28). The three Muk proteins form a complex in vitro (29). Purified MukB protein has a DNA binding activity, an ATP and GTP binding activity (15), and a Mg 2ϩ -dependent ATPase activity (12,29). The N-terminal globular domain of MukB binds to filaments of the FtsZ protein polymer (12) and to eukaryotic microtubules in vitro (13). To analyze the in vivo function of MukB, various suppressor mutations and syntheticlethal mutations have been identified (10,(25)(26)(27). Mutations of the topA gene, encoding topoisomerase I, suppress the temperature-sensitive growth and anucleate cell production caused by null mutation of each muk gene. The suppression correlates with excess negative supercoiling by DNA gyrase, because the gyrase inhibitor coumermycin reverses the suppression caused by the topA mutations, suggesting that muk mutations cause a defect in chromosome folding and DNA condensation (20).DNA is fully methylated by DNA adenine methyltransferase (Dam methylase) in E. coli wild-type cells (1, 2, 5). Following initiation from the chromosomal origin (oriC), newly synthesized nascent DNA strands acquire a hemimethylated state at Dam methylation sites. The seqA gene is essential for control of synchronous initiation of chromosome replication (3, 14, 23). The purified SeqA protein preferentially binds GATC sequences in hemimethylated DNA (4,22). SeqA is localized as discrete foci in exponentially growing wild-type cells of E. coli (7,18). Formation of the visible SeqA foci depends on Dam methylation (7, 18) and ongoing replication (8), suggesting clusters of SeqA molecules which bind to hemimethylated nascent DNA strands. A single SeqA focus localized at midcell seems to separate into two foci, and these foci subsequently migrate rapidly in opposite directions to 1/4 and 3/4 positions of the cell (7,8,18). In the mukB null mutant, SeqA clusters are abnormal in size and subcellular localization (7, 18), suggesting that MukB may participate in separation or migration of SeqA-DNA clusters. Interestingly, E. coli and related bacteria possess MukF, MukE, and MukB together with SeqA, MutH, and Dam methylase (8).Weitao et al. (24) showed...

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Multicopy suppresors, mssA and mssB, of an smbA mutation of Escherichia coli

Cited by 34 publications

References 29 publications

Selective mRNA Degradation by Polynucleotide Phosphorylase in Cold Shock Adaptation in Escherichia coli

Selective mRNA Degradation by Polynucleotide Phosphorylase in Cold Shock Adaptation in Escherichia coli

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

Null Mutation of thedamorseqAGene Suppresses Temperature-Sensitive Lethality but Not Hypersensitivity to Novobiocin ofmukNull Mutants

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