Multicopy suppression of cold-sensitive sec mutations in Escherichia coli.

ABSTRACT The BipA protein of Escherichia coli has intriguing similarities to the elongation factor subfamily of GTPases, including EF-Tu, EF-G, and LepA. In addition, phenotypes of a bipA deletion mutant suggest that BipA is involved in regulation of a variety of pathways. These two points have led to speculation that BipA may be a novel regulatory protein that affects efficient translation of target genes through direct interaction with the ribosome. We isolated and characterized suppressors of the cold-sensitive growth phenotype exhibited by ΔbipA strains and identified insertion mutations in rluC. The rluC gene encodes a pseudouridine synthase responsible for pseudouridine modification of 23S rRNA at three sites, all located near the peptidyl transferase center. Deletion of rluC not only suppressed cold sensitivity but also alleviated the decrease in capsule synthesis exhibited by bipA mutants, suggesting that the phenotypic effects of BipA are manifested through an effect on the ribosome. The suppressor effect is specific to rluC, as deletion of other rlu genes did not relieve cold sensitivity, and further, more than a single pseudouridine residue is involved, as alteration of single residues did not produce suppressors. These results are consistent with a role for BipA in either the structure or the function of the ribosome and imply that wild-type ribosomes are dependent on BipA for efficient expression of target mRNAs and that the lack of pseudouridylation at these three sites renders the ribosomes BipA independent.

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Dissection of 16S rRNA Methyltransferase (KsgA) Function in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.01259-07 ◽

2007 ◽

Vol 189 (23) ◽

pp. 8510-8518 ◽

Cited By ~ 17

Author(s):

Koichi Inoue ◽

Soumit Basu ◽

Masayori Inouye

Keyword(s):

Escherichia Coli ◽

16S Rrna ◽

Growth Defect ◽

Methyltransferase Activity ◽

Additional Function ◽

Multicopy Suppressor ◽

E Coli ◽

Acid Shock ◽

Cold Sensitive ◽

Increased Sensitivity

ABSTRACT A 16S rRNA methyltransferase, KsgA, identified originally in Escherichia coli is highly conserved in all living cells, from bacteria to humans. KsgA orthologs in eukaryotes possess functions in addition to their rRNA methyltransferase activity. E. coli Era is an essential GTP-binding protein. We recently observed that KsgA functions as a multicopy suppressor for the cold-sensitive cell growth of an era mutant [Era(E200K)] strain (Q. Lu and M. Inouye, J. Bacteriol. 180:5243-5246, 1998). Here we observed that although KsgA(E43A), KsgA(G47A), and KsgA(E66A) mutations located in the S-adenosylmethionine-binding motifs severely reduced its methyltransferase activity, these mutations retained the ability to suppress the growth defect of the Era(E200K) strain at a low temperature. On the other hand, a KsgA(R248A) mutation at the C-terminal domain that does not affect the methyltransferase activity failed to suppress the growth defect. Surprisingly, E. coli cells overexpressing wild-type KsgA, but not KsgA(R248A), were found to be highly sensitive to acetate even at neutral pH. Such growth inhibition also was observed in the presence of other weak organic acids, such as propionate and benzoate. These chemicals are known to be highly toxic at acidic pH by lowering the intracellular pH. We found that KsgA-induced cells had increased sensitivity to extreme acid conditions (pH 3.0) compared to that of noninduced cells. These results suggest that E. coli KsgA, in addition to its methyltransferase activity, has another unidentified function that plays a role in the suppression of the cold-sensitive phenotype of the Era(E200K) strain and that the additional function may be involved in the acid shock response. We discuss a possible mechanism of the KsgA-induced acid-sensitive phenotype.

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Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00044-08 ◽

2008 ◽

Vol 190 (15) ◽

pp. 5368-5381 ◽

Cited By ~ 46

Author(s):

Kazuyuki Fujimitsu ◽

Masayuki Su'etsugu ◽

Yoko Yamaguchi ◽

Kensaku Mazda ◽

Nisi Fu ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Cell Cycle Progression ◽

Synthetic Lethality ◽

Active Form ◽

Dependent Manner ◽

Chromosomal Replication ◽

Independent Manner ◽

Multiple Copies ◽

Cold Sensitive

ABSTRACT The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the β clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25°C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25°C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42°C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25°C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25°C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.

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Genetics and Regulation of the Major Enzymes of Alanine Synthesis in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00738-10 ◽

2010 ◽

Vol 192 (20) ◽

pp. 5304-5311 ◽

Cited By ~ 37

Author(s):

Sok Ho Kim ◽

Barbara L. Schneider ◽

Larry Reitzer

Keyword(s):

Escherichia Coli ◽

Carbon Source ◽

Genetic Analysis ◽

Regulatory Protein ◽

Carbon Sources ◽

Optimal Growth ◽

Expression Studies ◽

Alanine Synthesis ◽

Multicopy Suppression

ABSTRACT Genetic analysis of alanine synthesis in the model genetic organism Escherichia coli has implicated avtA, the still uncharacterized alaA and alaB genes, and probably other genes. We identified alaA as yfbQ. We then transferred mutations in several transaminase genes into a yfbQ mutant and isolated a mutant that required alanine for optimal growth. For cells grown with carbon sources other than pyruvate, the major alanine-synthesizing transaminases are AvtA, YfbQ (AlaA), and YfdZ (which we designate AlaC). Growth with pyruvate as the carbon source and multicopy suppression suggest that several other transaminases can contribute to alanine synthesis. Expression studies showed that alanine modestly repressed avtA and yfbQ but had no effect on yfdZ. The leucine-responsive regulatory protein (Lrp) mediated control by alanine. We purified YfbQ and YfdZ and showed that both are dimers with Km s for pyruvate within the intracellular range of pyruvate concentration.

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