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1.
J Bacteriol ; 204(4): e0057121, 2022 04 19.
Article in English | MEDLINE | ID: mdl-35343794

ABSTRACT

In Escherichia coli, three isoforms of the essential translation initiation factor IF2 (IF2-1, IF2-2, and IF2-3) are generated from separate in-frame initiation codons in infB. The isoforms have earlier been suggested to additionally participate in DNA damage repair and replication restart. It is also known that the proteins RecA and RecBCD are needed for repair of DNA double-strand breaks (DSBs) in E. coli. Here, we show that strains lacking IF2-1 are profoundly sensitive to two-ended DSBs in DNA generated by radiomimetic agents phleomycin or bleomycin, or by endonuclease I-SceI. However, these strains remained tolerant to other DSB-generating genotoxic agents or perturbations to which recA and recBC mutants remained sensitive, such as to mitomycin C, type-2 DNA topoisomerase inhibitors, or DSB caused by palindrome cleavage behind a replication fork. Data from genome-wide copy number analyses following I-SceI cleavage at a single chromosomal locus suggested that, in a strain lacking IF2-1, the magnitude of recombination-dependent replication through replication restart mechanisms is largely preserved but the extent of DNA resection around the DSB site is reduced. We propose that in the absence of IF2-1 it is the synapsis of a RecA nucleoprotein filament to its homologous target that is weakened, which in turn leads to a specific failure in assembly of Ter-to-oriC directed replisomes needed for consummation of two-ended DSB repair. IMPORTANCE Double-strand breaks (DSBs) in DNA are major threats to genome integrity. In Escherichia coli, DSBs are repaired by RecA- and RecBCD-mediated homologous recombination (HR). This study demonstrates a critical role for an isoform (IF2-1) of the translation initiation factor IF2 in the repair of two-ended DSBs in E. coli (that can be generated by ionizing radiation, certain DNA-damaging chemicals, or endonuclease action). It is proposed that IF2-1 acts to facilitate the function of RecA in the synapsis between a pair of DNA molecules during HR.


Subject(s)
DNA Breaks, Double-Stranded , Escherichia coli , DNA/metabolism , DNA Repair , DNA Replication , Escherichia coli/genetics , Escherichia coli/metabolism , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism
2.
J Bacteriol ; 204(4): e0056921, 2022 04 19.
Article in English | MEDLINE | ID: mdl-35343793

ABSTRACT

Homologous recombination (HR) is critically important for chromosomal replication, as well as DNA damage repair in all life forms. In Escherichia coli, the process of HR comprises (i) two parallel presynaptic pathways that are mediated, respectively, by proteins RecB/C/D and RecF/O/R/Q; (ii) a synaptic step mediated by RecA that leads to generation of Holliday junctions (HJs); and (iii) postsynaptic steps mediated sequentially by HJ-acting proteins RuvA/B/C followed by proteins PriA/B/C of replication restart. Combined loss of RuvA/B/C and a DNA helicase UvrD is synthetically lethal, which is attributed to toxicity caused by accumulated HJs since viability in these double mutant strains is restored by removal of the presynaptic or synaptic proteins RecF/O/R/Q or RecA, respectively. Here we show that, as in ΔuvrD strains, ruv mutations confer synthetic lethality in cells deficient for transcription termination factor Rho, and that loss of RecFORQ presynaptic pathway proteins or of RecA suppresses this lethality. Furthermore, loss of IF2-1 (which is one of three isoforms [IF2-1, IF2-2, and IF2-3] of the essential translation initiation factor IF2 that are synthesized from three in-frame initiation codons in infB) also suppressed uvrD-ruv and rho-ruv lethalities, whereas deficiency of IF2-2 and IF2-3 exacerbated the synthetic defects. Our results suggest that Rho deficiency is associated with an increased frequency of HR that is mediated by the RecFORQ pathway along with RecA. They also lend support to earlier reports that IF2 isoforms participate in DNA transactions, and we propose that they do so by modulation of HR functions. IMPORTANCE The process of homologous recombination (HR) is important for maintenance of genome integrity in all cells. In Escherichia coli, the RecA protein is a critical participant in HR, which acts at a step common to and downstream of two HR pathways mediated by the RecBCD and RecFOR proteins, respectively. In this study, an isoform (IF2-1) of the translation initiation factor IF2 has been identified as a novel facilitator of RecA's function in vivo during HR.


Subject(s)
Escherichia coli Proteins , Escherichia coli , Bacterial Proteins/metabolism , DNA Helicases/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Homologous Recombination , Humans , Mutation , Peptide Initiation Factors/genetics , Peptide Initiation Factors/metabolism , Protein Isoforms/genetics
3.
J Bacteriol ; 203(17): e0019521, 2021 08 09.
Article in English | MEDLINE | ID: mdl-34124945

ABSTRACT

Topoisomerase I (Topo I) of Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here, we show that several topA alleles, including ΔtopA, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in the absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain (MDS42) or (ii) by an RNA polymerase (RNAP) mutation, rpoB*35, that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) one of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn leads to transcription-replication conflicts and to cSDR. IMPORTANCE In all life forms, double-helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli. That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability and suggests that in its absence, aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.


Subject(s)
Bacterial Proteins/metabolism , Chromosomes, Bacterial/genetics , DNA Topoisomerases, Type I/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Transcription, Genetic , Bacterial Proteins/genetics , Chromosomes, Bacterial/metabolism , DNA Replication , DNA Topoisomerases, Type I/genetics , DNA, Bacterial/genetics , DNA-Binding Proteins/genetics , Escherichia coli/enzymology , Escherichia coli/metabolism , Genome, Bacterial
6.
Microbiology (Reading) ; 165(4): 463-474, 2019 04.
Article in English | MEDLINE | ID: mdl-30724731

ABSTRACT

H-NS is an abundant nucleoid-associated protein in the enterobacteria that mediates both chromatin compaction and transcriptional silencing of numerous genes, especially those that have been acquired by horizontal transfer or that are involved in virulence functions. With two dimerization domains (N-terminal and central) and a C-terminal DNA-binding domain, the 15 kDa H-NS polypeptide can assemble as long polymeric filaments on DNA, and mutations in any of the three domains confer a dominant-negative phenotype in vivo by a subunit-poisoning mechanism. Here we confirm that several of these mutants [L26P, I119T and a truncation beyond residue 92(Δ93)] are also dominant-negative in vitro, in that they reverse the inhibition imposed by native H-NS in two different transcription assay formats (initiation+elongation, or elongation alone). On the other hand, another dominant-negative truncation mutant Δ64 (which possesses only the protein's N-terminal domain) per se completely and unexpectedly inhibited transcription in both assay formats. The Hha protein, which is a paralogue of H-NS and resembles its isolated N-terminal domain, also behaved like Δ64 as an inhibitor of transcription in vitro. We propose that under certain growth conditions, Escherichia coli RNA polymerase may be the direct inhibitory target of Hha, and that this effect is experimentally mimicked by the isolated N-terminal domain of H-NS.


Subject(s)
Bacterial Proteins/metabolism , DNA-Binding Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Transcription, Genetic/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Directed RNA Polymerases/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Gene Silencing , Mutation , Protein Domains , Protein Multimerization
7.
Transcription ; 4(4): 153-7, 2013.
Article in English | MEDLINE | ID: mdl-23756343

ABSTRACT

Nascent untranslated transcripts in bacteria are prone to generating RNA-DNA hybrids (R-loops); Rho-dependent transcription termination acts to reduce their prevalence. Here we discuss the mechanisms of R-loop formation and growth inhibition in bacteria.


Subject(s)
Bacteria/metabolism , DNA, Bacterial/metabolism , RNA, Bacterial/metabolism , Bacteria/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Replication , DNA-Directed RNA Polymerases/metabolism , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Rho Factor/genetics , Rho Factor/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic
8.
Proc Natl Acad Sci U S A ; 110(1): 258-63, 2013 Jan 02.
Article in English | MEDLINE | ID: mdl-23251031

ABSTRACT

Two pathways of transcription termination, factor-independent and -dependent, exist in bacteria. The latter pathway operates on nascent transcripts that are not simultaneously translated and requires factors Rho, NusG, and NusA, each of which is essential for viability of WT Escherichia coli. NusG and NusA are also involved in antitermination of transcription at the ribosomal RNA operons, as well as in regulating the rates of transcription elongation of all genes. We have used a bisulfite-sensitivity assay to demonstrate genome-wide increase in the occurrence of RNA-DNA hybrids (R-loops), including from antisense and read-through transcripts, in a nusG missense mutant defective for Rho-dependent termination. Lethality associated with complete deficiency of Rho and NusG (but not NusA) was rescued by ectopic expression of an R-loop-helicase UvsW, especially so on defined growth media. Our results suggest that factor-dependent transcription termination subserves a surveillance function to prevent translation-uncoupled transcription from generating R-loops, which would block replication fork progression and therefore be lethal, and that NusA performs additional essential functions as well in E. coli. Prevention of R-loop-mediated transcription-replication conflicts by cotranscriptional protein engagement of nascent RNA is emerging as a unifying theme among both prokaryotes and eukaryotes.


Subject(s)
DNA/chemistry , Escherichia coli/genetics , Genome, Bacterial/genetics , Nucleic Acid Conformation , RNA/chemistry , Rho Factor/metabolism , Transcription Termination, Genetic/physiology , Base Sequence , DNA/genetics , DNA Helicases/genetics , Escherichia coli Proteins/genetics , Models, Genetic , Molecular Sequence Data , Peptide Elongation Factors/genetics , RNA/genetics , Sequence Analysis, DNA , Sulfites , Transcription Factors/genetics , Transcriptional Elongation Factors , Viral Proteins/genetics
9.
J Bacteriol ; 194(20): 5657-66, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22904281

ABSTRACT

The protein-gene pairs ArgP-argO of Escherichia coli and LysG-lysE of Corynebacterium glutamicum are orthologous, with the first member of each pair being a LysR-type transcriptional regulator and the second its target gene encoding a basic amino acid exporter. Whereas LysE is an exporter of arginine (Arg) and lysine (Lys) whose expression is induced by Arg, Lys, or histidine (His), ArgO exports Arg alone, and its expression is activated by Arg but not Lys or His. We have now reconstituted in E. coli the activation of lysE by LysG in the presence of its coeffectors and have shown that neither ArgP nor LysG can regulate expression of the noncognate orthologous target. Of several ArgP-dominant (ArgP(d)) variants that confer elevated Arg-independent argO expression, some (ArgP(d)-P274S, -S94L, and, to a lesser extent, -P108S) activated lysE expression in E. coli. However, the individual activating effects of LysG and ArgP(d) on lysE were mutually extinguished when both proteins were coexpressed in Arg- or His-supplemented cultures. In comparison with native ArgP, the active ArgP(d) variants exhibited higher affinity of binding to the lysE regulatory region and less DNA bending at both argO and lysE. We conclude that the transcription factor LysG from a Gram-positive bacterium, C. glutamicum, is able to engage appropriately with the RNA polymerase from a Gram-negative bacterium, E. coli, for activation of its cognate target lysE in vivo and that single-amino-acid-substitution variants of ArgP can also activate the distantly orthologous target lysE, but by a subtly different mechanism that renders them noninterchangeable with LysG.


Subject(s)
Amino Acid Transport Systems, Basic/biosynthesis , Amino Acid Transport Systems/biosynthesis , Bacterial Proteins/biosynthesis , Corynebacterium glutamicum/genetics , DNA-Binding Proteins/biosynthesis , Escherichia coli Proteins/biosynthesis , Escherichia coli/genetics , Periplasmic Binding Proteins/biosynthesis , Transcription, Genetic , Amino Acid Transport Systems/genetics , Amino Acid Transport Systems, Basic/genetics , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial , Metabolic Engineering , Periplasmic Binding Proteins/genetics , Recombination, Genetic , Transcriptional Activation
10.
Mol Microbiol ; 82(6): 1330-48, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22026368

ABSTRACT

The endonuclease RNase E of Escherichia coli is essential for viability, but deletion of its C-terminal half (CTH) is not lethal. RNase E preferentially acts on 5'-monophosphorylated RNA whose generation from primary transcripts is catalysed by RppH, but ΔRppH strains are viable. Here we show that the RNase E-ΔCTH ΔRppH combination is lethal, and that the lethality is suppressed by rho or nusG mutations impairing Rho-dependent transcription termination. Lethality was correlated with defects in bulk mRNA decay and tRNA processing, which were reversed by the rho suppressor. Lethality suppression was dependent on RNase H1 or the helicase UvsW of phage T4, both of which act to remove RNA-DNA hybrids (R-loops). The rho and nusG mutations also rescued inviability of a double alteration R169Q (that abolishes 5'-sensing) with ΔCTH in RNase E, as also that of conditional RNase E deficiency. We suggest that the ΔCTH alteration leads to loss of a second 5'-end-independent pathway of RNase E action. We further propose that an increased abundance of R-loops in the rho and nusG mutants, although ordinarily inimical to growth, contributes to rescue the lethality associated with loss of the two RNase E cleavage pathways by providing an alternative means of RNA degradation.


Subject(s)
Endoribonucleases/metabolism , Escherichia coli Proteins/genetics , Escherichia coli/enzymology , Peptide Elongation Factors/genetics , Rho Factor/metabolism , Transcription Factors/genetics , Transcription, Genetic , Acid Anhydride Hydrolases/genetics , Acid Anhydride Hydrolases/metabolism , Endoribonucleases/chemistry , Endoribonucleases/genetics , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Mutation , Peptide Elongation Factors/metabolism , Protein Structure, Tertiary , RNA Stability , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , Rho Factor/genetics , Transcription Factors/metabolism
11.
J Bacteriol ; 193(21): 5985-96, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21890697

ABSTRACT

Initially identified as an inhibitor of oriC-initiated DNA replication in vitro, the ArgP or IciA protein of Escherichia coli has subsequently been described as a nucleoid-associated protein and also as a transcriptional regulator of genes involved in DNA replication (dnaA and nrdA) and amino acid metabolism (argO, dapB, and gdhA [the last in Klebsiella pneumoniae]). ArgP mediates lysine (Lys) repression of argO, dapB, and gdhA in vivo, for which two alternative mechanisms have been identified: at the dapB and gdhA regulatory regions, ArgP binding is reduced upon the addition of Lys, whereas at argO, RNA polymerase is trapped at the step of promoter clearance by Lys-bound ArgP. In this study, we have examined promoter-lac fusions in strains that were argP(+) or ΔargP or that were carrying dominant argP mutations in order to identify several new genes that are ArgP-regulated in vivo, including lysP, lysC, lysA, dapD, and asd (in addition to argO, dapB, and gdhA). All were repressed upon Lys supplementation, and in vitro studies demonstrated that ArgP binds to the corresponding regulatory regions in a Lys-sensitive manner (with the exception of argO, whose binding to ArgP was Lys insensitive). Neither dnaA nor nrdA was ArgP regulated in vivo, although their regulatory regions exhibited low-affinity binding to ArgP. Our results suggest that ArgP is a transcriptional regulator for Lys repression of genes in E. coli but that it is noncanonical in that it also exhibits low-affinity binding, without apparent direct regulatory effect, to a number of additional sites in the genome.


Subject(s)
DNA-Binding Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Lysine/metabolism , Artificial Gene Fusion , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , Escherichia coli Proteins/genetics , Gene Deletion , Genes, Reporter , Protein Binding , Regulon , Repressor Proteins/genetics , Repressor Proteins/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
12.
J Bacteriol ; 193(15): 3832-41, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21602341

ABSTRACT

Nascent transcripts in Escherichia coli that fail to be simultaneously translated are subject to a factor-dependent mechanism of termination (also termed a polarity) that involves the proteins Rho and NusG. In this study, we found that overexpression of YdgT suppressed the polarity relief phenotypes and restored the efficiency of termination in rho or nusG mutants. YdgT and Hha belong to the H-NS and StpA family of proteins that repress a large number of genes in Gram-negative bacteria. Variants of H-NS defective in one or the other of its two dimerization domains, but not those defective in DNA binding alone, also conferred a similar suppression phenotype in rho and nusG mutants. YdgT overexpression was associated with derepression of proU, a prototypical H-NS-silenced locus. Polarity relief conferred by rho or nusG was unaffected in a derivative completely deficient for both H-NS and StpA, although the suppression effects of YdgT or the oligomerization-defective H-NS variants were abolished in this background. Transcription elongation rates in vivo were unaffected in any of the suppressor-bearing strains. Finally, the polarity defects of rho and nusG mutants were exacerbated by Hha and YdgT deficiency. A model is proposed that invokes a novel role for the polymeric architectural scaffold formed on DNA by H-NS and StpA independent of the gene-silencing functions of these nucleoid proteins, in modulating Rho-dependent transcription termination such that interruption of the scaffold (as obtained by expression either of the H-NS oligomerization variants or of YdgT) is associated with improved termination efficiency in the rho and nusG mutants.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Fimbriae Proteins/metabolism , Gene Expression Regulation, Bacterial , Transcription, Genetic , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Fimbriae Proteins/genetics , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Multigene Family , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
13.
J Bacteriol ; 193(15): 3842-50, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21602355

ABSTRACT

The proteins NusA and NusG, which are essential for the viability of wild-type Escherichia coli, participate in various postinitiation steps of transcription including elongation, antitermination, and termination. NusG is required, along with the essential Rho protein, for factor-dependent transcription termination (also referred to as polarity), but the role of NusA is less clear, with conflicting reports that it both promotes and inhibits the process. In this study, we found that a recessive missense nusA mutant [nusA(R258C)] exhibits a transcription termination-defective (that is, polarity-relieved) phenotype, much like missense mutants in rho or nusG, but is unaffected for either the rate of transcription elongation or antitermination in λ phage. Various combinations of the rho, nusG, and nusA mutations were synthetically lethal, and the lethality was suppressed by expression of the N-terminal half of nucleoid protein H-NS. Our results suggest that NusA function is indeed needed for factor-dependent transcription termination and that an entire spectrum of termination efficiencies can be generated by perturbations of the Rho, NusG, NusA, and H-NS family of proteins, with the corresponding phenotypes extending from polarity through polarity relief to lethality.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Fimbriae Proteins/metabolism , Gene Expression Regulation, Bacterial , Peptide Elongation Factors/metabolism , Rho Factor/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Fimbriae Proteins/genetics , Mutation , Peptide Elongation Factors/genetics , Rho Factor/genetics , Transcription Factors/genetics , Transcriptional Elongation Factors
14.
Antonie Van Leeuwenhoek ; 95(1): 91-100, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18985430

ABSTRACT

The PhoP-PhoQ two-component system of Yersinia pseudotuberculosis, a Gram-negative enteric pathogen which causes a variety of gastrointestinal and extraintestinal infections in humans, has been shown to be necessary for virulence. A phoP-phoQ null mutant of a strain of Y. pseudotuberculosis cured of its native plasmid pYV was obtained and studied for generation of immune response in mouse model following intravenous inoculation. The phoP-phoQ null mutant elicited much weaker IgG antibody response to whole cell sonicated (WCS) antigen, in particular that of IgG2a isotype. Interferon-gamma levels were also significantly reduced in cultured splenocytes of mice immunized with phoP-phoQ null mutant. The null mutant was found to be about 72-fold less virulent than the parent isogenic strain of Y. pseudotuberculosis. Average counts in spleen of mice inoculated with the null mutant were observed to reduce by at least four logs when compared with the counts in the spleen of mice inoculated with parent isogenic strain. We can thus suggest that the Th1-type immune response of the phoP-phoQ null mutant of Y. pseudotuberculosis is diminished in mice.


Subject(s)
Bacterial Proteins/immunology , Mutation , Plasmids/genetics , Th1 Cells/immunology , Yersinia pseudotuberculosis Infections/immunology , Yersinia pseudotuberculosis/immunology , Yersinia pseudotuberculosis/pathogenicity , Animals , Antibodies, Bacterial/blood , Bacterial Proteins/genetics , Cytokines/analysis , Female , Humans , Immunoglobulin G/blood , Mice , Virulence , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis Infections/microbiology
15.
Mol Microbiol ; 54(3): 598-603, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15491353

ABSTRACT

Active mechanisms exist to prevent transcription that is uncoupled from translation in the protein-coding genes of bacteria, as exemplified by the phenomenon of nonsense polarity. Bacterial transcription-translation coupling may be viewed as one among several co-transcriptional processes, including those for mRNA processing and export in the eukaryotes, that operate in the various life forms to render the nascent transcript unavailable for formation of otherwise deleterious R-loops in the genome.


Subject(s)
Bacteria/genetics , Nucleic Acid Conformation , Protein Biosynthesis , Transcription, Genetic , Bacteria/metabolism , Cold Temperature , DNA, Bacterial/chemistry , Gene Expression Regulation, Bacterial
16.
J Bacteriol ; 186(19): 6391-9, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15375119

ABSTRACT

An ampicillin enrichment strategy following transposon insertion mutagenesis was employed to obtain NaCl-sensitive mutants of a gltBD (glutamate synthase [GOGAT]-deficient) strain of Escherichia coli. It was reasoned that the gltBD mutation would sensitize the parental strain even to small perturbations affecting osmotolerance. Insertions conferring an osmosensitive phenotype were identified in the proU, argP (formerly iciA), and glnE genes encoding a glycine betaine/proline transporter, a LysR-type transcriptional regulator, and the adenylyltransferase for glutamine synthetase, respectively. The gltBD+ derivatives of the strains were not osmosensitive. The argP mutation, but not the glnE mutation, was associated with reduced glutamate dehydrogenase activity and a concomitant NH4+ assimilation defect in the gltBD strain. Supplementation of the medium with lysine or a lysine-containing dipeptide phenocopied the argP null mutation for both osmosensitivity and NH4+ assimilation deficiency in a gltBD background, and a dominant gain-of-function mutation in argP was associated with suppression of these lysine inhibitory effects. Osmosensitivity in the gltBD strains, elicited either by lysine supplementation or by introduction of the argP or glnE mutations (but not proU mutations), was also correlated with a reduction in cytoplasmic glutamate pools in cultures grown at elevated osmolarity. We propose that an inability to accumulate intracellular glutamate at high osmolarity underlies the osmosensitive phenotype of both the argP gltBD and glnE gltBD mutants, the former because of a reduction in the capacity for NH4+ assimilation into glutamate and the latter because of increased channeling of glutamate into glutamine.


Subject(s)
DNA Transposable Elements , DNA-Binding Proteins/physiology , Escherichia coli Proteins/physiology , Escherichia coli/metabolism , Glutamate Synthase/physiology , Nucleotidyltransferases/physiology , Sodium Chloride/pharmacology , Escherichia coli/genetics , Glutamic Acid/metabolism , Lysine/pharmacology , Quaternary Ammonium Compounds/metabolism
17.
J Bacteriol ; 186(11): 3539-46, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15150242

ABSTRACT

The anonymous open reading frame yggA of Escherichia coli was identified in this study as a gene that is under the transcriptional control of argP (previously called iciA), which encodes a LysR-type transcriptional regulator protein. Strains with null mutations in either yggA or argP were supersensitive to the arginine analog canavanine, and yggA-lac expression in vivo exhibited argP(+)-dependent induction by arginine. Lysine supplementation phenocopied the argP null mutation in that it virtually abolished yggA expression, even in the argP+ strain. The dipeptides arginylalanine and lysylalanine behaved much like arginine and lysine, respectively, to induce and to turn off yggA transcription. Dominant missense mutations in argP (argPd) that conferred canavanine resistance and rendered yggA-lac expression constitutive were obtained. The protein deduced to be encoded by yggA shares similarity with a basic amino acid exporter (LysE) of Corynebacterium glutamicum, and we obtained evidence for increased arginine efflux from E. coli strains with either the argPd mutation or multicopy yggA+. The null yggA mutation abolished the increased arginine efflux from the argPd strain. Our results suggest that yggA encodes an ArgP-regulated arginine exporter, and we have accordingly renamed it argO (for "arginine outward transport"). We propose that the physiological function of argO may be either to prevent the accumulation to toxic levels of canavanine (which is a plant-derived antimetabolite) or arginine or to maintain an appropriate balance between the intracellular lysine and arginine concentrations.


Subject(s)
Arginine/metabolism , DNA-Binding Proteins/physiology , Escherichia coli Proteins/physiology , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Open Reading Frames , Biological Transport , Canavanine/pharmacology , Transcription, Genetic
18.
Genetics ; 166(3): 1165-76, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15082538

ABSTRACT

Escherichia coli nusG and rho mutants, which are defective in transcription termination, are killed following transformation with several ColE1-like plasmids that lack the plasmid-encoded copy-number regulator gene rom because of uncontrolled plasmid replication within the cells. In this study, a mutation [dnaC1331(A84T)] in the dnaC gene encoding the replicative helicase-loading protein was characterized as a suppressor of this plasmid-mediated lethality phenotype. The mutation also reduced the copy number of the plasmids in otherwise wild-type strains. In comparison with the isogenic dnaC(+) strain, the dnaC mutant was largely unaffected for (i) growth on rich or minimal medium, (ii) tolerance to UV irradiation, or (iii) survival in the absence of the PriA, RecA, or RecB proteins. However, it was moderately SOS-induced and was absolutely dependent on both the Rep helicase and the PriC protein for its viability. A dnaC1331(A84T) dam mutant, but not its mutH derivative, exhibited sensitivity to growth on rich medium, suggestive of a reduced capacity in the dnaC1331(A84T) strains to survive chromosomal double-strand breaks. We propose that DnaC-A84T is proficient in the assembly of replication forks for both initiation of chromosome replication (at oriC) and replication restart via the Rep-PriC pathway, but that it is specifically defective for replication restart via the PriA-PriB pathway (and consequently also for replication of the Rom(-) ColE1-like plasmids).


Subject(s)
Bacterial Proteins/genetics , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , Escherichia coli/genetics , Gene Dosage , Mutation , Plasmids , Bacterial Proteins/metabolism , Chromosomes, Bacterial , DNA Replication , DNA-Binding Proteins/metabolism , Escherichia coli/growth & development , Escherichia coli/radiation effects , Gene Expression Regulation, Bacterial , Genes, Lethal , Replication Protein A , SOS Response, Genetics , Suppression, Genetic , Ultraviolet Rays
19.
Genetics ; 166(2): 681-92, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15020459

ABSTRACT

A conditional lethal galE(Ts)-based strategy was employed in Escherichia coli, first to eliminate all growth-associated chromosomal reversions in lacZ or forward mutations in lacI/lacO by incubation at the restrictive temperature and subsequently to recover (as papillae) spontaneous mutations that had arisen in the population of nondividing cells after shift to the permissive temperature. Data from lacZ reversion studies in mutator strains indicated that the products of all genes for mismatch repair (mutHLS, dam, uvrD), of some for oxidative damage repair (mutMT), and of that for polymerase proofreading (dnaQ) are required in dividing cells; some others for oxidative damage repair (mutY, nth nei) are required in both dividing and nondividing cells; and those for alkylation damage repair (ada ogt) are required in nondividing cells. The spectrum of lacI/lacO mutations in nondividing cells was distinguished both by lower frequencies of deletions and IS1 insertions and by the unique occurrence of GC-to-AT transitions at lacO +5. In the second approach to study mutations that had occurred in nondividing cells, lacI/lacO mutants were selected as late-arising papillae from the lawn of a galE+ strain; once again, transitions at lacO +5 were detected among the mutants that had been obtained from populations initially grown on poor carbon sources such as acetate, palmitate, or succinate. Our results indicate that the lacO +5 site is mutable only in nondividing cells, one possible mechanism for which might be that random endogenous alkylation (or oxidative) damage to DNA in these cells is efficiently corrected by the Ada Ogt (or Nth Nei) repair enzymes at most sites but not at lacO +5. Furthermore, the late-arising papillae from the second approach were composed almost exclusively of dominant lacI/lacO mutants. This finding lends support to "instantaneous gratification" models in which a spontaneous lesion, occurring at a random site in DNA of a nondividing cell, is most likely to be fixed as a mutation if it allows the cell to immediately exit the nondividing state.


Subject(s)
Escherichia coli/genetics , Lac Operon/genetics , Acetates/metabolism , Escherichia coli/drug effects , Genes, Dominant , Microbial Sensitivity Tests , Mutagens/pharmacology , Mutation , UDPglucose 4-Epimerase/genetics , UDPglucose 4-Epimerase/metabolism
20.
J Mol Biol ; 332(1): 31-46, 2003 Sep 05.
Article in English | MEDLINE | ID: mdl-12946345

ABSTRACT

Two Escherichia coli genes, rnhA and recG, encode products that disrupt R-loops by hydrolysis and unwinding, respectively. It is known that the propensity for R-loop formation in vivo is increased during growth at 21 degrees C. We have identified several links between rnhA, recG, and R-loop-dependent plasmid replication on the one hand, and genes rho and nusG involved in factor-dependent transcription termination on the other. A novel nusG-G146D mutation phenocopied a rho-A243E mutation in conferring global deficiency in transcription termination, and both mutants were killed at 21 degrees C following overexpression of rnhA(+). Mutant combinations rnhA-nusG or recG-rho were synthetically lethal at 21 degrees C, with the former being suppressed by recG(+) overexpression. rho and nusG mutants were killed following transformation with plasmids such as pACYC184 or pUC19 (which have R-loop replication intermediates) even at 30 degrees C or 37 degrees C, and the lethality was correlated with greatly increased content of supercoiled monomer species of these and other co-resident R-loop-dependent plasmids. Plasmid-mediated lethality in the mutants was suppressed by overexpression of rnhA(+) or recG(+). Two additional categories of trans-acting suppressors of the plasmid-mediated lethality were identified whose primary effects were, respectively, a reduction in plasmid copy number even in the wild-type strain, and a restoration of the proficiency of in vivo transcription termination in the nusG and rho mutant strains. The former category of suppressors included rom(+), and mutations in rpoB(Q513L), pcnB, and polA, whereas the latter included a mutation in rho (R221C) and several non-null mutations (E74K, L26P, and delta64-137) in the gene encoding the nucleoid protein H-NS. We propose that an increased occurrence of chromosomal R-loops in the rho and nusG mutants leads to titration of a cyloplasmic host factor(s) that negatively modulates the stability of plasmid R-loop replication intermediates and consequently to runaway plasmid replication.


Subject(s)
Chromosomes/genetics , DNA Replication , Escherichia coli/genetics , Nucleic Acid Conformation , Plasmids/genetics , Transcription, Genetic , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Mutation , Peptide Elongation Factors/genetics , Peptide Elongation Factors/metabolism , Plasmids/metabolism , Rho Factor/genetics , Rho Factor/metabolism , Ribonuclease H/genetics , Ribonuclease H/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
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