A Corynebacterium glutamicum rnhA recG double mutant showing lysozyme-sensitivity, temperature-sensitive growth, and UV-sensitivity

Corynebacterium glutamicum mutant KY9707 was originally isolated for lysozyme-sensitivity, and showed temperature-sensitive growth. Two DNA fragments from a wild-type C. glutamicum chromosomal library suppressed the temperature-sensitivity of KY9707. These clones also rescued the lysozyme-sensitivity of KY9707, although partially. One of them encodes a protein of 382 amino acid residues, the N-terminal domain of which was homologous to RNase HI. This gene suppressed the temperature-sensitive growth of an Escherichia coli rnhA rnhB double mutant. We concluded that this gene encodes a functional RNase HI of C. glutamicum and designated it as rnhA. The other gene encodes a protein of 707 amino acid residues highly homologous to RecG protein. The C. glutamicum recG gene complemented the UV-sensitivity of E. coli recG258::kan mutant. KY9707 showed increased UV-sensitivity, which was partially rescued by either the recG or rnhA gene of C. gluamicum. Point mutations were found in both recG and rnhA genes in KY9707. These suggest that temperature-sensitive growth, UV-sensitivity, and probably lysozyme-sensitivity also, of KY9707 were caused by mutations in the genes encoding RNase HI and RecG.     

Cloning of the Genes Concerned in Phenylalanine Biosynthesis in Corynebacterium glutamicum and its Application to Breeding of a Phenylalanine Producing Strain

The chorismate mutase and prephenate dehydratase genes of phenylalanine producing Corynebacterium glutamicum K38, which is resistant to p-fluorophenylalanine and m-fluorophenylalanine, were cloned into plasmid pCE53 in C. glutamicum KY9456, which lacks chorismate mutase and prephenate dehydratase. One of the resultant plasmids, pCmB4, contained a 9.4kb BamHI DNA fragment inserted into the unique BamHl site of pCE53. Plasmid pCmB4 complemented a phenylalanine and tyrosine double auxotroph of C. glutamicum KY9456. Introduction of pCmB4 into C. glutamicum RRL5 resulted in an about ten times increase in chorismate mutase activity. C. glutamicum K38 carrying the plasmid accumulated 19.0mg/ml of phenylalanine (50% increase over the yield of K38).     

RNase HI stimulates the activity of RnlA toxin in Escherichia coli

A type II toxin–antitoxin system in Escherichia coli, rnlA‐rnlB, functions as an anti‐phage mechanism. RnlA is a toxin with an endoribonuclease activity and the cognate RnlB inhibits RnlA toxicity in E. coli cells. After bacteriophage T4 infection, RnlA is activated by the disappearance of RnlB, resulting in the rapid degradation of T4 mRNAs and consequently no T4 propagation, when T4 dmd is defective: Dmd is an antitoxin against RnlA for promoting own propagation. Previous studies suggested that the activation of RnlA after T4 infection was regulated by multiple components. Here, we provide the evidence that RNase HI is an essential factor for activation of RnlA. The dmd mutant phage could grow on ΔrnhA (encoding RNase HI) cells, in which RnlA‐mediated mRNA cleavage activity was defective. RNase HI bound to RnlA in vivo and enhanced the RNA cleavage activity of RnlA in vitro. In addition, ectopic expression of RnlA in ΔrnlAB ΔrnhA cells has less effect on cell toxicity and RnlA‐mediated mRNA degradation than in ΔrnlAB cells. This is the first example of a direct factor for activation of a toxin.     

<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> RNase E/G-type endoribonuclease encoded by NCgl2281 is involved in the 5′ maturation of 5S rRNA

Corynebacterium glutamicum has one RNase E/G ortholog and one RNase J ortholog but no RNase Y. We previously reported that the C. glutamicum NCgl2281 gene encoding the RNase E/G ortholog complemented the rng::cat mutation in Escherichia coli but not the rne-1 mutation. In this study, we constructed an NCgl2281 knockout mutant and found that the mutant cells accumulated 5S rRNA precursor molecules. The processing of 16S and 23S rRNA, tRNA, and tmRNA was normal. Primer extension analysis revealed that the RNase E/G ortholog cleaved at the −1 site of the 5′ end of 5S rRNA. However, 3′ maturation was essentially unaffected. These findings showed that C. glutamicum NCgl2281 endoribonuclease is involved in the 5′ maturation of 5S rRNA. This is the first report showing the physiological function of the RNase E/G ortholog in bacteria having one RNase E/G and one RNase J but no RNase Y.     

TheamrG1 gene is involved in the activation of acetate inCorynebacterium glutamicum

During growth ofCorynebacterium glutamicum on acetate as its carbon and energy source, the expression of theptaack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genesamrG1 andamrG2 found in the deregulated transposon mutant C.glutamicum G25. TheamrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C.glutamicum. We constructed an in-frame deletion mutant and an overexpressing strain ofamrG1 in the C.glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, theamrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion, overexpression of theamrG1 gene in C.glutamicum 13032 had the adverse regulatory effect. These results indicate that theamrG1 gene encodes a repressor or co-repressor of theptaack operon.     

RecA, Tus protein and constitutive stable DNA replication inEscherichia coli rnhA mutants

Constitutive stable DNA replication (cSDR), which uniquely occurs inEscherichia coli rnhA mutants deficient in ribonuclease HI activity, requires RecA function. TherecA428 mutation, which inactivates the recombinase activity but imparts a constitutive coprotease activity, blocks cSDR inrnhA mutants. The result indicates that the recombinase activity of RecA, which promotes homologous pairing and strand exchange, is essential for cSDR. Despite the requirement for RecA recombinase activity, mutations inrecB, recD, recJ, ruvA andruvC neither inhibit nor stimulate cSDR. It was proposed that the property of RecA essential for homologous pairing and strand exchange is uniquely required for initiation of cSDR inrnhA mutants without involving the homologous recombination process. The possibility that RecA protein is necessary to counteract the action of Tus protein, a contra-helicase which stalls replication forks in theter region of the chromosome, was ruled out because introduction of thetus : :kan mutation, which inactivates Tus protein, did not alleviate the RecA requirement for cSDR.     

RNase HI Is Essential for Survival of Mycobacterium smegmatis

RNases H are involved in the removal of RNA from RNA/DNA hybrids. Type I RNases H are thought to recognize and cleave the RNA/DNA duplex when at least four ribonucleotides are present. Here we investigated the importance of RNase H type I encoding genes for model organism Mycobacterium smegmatis. By performing gene replacement through homologous recombination, we demonstrate that each of the two presumable RNase H type I encoding genes, rnhA and MSMEG4305, can be removed from M. smegmatis genome without affecting the growth rate of the mutant. Further, we demonstrate that deletion of both RNases H type I encoding genes in M. smegmatis leads to synthetic lethality. Finally, we question the possibility of existence of RNase HI related alternative mode of initiation of DNA replication in M. smegmatis, the process initially discovered in Escherichia coli. We suspect that synthetic lethality of double mutant lacking RNases H type I is caused by formation of R-loops leading to collapse of replication forks. We report Mycobacterium smegmatis as the first bacterial species, where function of RNase H type I has been found essential.     

TheamrG1 gene is involved in the activation of acetate inCorynebacterium glutamicum

During growth ofCorynebacterium glutamicum on acetate as its carbon and energy source, the expression of theptaack operon is induced, coding for the acetate-activating enzymes, which are phosphotransacetylase (PTA) and acetate kinase (AK). By transposon rescue, we identified the two genesamrG1 andamrG2 found in the deregulated transposon mutant C.glutamicum G25. TheamrG1 gene (NCBI-accession: AF532964) has a size of 732 bp, encoding a polypeptide of 243 amino acids and apparently is partially responsible for the regulation of acetate metabolism in C.glutamicum. We constructed an in-frame deletion mutant and an overexpressing strain ofamrG1 in the C.glutamicum ATCC13032 wildtype. The strains were then analyzed with respect to their enzyme activities of PTA and AK during growth on glucose, acetate and glucose or acetate alone as carbon sources. Compared to the parental strain, theamrG1 deletion mutant showed higher specific AK and PTA activities during growth on glucose but showed the same high specific activities of AK and PTA on medium containing acetate plus glucose and on medium containing acetate. In contrast to the gene deletion, overexpression of theamrG1 gene in C.glutamicum 13032 had the adverse regulatory effect. These results indicate that theamrG1 gene encodes a repressor or co-repressor of theptaack operon.     

Characterization of an adenylate cyclase gene (cyaB) deletion mutant of Corynebacterium glutamicum ATCC 13032

Genome analysis of C. glutamicum ATCC 13032 has showed one putative adenylate cyclase gene, cyaB (cg0375) which encodes membrane protein belonging to class III adenylate cyclases. To characterize the function of cyaB, a deletion mutant was constructed, and the mutant showed decreased level of intracellular cyclic AMP compared to that of wild-type. Interestingly, the cyaB mutant displayed growth defect on acetate medium, and this effect was reversed by complementation with cyaB gene. Similarly, it showed growth defect on glucose-acetate mixture minimal medium, and the utilization of glucose was retarded in the presence of acetate. The deletion mutant retained the activity of glyoxylate bypass enzymes. Additionally, the mutant could grow on ethanol but not on propionate medium. The data obtained from this study suggests that adenylate cyclase plays an essential role in the acetate metabolism of C. glutamicum, even though detailed regulatory mechanisms involving cAMP are not yet clearly defined. The observation that glyoxylate bypass enzymes are derepressed in cyaB mutant indicates the involvement of cAMP in the repression of aceB and aceA.     

A mutation in the Corynebacterium glutamicum ltsA gene causes susceptibility to lysozyme, temperature-sensitive growth, and L-glutamate production

The Corynebacterium glutamicum mutant KY9714, originally isolated as a lysozyme-sensitive mutant, does not grow at 37 degrees C. Complementation tests and DNA sequencing analysis revealed that a mutation in a single gene of 1,920 bp, ltsA (lysozyme and temperature sensitive), was responsible for its lysozyme sensitivity and temperature sensitivity. The ltsA gene encodes a protein homologous to the glutamine-dependent asparagine synthetases of various organisms, but it could not rescue the asparagine auxotrophy of an Escherichia coli asnA asnB double mutant. Replacement of the N-terminal Cys residue (which is conserved in glutamine-dependent amidotransferases and is essential for enzyme activity) by an Ala residue resulted in the loss of complementation in C. glutamicum. The mutant ltsA gene has an amber mutation, and the disruption of the ltsA gene caused lysozyme and temperature sensitivity similar to that in the KY9714 mutant. L-Glutamate production was induced by elevating growth temperature in the disruptant. These results indicate that the ltsA gene encodes a novel glutamine-dependent amidotransferase that is involved in the mechanisms of formation of rigid cell wall structure and in the L-glutamate production of C. glutamicum.     

Anaerobic growth of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> using nitrate as a terminal electron acceptor

Corynebacterium glutamicum, a gram-positive soil bacterium, has been regarded as an aerobe because its growth by fermentative catabolism or by anaerobic respiration has, to this date, not been demonstrated. In this study, we report on the anaerobic growth of C. glutamicum in the presence of nitrate as a terminal electron acceptor. C. glutamicum strains R and ATCC13032 consumed nitrate and excreted nitrite during growth under anaerobic, but not aerobic, conditions. This was attributed to the presence of a narKGHJI gene cluster with high similarity to the Escherichia coli narK gene and narGHJI operon. The gene encodes a nitrate/nitrite transporter, whereas the operon encodes a respiratory nitrate reductase. Transposonal inactivation of C. glutamicum narG or narH resulted in mutants with impaired anaerobic growth on nitrate because of their inability to convert nitrate to nitrite. Further analysis revealed that in C. glutamicum, narK and narGHJI are cotranscribed as a single narKGHJI operon, the expression of which is activated under anaerobic conditions in the presence of nitrate. C. glutamicum is therefore a facultative anaerobe.     

Functional relationship between Escherichia coli RNase E and the CafA protein

We analyzed the functional relationship between the Escherichia coli RNase E and the CafA protein, which show extensive sequence similarity. The temperature-sensitive growth of the RNase E mutant strain ams1 was partially suppressed by multicopy plasmids bearing the cafA gene. Introduction of a cafA::cat mutation enhanced the temperature sensitivity of the ams1 mutant. These results suggest that there is a functional homology between these two proteins. Received: 17 May 1996 / Accepted: 1 October 1996     

RecA,Tus protein and constitutive stable DNA replication inEscherichia coli rnhA mutants

Constitutive stable DNA replication (cSDR), which uniquely occurs inEscherichia coli rnhA mutants deficient in ribonuclease HI activity, requires RecA function. TherecA428 mutation, which inactivates the recombinase activity but imparts a constitutive coprotease activity, blocks cSDR inrnhA mutants. The result indicates that the recombinase activity of RecA, which promotes homologous pairing and strand exchange, is essential for cSDR. Despite the requirement for RecA recombinase activity, mutations inrecB, recD, recJ, ruvA andruvC neither inhibit nor stimulate cSDR. It was proposed that the property of RecA essential for homologous pairing and strand exchange is uniquely required for initiation of cSDR inrnhA mutants without involving the homologous recombination process. The possibility that RecA protein is necessary to counteract the action of Tus protein, a contra-helicase which stalls replication forks in theter region of the chromosome, was ruled out because introduction of thetus : :kan mutation, which inactivates Tus protein, did not alleviate the RecA requirement for cSDR.     

RecG Directs DNA Synthesis during Double-Strand Break Repair

Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability.     

Identification and characterization of a new Escherichia coli gene that is a dosage-dependent suppressor of a dnaK deletion mutation.

We report the isolation and characterization of a previously unidentified Escherichia coli gene that suppresses the temperature-sensitive growth and filamentation of a dnaK deletion mutant strain. Introduction of a multicopy plasmid carrying this wild-type gene into a dnaK deletion mutant strain rescued the temperature-sensitive growth of the dnaK deletion mutant strain at 40.5 degrees C and the filamentation, fully at 37 degrees C and partially at 40.5 degrees C. However, the inability of dnaK mutant cells to support bacteriophage lambda growth was not suppressed. This gene was also able to suppress the temperature-sensitive growth of a grpE280 mutant strain at 41 degrees C. Filamentation of the grpE280 mutant strain was suppressed at 37 degrees C but not at 41 degrees C. The dnaK suppressor gene, designated dksA, maps near the mrcB gene (3.7 min on the E. coli chromosome). DNA sequence analysis and in vivo experiments showed that dksA encodes a 17,500-Mr polypeptide. Gene disruption experiments indicated that dksA is not an essential gene.