Resolution of Holliday junctions in Escherichia coli: identification of the ruvC gene product as a 19-kilodalton protein.

The ruvC gene of Escherichia coli specifies a nuclease that resolves Holliday junction intermediates in genetic recombination (B. Connolly, C.A. Parsons, F.E. Benson, H.J. Dunderdale, G.J. Sharples, R.G. Lloyd, and S.C. West, Proc. Natl. Acad, Sci. USA 88:6063-6067, 1991). The gene was located between aspS and the ruvAB operon by DNA sequencing and deletion analysis of ruvC plasmids and was shown to encode a protein of 18,747 Da. Analysis of the DNA flanking ruvC indicated that the gene is transcribed independently of the LexA-regulated ruvAB operon and is not under direct SOS control. ruvC lies downstream of an open reading frame, orf-33, for a protein which migrates during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 33-kDa polypeptide. These two genes probably form an operon. However, expression of ruvC was found to be very poor relative to that of orf-33. A double ribosomal frameshift between these genes is proposed as a possible reason for the low level of RuvC. Two further open reading frames of unknown function were identified, one on either side of the orf-33-ruvC operon.     

Modulation of DNA repair and recombination by the bacteriophage lambda Orf function in Escherichia coli K-12

The orf gene of bacteriophage lambda, fused to a promoter, was placed in the galK locus of Escherichia coli K-12. Orf was found to suppress the recombination deficiency and sensitivity to UV radiation of mutants, in a Delta(recC ptr recB recD)::P(tac) gam bet exo pae cI DeltarecG background, lacking recF, recO, recR, ruvAB, and ruvC functions. It also suppressed defects of these mutants in establishing replication of a pSC101-related plasmid. Compared to orf, the recA803 allele had only small effects on recF, recO, and recR mutant phenotypes and no effect on a ruvAB mutant. In a fully wild-type background with respect to known recombination and repair functions, orf partially suppressed the UV sensitivity of ruvAB and ruvC mutants.     

The recR locus of Escherichia coli K-12: molecular cloning, DNA sequencing and identification of the gene product.

The recR gene of Escherichia coli, which is associated with recBC-independent mechanisms of recombination and DNA repair, has been located between dnaZX and htpG on a 6.4 kb EcoRI fragment of DNA that has been cloned and analysed in lambda and plasmid vectors. Nucleotide sequencing of this interval revealed two open reading frames that constitute an operon lying immediately downstream of dnaZX. The second of these two reading frames was identified as recR. It encodes a polypeptide with a predicted molecular weight of 21,965 Daltons that migrates on SDS gels as a 26 kDa protein. The first gene of the operon encodes a polypeptide of 12,015 daltons. Its function is not known.     

Phage operon involved in sensitivity to the Lactococcus lactis abortive infection mechanism AbiD1.

Phage bIL66 is unable to grow on Lactococcus lactis cells harboring the abortive infection gene abiD1. Spontaneous phage mutants able to grow on AbiD1 cells were used to study phage-Abi interaction. A 1.33-kb DNA segment of a mutant phage allowed growth of AbiD1s phages in AbiD1 cells when present in trans. Sequence analysis of this segment revealed an operon composed of four open reading frames, designated orf1 to orf4. The operon is transcribed 10 min after infection from a promoter presenting an extended -10 consensus sequence but no -35 sequence. Analysis of four independent AbiD1r mutants revealed a different point mutation localized in orf1, implying that this open reading frame is needed for sensitivity to AbiD1. However, the sensitivity is partly suppressed when orf3 is expressed in trans on a high-copy-number plasmid, suggesting that AbiD1 acts by decreasing the concentration of an available orf3 product.     

Organization and nucleotide sequence of a gene cluster coding for eight ribosomal proteins in the archaebacterium Halobacterium marismortui

A DNA fragment containing the genes for the eight ribosomal proteins HmaL3, HL6, HmaL23, HmaL2, HmaS19, HmaL22, HmaS3, and HmaL29 from Halobacterium marismortui has been cloned and sequenced. The organization of this gene cluster in general corresponds to the S10 operon of Escherichia coli although there exists some differences between them. The sequence analysis of the 5'- and 3'-region of the gene cluster revealed three open reading frames (orf1, orf2, and orf3) which do not code for any ribosomal protein whose structure is known. A putative promoter is located upstream of orf1. Out of the eight ribosomal proteins five have counterparts in eubacteria only, two in both eubacteria and eukaryotes, and one is exclusively related to an eukaryotic ribosomal protein.     

Sequence and characterization of the Escherichia coli genome between the ndk and gcpE genes

Abstract The region of the chromosome immediately upstream of the Escherichia coli gene gcpE has been cloned and sequenced. This region contains two functional open reading frames, orf 384 and orf 337, encoding proteins of 43082 and 36189 Da, respectively. Sequencing analysis (this paper) and the isolation of a DNA fragment containing a functional promoter (Talukder, A.A., Yanai, S., and Yamada, M. (1994) Biosci. Biotech. Biochem. 58, 117–120) indicate that orf 337 is in an operon with gcpE . The gene orf 384 is immediately downstream of the gene ndk , which encodes nucleoside diphosphate kinase.     

Recombination is essential for viability of an Escherichia coli dam (DNA adenine methyltransferase) mutant

Double mutants of Escherichia coli dam (DNA adenine methyltransferase) strains with ruvA, ruvB, or ruvC could not be constructed, whereas dam derivatives with recD, recF, recJ, and recR were viable. The ruv gene products are required for Holliday junction translocation and resolution of recombination intermediates. A dam recG (Holliday junction translocation) mutant strain was isolated but at a very much lower frequency than expected. The inviability of a dam lexA (Ind(-)) host was abrogated by the simultaneous presence of plasmids encoding both recA and ruvAB. This result indicates that of more than 20 SOS genes, only recA and ruvAB need to be derepressed to allow for dam mutant survival. The presence of mutS or mutL mutations allowed the construction of dam lexA (Ind(-)) derivatives. The requirement for recA, recB, recC, ruvA, ruvB, ruvC, and possibly recG gene expression indicates that recombination is essential for viability of dam bacteria probably to repair DNA double-strand breaks. The effect of mutS and mutL mutations indicates that DNA mismatch repair is the ultimate source of most of these DNA breaks. The requirement for recombination also suggests an explanation for the sensitivity of dam cells to certain DNA-damaging agents.     

大肠杆菌O23 O-抗原基因簇序列的破译及UDP-N-乙酰葡萄糖-C4异构酶的鉴定

采用鸟枪法破译大肠杆菌O23标准株的O-抗原基因簇序列,并用生物信息学的方法进行了基因注释和分析;采用基因缺失和互补的方法鉴定了O23的UDP-GlcNAc C4异构酶(Gne);用同源建模的方法构建了O23 Gne的高级结构并对其活性位点进行了分析;分析了不同血清型大肠杆菌O-抗原基因簇中gne基因的多样性;根据O23O-抗原基因簇中的特异基因筛选出了可用于大肠杆菌O23快速检测的特异DNA序列。     

Involvement in DNA repair of the ruvA gene of Escherichia coli

Summary The ruv operon of Escherichia coli consists of two genes, orfl1 and ruv, which encode 22 and 37 kilodalton proteins, respectively, and are regulated by the SOS system. Although the distal gene, ruv, is known to be involved in DNA repair, the function of orf1 has not been studied. To examine whether orf1 is also involved in DNA repair, we constructed a strain with a deletion of the entire ruv operon. The strain was sensitive to UV even after introduction of low copy number plasmids carrying either orf1 or ruv, but UV resistance was restored by introduction of a plasmid carrying both orfl and ruv. These results suggest that orf1 as well as ruv is involved in DNA repair. Therefore, orf1 and ruv should be renamed ruvA and ruvB, respectively.     

Characterization of the tol-pal region of Escherichia coli K-12: translational control of tolR expression by TolQ and identification of a new open reading frame downstream of pal encoding a periplasmic protein.

The TolQ, TolR, TolA, TolB, and Pal proteins appear to function in maintaining the integrity of the outer membrane, as well as facilitating the uptake of the group A colicins and the DNA of the infecting filamentous bacteriophages. Sequence data showed that these genes are clustered in a 6-kb segment of DNA with the gene order orf1 tolQ tolR tolA tolB pal orf2 (a newly identified open reading frame encoding a 29-kD9 protein). Like those containing orf1, bacteria containing an insertion mutation in this gene showed no obvious phenotype. Analysis of beta-galactosidase activity from fusion constructs in which the lac operon was fused to various genes in the cluster showed that the genes in this region constitute two separate operons: orf1 tolQRA and tolB pal orf2. In the orf1 tolQRA operon, translation of MR was dependent on translation of the upstream tolQ region. Consistent with this result, no functional ribosome-binding site for TolR synthesis was detected.     

Sigma-B, a putative operon encoding alternate sigma factor of Staphylococcus aureus RNA polymerase: molecular cloning and DNA sequencing.

We have identified a gene cluster located on the chromosomal SmaI I fragment of a highly methicillin resistant strain of Staphylococcus aureus, consisting of four open reading frames (ORFs), named after the number of deduced amino acid residues, in the sequential order orf333-orf108-orf159-orf256. The gene cluster showed close similarities to the Bacillus subtilis sigB operon both in overall organization and in primary sequences of the gene products. The complete gene cluster (provisionally named sigma-B or sigB) was preceded by an sigmaA-like promoter (PA) and had an internal sigmaB-like promoter sequence (PB) between orf333 and orf108, suggesting a complex regulatory mechanism. The polypeptides encoded by orf333, -108, -159, and -256 showed 62, 67, 71, and 77% homologies, respectively, with the RsbU, RsbV, RsbW, and SigB polypeptides encoded by the B. subtilis sigB operon. A Tn551 insertional mutant, RUSA168 (insert in orf256 of the staphylococcal sigma-B operon), showed drastic reduction in methicillin resistance (decrease in MIC from 1,600 microg ml-1 to 12 to 25 microg ml-1off     

Identification of the Escherichia coli lytB Gene, Which Is Involved in Penicillin Tolerance and Control of the Stringent Response

The Escherichia coli lytB gene, which is involved in penicillin tolerance and control of the stringent response, was identified as a previously described open reading frame designated orf316 located in the ileS-lsp operon (0.4 min on the linkage map).