Mutational inactivation of the Saccharomyces cerevisiae RAD4 gene in Escherichia coli.

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. When plasmids containing the wild-type gene were transformed into various Escherichia coli strains, transformation frequencies were drastically reduced. Most plasmids recovered from transformants showed deletions or rearrangements. A minority of plasmids recovered from E. coli HB101 showed no evidence of deletion or rearrangement, but when they were transformed into S. cerevisiae on centromeric vectors, little or no complementation of the UV sensitivity of rad4 mutants was observed. Deliberate insertional mutagenesis of the wild-type RAD4 allele before transformation of E. coli restored transformation to normal levels. Plasmids recovered from these transformants contained an inactive rad4 allele; however, removal of the inserted DNA fragment restored normal RAD4 function. These experiments suggest that expression of the RAD4 gene is lethal to E. coli and show that lethality can be prevented by inactivation of the gene before transformation. Stationary-phase cultures of some strains of E. coli transformed with plasmids containing an inactivated RAD4 gene showed a pronounced delay in the resumption of exponential growth, suggesting that the mutant (and, by inference, possibly wild-type) Rad4 protein interferes with normal growth control in E. coli. The rad4-2, rad4-3, and rad4-4 chromosomal alleles were leaky relative to a rad4 disruption mutant. In addition, overexpression of plasmid-borne mutant rad4 alleles resulted in partial complementation of rad4 strains. These observations suggest that the Rad4 protein is relatively insensitive to mutational inactivation.     

Incorporation of phi80 phage into unusual chromosome sites and isolation of a specialized transducing bacteriophage]

The mutant strain KS713 of Escherichia coli K-12 deleted for the normal insertion site and secondary preferable one was obtained. The insertion frequency of phage phi80 into the double deletion strain is reduced about 30-fold with respect to integration into the strain H47 with deletion of the primary phi80 attachment site and about 500-fold relative to integration into wild type Escherichia coli. Analysis of the rare abnormal lysogens of KS 713 strain indicates that there are secondary sites on the chromosome, which are utilized for prophage attachment if insertion at preferable secondary att80-II site is eliminated too. The insertion of phi80 phage into the bfe locus was obtained by the appropriate selection technique. Induced prophage excision from the bfe site was rather efficient and lysates contained phi80 phage particles that could specificically transduce the argH+ gene. Upon transduction into a recipient strain carrying recA, heterogenotes harbouring both the wild-type and the mutant argH genes were isolated. These heterogenotes were used for producing high-frequency transducing lysates.     

大肠杆菌ubiA基因的克隆及序列测定

以E.coli JM83染色体DNA为模板PCR扩增ubiA的结构基因,将PCR产物克隆于pUCm-T Vector,对PCR产物进行测序鉴定。与已发表的大肠杆菌ubiA基因序列比较,同源性达99％。     

Determination of plasmid DNA concentration maintained by nonculturable Escherichia coli in marine microcosms.

The concentration of plasmid pBR322 DNA in nonculturable Escherichia coli JM83 was measured to determine whether the plasmid concentration changed during survival of E. coli in marine and estuarine water. E. coli JM83 containing the plasmid pBR322 was placed in both sterile seawater and sterile estuarine water and analyzed for survival (i.e., culturability) and plasmid maintenance. The concentration of pBR322 DNA remained stable in E. coli JM83 for 28 days in an artificial seawater microcosm, even though nonculturability was achieved within 7 days. E. coli JM83 incubated in sterile natural seawater or sterile estuarine water did not reach nonculturability within 30 days. Under all three conditions, plasmid pBR322 DNA was maintained at approximately the initial concentration. Cloning of DNA into the plasmid pUC8 did not alter the ability of E. coli to maintain vector plasmid DNA, even when the culture was in the nonculturable state, but the concentration of plasmid DNA decreased with time in the microcosm. We conclude that E. coli is able to maintain plasmid DNA while in the nonculturable state and that the concentration at which the plasmid is maintained appears to be dependent upon the copy number of the plasmid and/or the presence of foreign DNA.     

Determination of plasmid DNA concentration maintained by nonculturable Escherichia coli in marine microcosms.

The concentration of plasmid pBR322 DNA in nonculturable Escherichia coli JM83 was measured to determine whether the plasmid concentration changed during survival of E. coli in marine and estuarine water. E. coli JM83 containing the plasmid pBR322 was placed in both sterile seawater and sterile estuarine water and analyzed for survival (i.e., culturability) and plasmid maintenance. The concentration of pBR322 DNA remained stable in E. coli JM83 for 28 days in an artificial seawater microcosm, even though nonculturability was achieved within 7 days. E. coli JM83 incubated in sterile natural seawater or sterile estuarine water did not reach nonculturability within 30 days. Under all three conditions, plasmid pBR322 DNA was maintained at approximately the initial concentration. Cloning of DNA into the plasmid pUC8 did not alter the ability of E. coli to maintain vector plasmid DNA, even when the culture was in the nonculturable state, but the concentration of plasmid DNA decreased with time in the microcosm. We conclude that E. coli is able to maintain plasmid DNA while in the nonculturable state and that the concentration at which the plasmid is maintained appears to be dependent upon the copy number of the plasmid and/or the presence of foreign DNA.     

枯草芽孢杆菌耐盐突变株proA基因克隆及proBA基因渗透压调节功能研究

利用TaKaRaLAPCRTM试剂盒扩增枯草芽孢杆菌 931 5 1耐盐突变株proA基因的未知下游序列。根据测序结果 ,设计引物 ,克隆出发菌株和突变株全长proBA基因。将出发菌株和突变株的proBA基因分别转化大肠杆菌JM83(proBA- ) ,均能够与其功能互补。SDS PAGE分析其表达产物 ,有两条分子量分别约为 4 0kD和 4 5kD的新蛋白带出现。测定 4种转化子 (分别含有出发菌株和突变株proB基因的大肠杆菌 1 1 2 5 2转化子及proBA基因的大肠杆菌JM83转化子 )的耐盐能力。发现含有突变株proB或proBA基因转化子的耐盐能力 ,均比相应的含有出发菌株proB或proBA基因的转化子高。另外含有出发菌株和突变株的proBA基因转化子的耐盐能力 ,也均比相应的仅含proB基因的转化子高 ,表明枯草芽孢杆菌的ProA比大肠杆菌的ProA更为有效。测定所有JM83转化子胞内自由脯氨酸 ,发现其含量随盐浓度的上升而提高 ,其中含突变菌株proBA基因的转化子提高更为显著     

Restriction enzyme cleavage sites surrounding the structural gene for the lipoprotein of the Escherichia coli outer membrane.

The purified messenger ribonucleic acid (mRNA) for the lipoprotein of the Escherichia coli outer membrane was hybridized with fragments obtained by digestion of E. coli chromosomal deoxyribonucleic acid (DNA) with eight different restriction enzymes. For each restriction enzyme digestion, one specific fragment separated by agarose gel electrophoresis was found to hybridize with the lipoprotein mRNA. From the analysis of restriction fragments generated by double digestions with various combinations of restriction enzymes, cleavage sites for the restriction enzymes near the locus of the lipoprotein structural gene (lpp) were mapped. No restriction fragments of DNA from the E. coli lpp-2 mutant hybridized with the lipoprotein mRNA, confirming that the mutant has a deletion mutation in the vicinity of the lpp gene.     

Mutational analysis of loxP sites for efficient Cre-mediated insertion into genomic DNA

The Cre/loxP system has been used in transgenic models primarily to excise DNA flanked by loxP sites for gene deletion. However, the insertion reaction is more difficult to control since the excision event is kinetically favored. Mutant loxP sites favoring integration were identified using a novel, bacterial screening system. Utilizing lambda integrase, mutant loxP sites were placed at the E. coli attB site and the excision-insertion ratios of incoming DNA plasmids carrying a second, complementary mutant loxP site were determined. Comparison of 50 mutant loxP sites combinations to the native loxP site revealed that mutations to the inner 6 bp of the Cre binding domain severely inhibited recombination, while those in the outer 8 bps were more tolerated. The most efficient loxP combinations resulted in 1421-fold and 1529-fold increases in relative integration rates over wild-type loxP sites. These loxP mutants could be exploited for site-directed "tag and insert" recombination experiments.     

Repair of damage in double-stranded phi X174 (RF) DNA due to radiation-induced water radicals

Experiments in which the yields of radiation-induced OH and H radicals were varied, showed that both types of water radicals inactivate phi X174 RF DNA to about the same extent as measured by transfection of the (irradiated) DNA to E. coli wild-type spheroplasts. On the other hand, using spheroplasts prepared from E. coli strains, deficient in one of the proteins involved in excision DNA repair (uvrA- or uvrC-) or in post-replication repair (recA-), clear differences between damage originating from OH or H radical attack were found. Part of the radiation damage due to H radicals appeared to be repairable by an uvrA-gene-dependent repair mechanism, whereas this repair pathway does not play an important role in the case of OH radical damage. The reverse applies to uvrC-gene-dependent repair, which only affects OH radical damage (obtained under anoxic conditions), but has no influence on damage due to H radicals. Irradiation of double-stranded phi X174 (RF) DNA in the presence of oxygen however, yields damage--due to OH radicals only--which appeared not to be sensitive to either uvrC- or uvrA-gene-dependent repair. Furthermore, post-replication repair (recA) has only very little effect on the amount of inactivation by H or OH radicals, when irradiation is carried out under anoxic conditions. We did not find significant inactivation due to hydrated electrons, whether the biological activity was determined by use of wild-type spheroplasts or of strains deficient in excision or post-replication repair proteins.     

Nonessential genes of phage phiYeO3-12 include genes involved in adaptation to growth on Yersinia enterocolitica serotype O:3

Bacteriophage phiYeO3-12 is a T7/T3-related lytic phage that naturally infects Yersinia enterocolitica serotype O:3 strains by using the lipopolysaccharide O polysaccharide (O antigen) as its receptor. The phage genome is a 39,600-bp-long linear, double-stranded DNA molecule that contains 58 genes. The roles of many of the genes are currently unknown. To identify nonessential genes, the isolated phage DNA was subjected to MuA transposase-catalyzed in vitro transposon insertion mutagenesis with a lacZ' gene-containing reporter transposon. Following electroporation into Escherichia coli DH10B and subsequent infection of E. coli JM109/pAY100, a strain that expresses the Y. enterocolitica O:3 O antigen on its surface, mutant phage clones were identified by their beta-galactosidase activity, manifested as a blue color on indicator plates. Transposon insertions were mapped in a total of 11 genes located in the early and middle regions of the phage genome. All of the mutants had efficiencies of plating (EOPs) and fitnesses identical to those of the wild-type phage when grown on E. coli JM109/pAY100. However, certain mutants exhibited altered phenotypes when grown on Y. enterocolitica O:3. Transposon insertions in genes 0.3 to 0.7 decreased the EOP on Y. enterocolitica O:3, while the corresponding deletions did not, suggesting that the low EOP was not caused by inactivation of the genes per se. Instead, it was shown that in these mutants the low EOP was due to the delayed expression of gene 1, coding for RNA polymerase. On the other hand, inactivation of gene 1.3 or 3.5 by either transposon insertion or deletion decreased phage fitness when grown on Y. enterocolitica. These results indicate that phiYeO3-12 has adapted to utilize Y. enterocolitica as its host and that these adaptations include the products of genes 1.3 and 3.5, DNA ligase and lysozyme, respectively.     

Construction and characterization of mutations in hupB, the gene encoding HU-beta (HU-1) in Escherichia coli K-12.

Plasmid pJMC21 contains Escherichia coli chromosomal DNA encoding Lon protease, HU-beta (HU-1), and an unidentified 67,000-dalton protein. A kanamycin resistance cassette was used in the construction of insertion and deletion mutations in hupB, the gene encoding HU-beta on plasmid pJMC21. The reconstructed plasmids were linearized and used to introduce hupB chromosomal mutations into JC7623 (recBC sbcBC). These mutations, as expected, mapped in the 9.8-min region of the E. coli chromosome by P1 transduction (16% linkage to proC+). Southern blot hybridization of chromosomal fragments verified that hupB+ was replaced by the mutant allele, with no indication of gene duplication. All the mutant strains had growth rates identical to that of wild-type E. coli, were resistant to UV irradiation and nitrofurantoin, and supported the in vivo transposition-replication of bacteriophage Mu, Mu lysogenization, Tn10 transposition from lambda 1098, and lambda replication-lysogenization. The only observable phenotypic variation was a reduced Mu plaque size on the hupB mutant strains; however, the yield of bacteriophage Mu in liquid lysates prepared from the mutant strains was indistinguishable from the yield for the wild type.     

Cloning of a Porphyromonas (Bacteroides) gingivalis protease gene and characterization of its product

A clone expressing a Porphyromonas gingivalis protease from the recombinant plasmid (pYS307) has been identified in a genomic library of P. gingivalis W83. The cloned gene was localized to a 2.4-kb DNA fragment between BamHI and HindIII sites. When a 3.2-kb HindIII fragment of pYS307 was used as a probe in Southern hybridization, HindIII-digested chromosomal DNA of P. gingivalis W83, as well as those of W50 and W12, showed a single 3.2-kb hybridizing band, while that of P. gingivalis 33277 showed a 5.0-kb band. Colonies of E. coli containing pYS307 showed pronounced proteolytic zones on skim milk agar plates only when incubated in an oxygen-free environment. BSA substrate zymography of whole cell extract of E. coli containing pYS307 revealed a protease of approx. 80 kDa which was active under reducing conditions. These results suggest that the cloned protease is thiol-dependent. Antiserum to P. gingivalis W50 reacted with a single band of 80 kDa when a cell lysate sample of an E. coli JM83 containing pYS307 was prepared for electrophoresis in the absence of beta-mercaptoethanol. When samples were solubilized in the presence of beta-mercaptoethanol prior to electrophoresis, the antiserum reacted with the bands of 50 and 38 kDa, but there was no reaction observed at 80 kDa. The activity of the cloned protease was inhibited by TLCK, TPCK, EDTA, PMSF, iodoacetic acid and ZnCl2.     

GATC sequences, DNA nicks and the MutH function in Escherichia coli mismatch repair.

Circular heteroduplex DNAs of bacteriophage phi X174 have been constructed carrying either a G:T (Eam+/Eam3) or a G:A (Bam+/Bam16) mismatch and containing either two, one or no GATC sequences. Mismatches were efficiently repaired in wild-type Escherichia coli transfected with phi X174 heteroduplexes only when two unmethylated GATC sequences were present in phi X174 DNA. The requirements for GATC sequences in substrate DNA and for the E. coli MutH function in E. coli mismatch repair can be alleviated by the presence of a persistent nick (transfection with nicked heteroduplex DNA in ligase temperature-sensitive mutant at 40 degrees C). A persistent nick in the GATC sequence is as effective in stimulating mutL- and mutS-dependent mismatch repair as a nick distant from the GATC sequence and from the mismatch. These observations suggest that the MutH protein participates in methyl-directed mismatch repair by recognizing unmethylated DNA GATC sequences and/or stimulating the nicking of unmethylated strands.     

Overcoming a defect in generalized recombination in the marine fish pathogen Vibrio anguillarum 775: construction of a recA mutant by marker exchange.

A defect in generalized recombination has prevented the use of marker exchange for the construction of specific chromosomal mutations in the marine fish pathogen Vibrio anguillarum 775. Through the use of large segments of homologous DNA, we were successful in overcoming this defect and used marker exchange to construct a recA mutant of V. anguillarum H775-3. A recombinant cosmid carrying the recA gene of V. anguillarum 775 in the center of a 25-kb cloned DNA insert was isolated by complementation of methyl methanesulfonate (MMS) sensitivity in Escherichia coli HB101. The recA gene was inactivated by inserting a kanamycin resistance gene into recA, and the mutant gene was subsequently introduced into V. anguillarum H775-3 by conjugal mobilization. Isolation of recombinants between cosmid-borne recA::kan sequences and chromosomal DNA was facilitated by the introduction of an incompatible plasmid, and Southern hybridization was used to verify the presence of recA::kan in the chromosomal DNA of the recA mutant. V. anguillarum carrying recA::kan was considerably more sensitive to UV radiation and to MMS than was its parent, and near wild-type levels of resistance to MMS and UV light were restored by introduction of cloned recA genes from both E. coli and V. anguillarum. These results indicate that recA is required for DNA repair in V. anguillarum and demonstrate the utility of this modified marker exchange technique for the construction of mutations in this economically important fish pathogen.     

A G43 to U43 mutation in E. coli tRNAtyrsu3+ which affects processing by RNase P.

A novel mutation in the anticodon stem of E. coli tRNA1Tyrsu3+ (G43 to U43) has been characterized. The gene coding for the mutant tRNA, carried by phage phi 80DHA61.3 a derivative of phi 80psu3+su0, produces only 20% of mature suppressor tRNA as compared with phi 80psu3+. Both the mutant tRNA precursor and mature tRNA have an altered conformation. The precursor tRNA coded for by phi 80DHA61.3 is processed by RNase P more slowly than the su3+ precursor and does not form as stable an enzyme-substrate complex as does su3+ precursor. phi 80 DHA61.3 also contains a large deletion which begins in the spacer region between the su3+ gene and the su0- gene, extends through the su0- gene and includes most of the repeated region following the tRNA genes.     

Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants.

Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA. Endonuclease III is encoded by the nth gene. Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics. The gene nei is located at 16 min on the E. coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins. A nei partial deletion replacement mutant was constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis. nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo). Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type. Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay. In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type.