The Bacillus megaterium comE locus encodes a functional DNA uptake protein

From Bacillus megaterium, a genomic region was isolated and structurally characterized which strongly resembles the Bacillus subtilis competence locus comE encoding proteins involved in DNA uptake. Functionality of the B. megaterium comEA gene was proven by complementing a DNA-receptor mutant of B. subtilis. This finding provides first evidence for a latent ability of B. megaterium to develop natural competence, although such physiological state has not as yet been identified in this organism.     

Decreased mutant frequency in embryonic brain of DNA polymerase beta null mice

DNA polymerase beta (Polbeta) knockout mouse embryos exhibit extensive apoptosis in postmitotic neuronal cells and die immediately after birth. In contrast, no apoptosis has been observed in other tissues as well as liver in the mutant embryos. To study the relationship of Polbeta deficiency and mutagenesis during development and neurogenesis, we examined spontaneous mutations in Polbeta null (Polbeta-/-) and wild-type (Polbeta+/+) mouse embryos, by using the transgenic mutation detection system consisting of a pSSW shuttle vector with the Escherichia coli rpsL reporter gene. Unexpectedly, we found a significant decrease in the mutant frequency of Polbeta-/- brain (1.63+/-0.67x10(-5)) compared with wild-type controls (3.12+/-0.83x10(-5)) (P<0.001). In contrast, no such difference was found between livers from Polbeta-/- (0.92+/-0.38x10(-5)) and wild-type (0.71+/-0.31x10(-5)) embryos. Analysis of mutation spectra revealed that mutations in brains from the two genotypes were almost exclusively single-base deletions and that these sites fell within runs of 2-6 identical bases and a two base repeat in the rpsL sequence, while mutations in the corresponding livers contained base substitutions as well as single-base deletions. Taken together with the extensive neuronal apoptosis associated with Polbeta deficiency, we suggest that the lower mutant frequency observed in Polbeta-/- embryonic brain may be caused by the elimination of neuronal cells with unrepaired DNA damage through apoptosis.     

二元信号系统ComD/ComE与肺炎链球菌对β-内酰胺类抗生素耐药的相关性

目的 构建肺炎链球菌comD基因敲除突变株,了解comD基因与细菌β-内酰类抗生素耐药性的相关性,探讨氯氰碘柳胺下调comD、comE和comC基因mRNA的作用.方法 构建用于comD基因敲除的自杀质粒pEVP3comD,通过同源重组及插入失活获得肺炎链球菌ATCC6306株comD基因敲除突变株comD-.采用PCR及免疫荧光法对comD-突变株进行鉴定.采用实时荧光定量RT-PCR检测氯氰碘柳胺处理前后comD-突变株及野生株comD、comE和comC基因mRNA水平变化.采用二倍琼脂稀释法测定comD-突变株及野生株对青霉素G和头孢噻肟的敏感性.结果 测序及免疫荧光试验结果证实,所构建的comD-突变株染色体DNA中comD基因被插入失活.50μmol/L或100 μmol/L的氯氰碘柳胺能明显下调comD、comE和comC基因mRNA水平(P＜0.05),25μmol/L的氯氰碘柳胺则否.comD-突变株对青霉素及头孢噻肟最低抑菌浓度(MIC)值均为32μg/ml,明显高于野生株的0.06 μg/ml和1 μg/ml.结论 本研究成功地构建了肺炎链球菌comD基因敲除突变株.comD基因与细菌对β-内酰类抗生素耐药性密切相关.氯氰碘柳胺可通过下调comD、comE和comC基因的转录水平,从而对细菌感受态形成产生影响.     

Construction of transposition insertion libraries and specific gene inactivation in the pathogen Lactococcus garvieae

This paper reports the development of genetic tools in Lactococcus garvieae, an important Gram-positive bacterial pathogen affecting both fish and mammals. The vector pGKV210, a broad host range vector, was introduced by electroporation into L. garvieae UNIUD074. The maximal frequency obtained was 3.2 x 10(5) transformants/mug of DNA. Moreover, this effect is highly reproducible and appears to be constant, since all L. garvieae strains tested were transformed. Once the optimal transformation procedure was established, it was used to generate isogenic and transposition mutants. Insertional mutagenesis of the L. garvieae SA9H10L gene, similar to a Streptococcus pyogenes gene encoding the M protein (emm64), was carried out using the conditional replication plasmid pORI19. Transposition mutagenesis using the streptococcal temperature-sensitive suicide vector pTV408 to deliver Tn917 into the chromosome of L. garvieae was also achieved at a frequency of ca. 10(-4). Transposon flanking DNA sequences were obtained by plasmid rescue in Escherichia coli and their sequencing analysis demonstrated that the transposon was inserted at different chromosomal loci. Tn917 also made it possible to select a mutant in the operon involved in mannitol fermentation in this microorganism. The results obtained in the present study lay the foundation for future research on the virulence mechanisms of L. garvieae.     

Induction of mutant lymphocytes in cyclophosphamide- and chlorambucil-treated patients

Monitoring patients treated with single antineoplastic agents is aiding our understanding of what hazard these drugs pose in vivo. In this study, the frequency of mutant 6-thioguanine-resistant (TG(R)) peripheral blood lymphocytes was monitored before treatment and for < or =35 weeks after treatment of patients with cyclophosphamide (CP) or chlorambucil (CAB). The mean mutant frequency before treatment for six multiple sclerosis patients treated with high-dose CP was 2.53 x 10(-5) and increased after treatment to 4.61 x 10(-5) (P = 0.08, paired t-test). Using each patient as their own control, there were significant increases (each at P < 0.04) detectable within 2-4 weeks in four of the multiple sclerosis patients treated with CP. There was no increase in an untreated control monitored over the same period. In a patient receiving five sequential CP treatments at 1 month intervals, there were cumulative increases in the frequency of mutant cells. The mutant frequency increased from 0.31 x 10(-5) before treatment to 3.64 x 10(-5) after the final treatment and had decreased to 0.53 x 10(-5) at 35 weeks after treatment. In one of two CAB-treated patients with indolent non-Hodgkin's lymphoma, there was a significant increase in mutant frequency (P < 0.03) after treatment. Freshly isolated peripheral blood lymphocytes treated with 4-hydroperoxy-CP in vitro demonstrate a dose-dependent increase in mutant frequency. The increment in mutant frequency observed in vivo is of the order expected from the in vitro experiments. Although this study demonstrates that single or multiple doses of a single antineoplastic agent are mutagenic in vivo for some patients, further studies are needed to determine the extent and mechanism of the inter-individual variations in mutagenic response.     

Comparative analysis of HPRT mutant frequency in children with cancer

The link between exposure to environmental mutagens and the development of cancer is well established. Yet there is a paucity of data on the relationship between gene-environment interactions and the mechanisms associated with the somatic mutational events involved with malignant transformation, especially in children. To gain insight into somatic mutational mechanisms in children who develop cancer, we determined the background mutant frequency (Mf) in the hypoxanthine phosphoribosyl transferase (HPRT) reporter gene of peripheral blood lymphocytes from pediatric cancer patients at the time of diagnosis and prior to therapeutic intervention. We studied 23 children with hematologic malignancies and 31 children with solid tumors prior to initial therapeutic intervention. Children with solid tumors, specifically sarcomas, and Hodgkin's disease were significantly older and had elevated HPRT Mfs (6.1 x 10(-6) and 3.7 x 10(-6), respectively) at the time of diagnosis, compared to normal controls (2.3 x 10(-6)) and other pediatric tumor groups including children with acute lymphocytic leukemia and non-Hodgkin's lymphoma (ALL/NHL, 1.7 x 10(-6)), central nervous system tumors (CNS, 3.6 x 10(-6)), and neuroblastoma (1.9 x 10(-6)). Of importance is that the significant differences observed in HPRT Mfs between these groups no longer existed after correcting for the effects of age. These data demonstrate that in children who develop cancer there appears to be no significant increase in background HPRT Mf that would indicate significant exposure to genotoxic chemicals or an underlying DNA repair defect resulting in genomic instability. In addition, these data demonstrate the importance of correcting for the effect of age when comparing the frequency of somatic mutations in children and should provide baseline data for future longitudinal biomonitoring studies on the genetic effects of chemotherapy in children treated for cancer.     

Differences in the response to oxidative stress and mutant frequency in CD (Sprague-Dawley) and Fisher 344 rats due to an induced inflammatory response

In this study, the rodent air pouch model was used to examine the production and processing of oxidative DNA damage in two strains of rats commonly used in toxicity testing. An inflammatory response was induced by injecting zymosan A (50 mg) into an air pouch on male CD (Sprague-Dawley [S-D]) and Fisher 344 (F-344) rats, and the animals were then sacrificed at 1, 3, 7, 14, and 28 days (n = 6 per time point per strain). Tissues from the lining of the air pouch were collected for 8-hydroxy-2'-deoxyguanosine (8-OH-dG) analysis and for paraffin embedding. Significant (P < 0.01) increases in 8-OH-dG were observed after 1 day in the DNA from cells lining the air pouch of zymosan A-treated versus control S-D (101.5 +/- 27.1 vs. 23.1 +/- 2. 7 8-OH-dG/dG x 10(5)) and F-344 (51.4 +/- 5.3 vs. 14.4 +/- 0.6 8-OH-dG/dG x 10(5)) rats. By 28 days, 8-OH-dG levels had returned to background in S-D rats, but remained elevated in F-344 rats. The frequency of apoptosis was evaluated using the in situ end-labeling (TUNEL) assay, which revealed that zymosan A-treated S-D rats had a significantly (P < 0.05) higher frequency of apoptosis compared to zymosan A-treated F-344 rats. To examine the potential consequences of these differences in endogenously produced DNA damage and apoptosis, we measured mutations at the hprt locus in fibroblasts of the pouch lining and observed a significant (P < 0.05) increase in the mutant frequency at day 28 in F-344 rats (54.2 +/- 13.6 mutants per 10(6) cells) compared to controls (4.5 +/- 2.0 mutants per 10(6) cells). The mutant frequency was not increased in S-D rats. These data demonstrate that strain differences in the production and processing of oxidative DNA damage due to an inflammatory response may impact the long-term pathologic consequences of chronic inflammation. Environ. Mol. Mutagen. 35:336-342, 2000 Published 2000 Wiley-Liss, Inc.     

Frequency and spectrum of ENU-induced mutation in the Phi X174 transgene in mouse splenic lymphocytes and their significance to spontaneous transgenic rodent mutation frequencies

A perceived disadvantage of transgenic rodent mutation assays is that spontaneous mutant frequencies are high compared to those of endogenous genes and may consequently reduce sensitivity to induced mutation. We have previously argued that unrepaired G:T mismatches from spontaneous deamination of 5-methylcytosine at CpG sites could be converted to apparent in vivo mutations in the bacterial recovery systems because of rapid, random, mismatch repair in Escherichia coli. In this study, we have measured mutation frequencies in spleen of male mice induced by N-ethyl-N-nitrosourea (ENU) using the PhiX174 transgene, which is not subject to mismatch repair in E.coli, using single-burst analysis, a unique method to identify in vivo mutation. In order to compare our results to those using the lacI and cII transgenes, we converted all mutant frequencies to base pair substitution (bps) mutation frequencies per nucleotide based on mutant spectra from this study and published literature. We found this frequency in control spleen to be similar for lacI (3.8 +/- 0.7 x 10(-8)) and PhiX174 (3.1 +/- 1.2 x 10(-8)) at 6 weeks of age. We found a strong age dependence for spontaneous lacI mutation that extrapolated to a value at conception (1.8 +/- 0.9 x 10(-8)) that was not significantly different from the human germ line bps mutation frequency per nucleotide of 1.7 +/- 0.2 x 10(-8). These two transgenes provided similar mutational responses to 40 mg/kg ENU, 7- to 9-fold. In contrast, the cII target gene in the same tissue produces both spontaneous and induced mutation frequencies approximately 10 times higher, for unknown reasons. We conclude that the spontaneous mutant frequencies measured by the lacI and PhiX174 transgenes in this moderately dividing tissue accurately measure in vivo mutation frequencies at early ages. For these two transgenes, seemingly high mutant frequencies may reflect the expected accumulation of somatic mutation with age.     

Role of zinc-finger motif in redox regulation of human replication protein A

Replication protein A (RPA) is a heterotrimeric zinc-finger protein complex involved in DNA replication, repair, and genetic recombination. Unlike other zinc-finger proteins, RPA's zinc-finger motif is not essential for its single-stranded DNA (ssDNA) binding activity, but is involved in redox regulation of its single-stranded DNA (ssDNA) binding activity. To get an insight into the regulation of RPA-ssDNA interaction, wild-type RPA (wt-RPA) and zinc-finger mutant were examined for ssDNA binding activity using surface plasmon resonance technique. Interaction of wt-RPA with ssDNA under nonreducing conditions was very weak (KD x 2.33 x 10(-8) M) compared with that under reducing conditions (KD = 7.35 x 10(-11) M), whereas ssDNA binding affinity of the zinc-finger mutant was not affected by redox. The divalent ion chelator, o-phenanthroline, significantly reduced wt-RPA-ssDNA interaction, but had no effect on the zinc-finger mutant. The inhibitory effect of o-phenanthroline on RPA-ssDNA interaction was reversed by Zn(II), but not by other divalent cations, suggesting that Zn(II) is the unique metal coordinating the zinc-finger cysteines in redox regulation of RPA-ssDNA interaction. In DNA repair, redox affected RPA's interaction with damaged DNA, but not its role in stabilizing the xeroderma pigmentosum group A (XPA)-damaged DNA complex, suggesting that the zinc-finger motif may mediate the transition of RPA-XPA interaction to a stable RPA-XPA-damaged DNA complex in a redox-dependent manner.     

Comparison of hprt and lacI mutant frequency with DNA adduct formation in N-hydroxy-2-acetylaminofluorene-treated Big Blue rats

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF) is the proximate carcinogenic metabolite of the powerful rat liver carcinogen 2-acetylaminofluorene. In this study, transgenic Big Blue(R) rats were used to examine the relationship between in vivo mutagenicity and DNA adduct formation by N-OH-AAF in the target liver compared with that in nontarget tissues. Male rats were given one, two, or four doses of 25 mg N-OH-AAF/kg body weight by i.p. injection at 4-day intervals, and groups of treated and control rats were euthanized up to 10 weeks after beginning the dosing. Mutant frequencies were measured in the spleen lymphocyte hprt gene, and lacI mutant frequencies were determined in the liver and spleen lymphocytes. At 6 weeks after beginning the dosing, the hprt mutant frequency in spleen lymphocytes from the four-dose group was 16.5 x 10(-6) compared with 3.2 x 10(-6) in control animals. Also at 6 weeks, rats given one, two, or four doses of N-OH-AAF had lacI mutant frequencies in the liver of 97.6, 155.6, and 406.8 x 10(-6), respectively, compared with a control frequency of 25.7 x 10(-6); rats given four doses had lacI mutant frequencies in spleen lymphocytes of 55.8 x 10(-6) compared with a control frequency of 20.4 x 10(-6). Additional rats were evaluated for DNA adduct formation in the liver, spleen lymphocytes, and bone marrow by (32)P-postlabeling. Adduct analysis was conducted 1 day after one, two, and four treatments with N-OH-AAF, 5 days after one treatment, and 9 days after two treatments. N-(Deoxyguanosin-8-yl)-2-aminofluorene was the major DNA adduct identified in all the tissues examined. Adduct concentrations increased with total dose to maximum values in samples taken 1 day after two doses, and remained essentially the same after four doses. In samples taken after four doses, adduct levels were 103, 28, and 7 fmol/microg of DNA in liver, spleen lymphocytes, and bone marrow, respectively. The results indicate that the extent of both DNA adduct formation and mutant induction correlates with the organ specificity for N-OH-AAF carcinogenesis in the rat. Environ. Mol. Mutagen. 37:195-202, 2001. Published 2001 Wiley-Liss, Inc.     

Conditions for transformation of Pasteurella multocida by electroporation.

Conditions for electroporation of plasmid DNA into Pasteurella multocida were determined for use in developing a cloning system to study virulence factors of P. multocida. The highest efficiency of transformation (1.25 x 10(7) cfu/micrograms DNA) was obtained when 7.6 x 10(10) cells of P. multocida strain R473 were electroporated at 12.5 kV/cm (10 ms, 5 ng of pVM109). Transformation efficiencies of cells prepared at mid-log-phase were approximately 0.5 log10 lower than early, late, or stationary phases. Neither pBR322 nor pUC-19 were able to transform strain R473 under these conditions, even when DNA concentrations were increased to 1 microgram. When pBR322 was ligated with a Pasteurella plasmid, pLAR-1, the hybrid was able to transform strain R473 at an efficiency between 4.5 x 10(2) and 8 x 10(4) cfu/micrograms DNA. Six strains of P. multocida including serotypes A, B, D, and E were transformed successfully.     

A RecB-like helicase in Helicobacter pylori is important for DNA repair and host colonization

The human gastric pathogen Helicobacter pylori encounters frequent oxidative and acid stress in its specific niche, and this causes bacterial DNA damage. H. pylori exhibits a very high degree of DNA recombination, which is required for repairing both DNA double-stranded (ds) breaks and blocked replication forks. Nevertheless, few genes encoding components of DNA recombinational repair processes have been identified in H. pylori. An H. pylori mutant defective in a putative helicase gene (HP1553) was constructed and characterized herein. The HP1553 mutant strain was much more sensitive to mitomycin C than the WT strain, indicating that HP1553 is required for repair of DNA ds breaks. Disruption of HP1553 resulted in a significant decrease in the DNA recombination frequency, suggesting that HP1553 is involved in DNA recombination processes, probably functioning as a RecB-like helicase. HP1553 was shown to be important for H. pylori protection against oxidative stress-induced DNA damage, as the exposure of the HP1553 mutant cells to air for 6 h caused significant fragmentation of genomic DNA and led to cell death. In a mouse infection model, the HP1553 mutant strain displayed a greatly reduced ability to colonize the host stomachs, indicating that HP1553 plays a significant role in H. pylori survival/colonization in the host.     

Low-level pilin expression allows for substantial DNA transformation competence in Neisseria gonorrhoeae

The gonococcal pilus is a major virulence factor that has well-established roles in mediating epithelial cell adherence and DNA transformation. Gonococci expressing four gonococcal pilin variants with distinct piliation properties under control of the lac regulatory system were grown in different levels of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG). These pilin variants expressed various levels of pilin message and pilin protein in response to the level of IPTG in the growth medium. Moreover, posttranslational modifications of the variant pilin proteins were detected, including S-pilin production and glycosylation. The ratio of the modified and unmodified pilin forms did not substantially change with different levels of pilin expression, showing that these modifications are not linked to pilin expression levels. DNA transformation competence was also influenced by IPTG levels in the growth medium. Substantial increases in transformation competence over an isogenic, nonpiliated mutant were observed when limited amounts of three of the pilin variants were expressed. Immunoelectron microscopy showed that when limited amounts of pilin are expressed, pili are rare and do not explain the pilin-dependent transformation competence. This pilin-dependent transformation competence required prepilin processing, the outer membrane secretin PilQ, and the twitching-motility-regulating protein PilT. These requirements show that a fully functional pilus assembly apparatus is required for DNA uptake when limited pilin is produced. We conclude that the pilus assembly apparatus functions to import DNA into the bacterial cell in a pilin-dependent manner but that extended pili are not required for transformation competence.     

肺炎链球菌感受态的形成影响毒力因子mRNA的表达

目的：通过体外实验诱导的转化过程，探索肺炎链球菌（Streptococcus pneumoniae,S.pn)毒力因子的表达是否与环境诱导细菌感受态形成与转化有关。方法：采用插入失活的方式断裂S.pn感受态形成的关键基因comE，获得感受态缺陷菌株。以实时定量RT－PCR测定毒力因子在感受态缺陷菌与野生菌的表达是否存在差异。所测毒力因子有自溶酶、溶血素、胆碱结合蛋白、S-IgA、神经氨酸酶、透明质酸酶。结果：S.pn在540nm处吸光度（A）值＝0.044－0.127之间产生感受态。自溶酶、神经氨酸酶、透明质酸酶3种毒力因子的表达，野生菌组表达高于缺陷菌组，两均数间比较的t值分别为2．96（P＜0．05）、2．8（P＜0．05）、4．56（P＜0．05）。结论：细菌感受态能启动自溶酶、神经氨酸酶、透明质酸酶3种毒力因子的表达，提示S.pn的感受态能影响其毒力因子的表达。     

Influence of biological parameters and gene transfer technique on transformation of Fusarium oxysporum

Summary An improved method for PEG-mediated transformation of the fungal pathogen Fusarium oxysporum was developed which led to at least a 10-fold increase in transformation frequency. This improvement was gained through the analysis of biological factors, viz., protoplast origin, plasmid modifications and protoplast/plasmid ratio. Elecroporation, a recently developed method for introducing DNA into many cell types, was successfully applied. It gave similar transformation efficiencies to those obtained with the chemical method, and thus appeared a valuable alternative. Qualitative features such as integration events were also analysed. Molecular analysis of transformants revealed that a single copy of plasmid DNA was preferentially integrated by electroporation. The respective advantages of the two DNA transfer methods are discussed.Abbreviations (CM) complete medium (Daboussi 1980) - (MM) minimal medium (Daboussi 1980) - (MS) 10 mM MOPS, pH 6.3, 1 M sorbitol - (MSC) MS with 10 mM CaCl₂ - (HS) 5 mM HEPES, pH 6.3, 20% sucrose - (PEG) polyethylene glycol - (FD) microfarad     

Chitin-induced carbotype conversion in Vibrio vulnificus

As an etiological agent of bacterial sepsis and wound infections, Vibrio vulnificus is unique among the Vibrionaceae. The most intensely studied of its virulence factors is the capsular polysaccharide (CPS). Over 100 CPS types have been identified, yet little is known about the genetic mechanisms that drive such diversity. Chitin, the second-most-abundant polysaccharide in nature, is known to induce competence in Vibrio species. Here, we show that the frequency of chitin-induced transformation in V. vulnificus varies by strain and that (GlcNAc)(2) is the shortest chitin-derived polymer capable of inducing competence. Transformation frequencies (TFs) increased 8-fold when mixed-culture biofilms were exposed to a strain-specific lytic phage, suggesting that the lysis of dead cells during lytic infection increased the amount of extracellular DNA within the biofilm that was available for transfer. Furthermore, we show that V. vulnificus can undergo chitin-dependent carbotype conversion following the uptake and recombination of complete cps loci from exogenous genomic DNA (gDNA). The acquisition of a partial locus was also demonstrated when internal regions of homology between the endogenous and exogenous loci existed. This suggested that the same mechanism governing the transfer of complete cps loci also contributed to their evolution by generating novel combinations of CPS biosynthesis genes. Since no evidence that cps loci were preferentially acquired during natural transformation (random transposon-tagged DNA was readily taken up in chitin transformation assays) exists, the phenomenon of chitin-induced transformation likely plays an important but general role in the evolution of this genetically promiscuous genus.     

Non-essential loci in the BamHI-I and -F fragments of the HVT FC126 genome

The sequence of BamHI-I fragment of the herpesvirus of turkeys (HVT) FC126 strain DNA was analyzed for the presence of potential open reading frames (ORFs). Four complete (ORFs 2 to 5) and 2 partial ORFs (ORFs 1 and 6) were detected. ORFs 2 and 3 were homologous to the HSV-1 UL55 and the EHV-1 gene 3, respectively. The ORF 6 was already partially sequenced by Smith et al. (Virology 207, 205-216, 1995), and was homologous to a Marek's disease virus (MDV) ORF located in a similar position (ORF 21; Ross et al., Virus Genes 7, 33-51, 1993a). No significant homology was found for the other ORFs. ORF 4 was antisense to ORF 3. Two HVT recombinants having an expression cassette inserted into two intergenic sites were generated and tested for viremia in chickens. Results demonstrated that these 2 intergenic loci are non-essential for in vitro and in vivo HVT replication. A 650 bp deletion in the repeat region flanking UL (TRL and IRL (BamHI-F)) has been identified in some DNA molecules of HVT FC126 strain. This deletion covers the entire truncated pp38 homologous ORF and the N-terminus of a small ORF which has no detectable homology with any known gene. Our results indicate that (1) this genomic region including the HVT pp38 homologue was not essential for in vitro and in vivo growth of HVT, and (2) this deletion had no apparent effect on Marek's disease (MD) protection induced by HVT.