Influence of DNA end structure on the mechanism of initiation of DNA unwinding by the Escherichia coli RecBCD and RecBC helicases

Escherichiacoli RecBCD is a bipolar DNA helicase possessing two motor subunits (RecB, a 3′-to-5′ translocase, and RecD, a 5′-to-3′ translocase) that is involved in the major pathway of recombinational repair. Previous studies indicated that the minimal kinetic mechanism needed to describe the ATP-dependent unwinding of blunt-ended DNA by RecBCD in vitro is a sequential n-step mechanism with two to three additional kinetic steps prior to initiating DNA unwinding. Since RecBCD can “melt out” ∼ 6 bp upon binding to the end of a blunt-ended DNA duplex in a Mg²⁺-dependent but ATP-independent reaction, we investigated the effects of noncomplementary single-stranded (ss) DNA tails [3′-(dT)₆ and 5′-(dT)₆ or 5′-(dT)₁₀] on the mechanism of RecBCD and RecBC unwinding of duplex DNA using rapid kinetic methods. As with blunt-ended DNA, RecBCD unwinding of DNA possessing 3′-(dT)₆ and 5′-(dT)₆ noncomplementary ssDNA tails is well described by a sequential n-step mechanism with the same unwinding rate (mk_U = 774 ± 16 bp s^− 1) and kinetic step size (m = 3.3 ± 1.3 bp), yet two to three additional kinetic steps are still required prior to initiation of DNA unwinding (k_C = 45 ± 2 s^− 1). However, when the noncomplementary 5′ ssDNA tail is extended to 10 nt [5′-(dT)₁₀ and 3′-(dT)₆], the DNA end structure for which RecBCD displays optimal binding affinity, the additional kinetic steps are no longer needed, although a slightly slower unwinding rate (mk_U = 538 ± 24 bp s^− 1) is observed with a similar kinetic step size (m = 3.9 ± 0.5 bp). The RecBC DNA helicase (without the RecD subunit) does not initiate unwinding efficiently from a blunt DNA end. However, RecBC does initiate well from a DNA end possessing noncomplementary twin 5′-(dT)₆ and 3′-(dT)₆ tails, and unwinding can be described by a simple uniform n-step sequential scheme, without the need for the additional k_C initiation steps, with a similar kinetic step size (m = 4.4 ± 1.7 bp) and unwinding rate (mk_obs = 396 ± 15 bp s^− 1). These results suggest that the additional kinetic steps with rate constant k_C required for RecBCD to initiate unwinding of blunt-ended and twin (dT)₆-tailed DNA reflect processes needed to engage the RecD motor with the 5′ ssDNA.     

Novel ecological niche of Cetobacterium somerae, an anaerobic bacterium in the intestinal tracts of freshwater fish

AIMS: This study was conducted to clarify the taxonomic status of Bacteroides type A strains with high vitamin B(12)-producing ability that is widely distributed in the intestinal tracts of freshwater fish. METHODS AND RESULTS: Seventeen strains of Bacteroides type A isolated from five fish species were all rod-shaped and gram-negative. The strains were positive for esculin hydrolysis, nitrate reduction, resistance to bile, acid phosphatase, and negative for the production of catalase and urease and the susceptibility to vancomycin. The G+C content of DNA from the 17 strains was 29 x 1-31 x 9 mol%, and 16S rDNA sequence analysis revealed a close phylogenetic relationship between Bacteroides type A strains and Cetobacterium somerae sharing 99 x 7-100% sequence similarity. In addition, strains were capable of producing vitamin B(12) at a rate of 1 x 82-13 x 98 ng ml(-1) in 48 h. CONCLUSION: Phenotypic and phylogenetic characteristics indicated that all isolates previously classified as Bacteroides type A strains belong to C. someare. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided the important finding of novel niche of vancomycin-resistant bacteria such as C. somerae in the intestinal tract of freshwater fish.     

Cytokine release and expression induced by OmpA proteins from the Gram-negative anaerobes, Porphyromonas asaccharolytica and Bacteroides fragilis

OmpA proteins from Gram-negative anaerobes Porphyromonas asaccharolytica and Bacteroides fragilis induced release and expression of IL-1alpha, tumor necrosis factor (TNF)-alpha, IFN-gamma, IL-6, and IL-10 from murine splenocytes in vitro in a dose-dependent fashion. The release of the cytokines induced by B. fragilis Bf-OmpA was at much lower levels compared with P. asaccharolytica Omp-PA; Bf-OmpA did not induce release of IL-10. Omp-PA and Bf-OmpA were able to upregulate mRNA expression of the tested cytokines. The results obtained with refolded Bf-OmpA were similar to those with native Bf-OmpA. The data presented in this research demonstrate for the first time that Omps from anaerobic bacteria can induce the release of cytokines, suggesting that Omp-PA and Bf-OmpA may play important roles in the pathogenic processes of these bacteria.     

天然砂与修饰砂对病毒的吸附与去除

分别在不同pH、含有不同金属阳离子及腐殖酸的脱氯自来水与蒸馏水中,研究了砂与金属氢氧化物修饰砂吸附与去除Polivirus 1(PV1)和Bacteroides fragilis phage(B．fp)的不同效果。结果显示,天然砂吸附病毒效率随水体pH的降低而增加,但当砂表面经氧氧化铝、氢氧化铁沉积修饰后,却在中性pH具有较高的吸附率。金属阳离子可促进天然砂吸附病毒,并随其浓度和价态的增加而增加,但对修饰砂作用不明显。腐殖酸对砂及修饰砂吸附病毒的影响也不同,修饰砂吸附病毒不受腐殖酸的存在及其浓度影响,而天然砂会因腐殖酸的存在及其浓度的提高而降低吸附病毒效率。在任一实验条件下,砂与修饰砂在自来水中的吸附病毒效率高于在蒸馏水中的吸附效率,扫描电镜观察显示砂与修饰砂具有明显不同的表面结构特征,这是两者具有不同吸附效率的基础。从砂与修饰砂吸附PV1的效率高于B．fp的现象说明B．fp是一个更为合适的病毒去除指示生物。本实验结果表明了金属氢氧化物修饰砂可为传统的净水工艺提供高效的病毒去除过滤介质与途径。     

培养基成分对口腔福赛类杆菌生长影响的研究

目的比较不同培养基成分对福赛类杆菌ATCC43037生长的影响,寻找一种有效促进福赛类杆菌生长的培养基.方法将福赛类杆菌ATCC430 37 接种到5种不同培养基(包括琼脂板和液体培养基)中,在厌氧培养箱内37℃厌氧培养7 d,观察平板上菌落生长情况,在λ=550 nm处每24 h测定液体培养菌液A值.细菌通过革兰染色和PCR法鉴定.结果1.生长在血平板上的福赛类杆菌ATCC43037菌落形态呈粉红色或白色小斑点状,在以BHI 为基础培养基中添加氯化血红素、维生素K3、N-乙酰胞壁酸的No2血琼脂培养基生长最好,而在无氯化血红素、维生素K3及血的No4琼脂板上不生长.2.福赛类杆菌ATCC43037在以TSB 为基础培养基中添加酵母提取物、植物蛋白胨、氯化血红素、维生素K3、N-乙酰胞壁酸和D TT的No1液体培养基生长最好,培养7 d后A值＞0.8,在缺乏N-乙酰胞壁酸的No5液体培养基中几乎不生长.3.菌落细菌革兰染色为G 梭状杆菌,PCR法鉴定为福赛类杆菌.结论福赛类杆菌在血平板和液体培养基中存在不同的生长特性,N-乙酰胞壁酸是福赛类杆菌在液体培养基中生长最重要的成分,DTT有益于细菌生长.     

Purification and partial characterization of an iron regulated outer membrane protein of B. fragilis under non-denaturing conditions

The CHAPS-PAGE gelsystem we applied gave a good separation of the proteins of Bacteroides fragilis under non-denaturing conditions. We succeeded with preparative CHAPS-PAGE in purifying an iron regulated outer membrane protein (a 44 kDa polypeptide on SDS-PAGE) of B. fragilis. This integral membrane protein proved to be a lipopolysaccharide binding protein with an isoelectric point of approximately pH 5.5. This method of purifying membrane proteins could be an important step in research into the function of membrane proteins.     

Metabolism of cellobiose and cellulose byBacteroides cellulosolvens

Summary The metabolism ofBacteroides cellulosolvens was studied on cellobiose and cellulose as energy and carbon sources. The growth rate was faster on cellobiose; however, growth on cellulose resulted in consumption of 55% more hexose equivalents, and in production of 49% more biomass, and 30% more metabolites (ethanol, acetate, and lactate). On each substrateB. cellulosolvens exhibited two distinct ranges of molar growth yields (Y _H g cells/mol hexose). At low substrate concentrations (less than 30 mmol) hexoseY _H values were 25.5 for cellulose and 28.5 for cellobiose, while at hexose levels greater than 30 mmolY _H values were 13.5 and 15, respectively. Shifts in metabolism towards greater lactic acid production resulted in decreased ATP production; however, this did not cause early growth cessation, as these shifts occurred after the drop inY _H.Issued as NRCC No. 27409.     

In Vivo Metal Substitution in Bacteroides Fragilis Superoxide Dismutase

Bacteroides fragilis. an obligate anaerobe, synthesizes an azide-inhibitable iron-containing superoxide dismutase when grown in complex medium. Cells grown anaerobically in complex media containing dcsferrioxamine (Desferal^TM, Ciba-Geigy) and graded concentrations of Mn synthesize the azide-resistant manganese-containing SOD. The fraction of MnSOD activity in dialyzed cell extracts increased prograsively as the Mn concentration in the medium increased. The fraction of MnSOD activity also increased in extracts of cells grown in the medium with I mM Mn but with graded concentrations of desferrioxamine (0–10 micromolar). The SOD activity in the cells grown under the various conditions varied but not in a causal relationship with either Mn or desferrioxamine concentration. Electrophoresis reveaicd that the SOD activity in cells grown in the absence or presence of I mM Mn migrated with the same relative mobility and exhibited identical activity patterns when examined separately or as a mixture. These data are consistent with substitution of Mn for Fe in the B. fragilis apoprotein under anaerobic conditions and support the model of a single protein binding either Fe or Mn.     

Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli

A major immunodominant surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis 381 has been purified and its amino-terminal amino acid sequence has been determined. Using oligonucleotide probes corresponding to the sequence, we identified a recombinant plasmid clone carrying a single 4.2-kb BamHI fragment from pUC19 libraries of P. gingivalis. The BamHI fragment transferred to the bacteriophage T7 RNA polymerase/promoter expression vector system produced a slightly larger (77-kDa) protein, a precursor form, immunoreactive to the antibody against the 75-kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 75-kDa protein. Genomic Southern analysis revealed a single copy of the 75-kDa protein gene per genome among all P. gingivalis strains tested, and that no homologous genes are present in other black-pigmented Bacteroides species. These observations suggest that the 75-kDa protein gene may be useful as a specific DNA probe to classify or to detect this organism.     

Isolation and characterization of a porin-like protein of 45 kilodaltons from Bacteroides fragilis

Resistance to the combination of amoxicillin and clavulanic acid in some Bacteroides fragilis strains may be associated with a lack of porin proteins. Comparison of outer membrane protein profiles from one resistant strain ( B. fragilis CFPL 358) and two susceptible strains of B . fragilis (ATCC 25285 and CFPL 92125) showed that a few proteins were missing in the resistant strain, especially a 45-kDa protein. To determine whether this protein was a porin-like protein, we attempted to isolate it from the two susceptible strains by using gel filtration (Sephacryl S-200, Superose 6) and ion exchange chromatographies (DEAE Trisacryl, DEAE Sepharose Fast Flow). Elution from DEAE resins was poor compared to the 60–67-kDa region, which suggested that the 45-kDa protein exhibited stronger cationic forms. The use of sodium dodecyl sulfate during elution improved the recovery of the 45-kDa protein, showing that detergent modified its conformation and its ionic bounds with the chromatographic matrices but it was not sufficient for good purification. Superose 6 gel filtration also failed to separate this protein from the 60–67-kDa region. The only method resulting in the positive recovery of a purified 45-kDa band from both susceptible B. fragilis strains was electroelution from SDS-PAGE. The swelling assay showed that the 45-kDa protein was a porin-like protein. From this study, we concluded that the 45-kDa protein from B. fragilis was a porin-like protein which might be involved in the antibiotic resistance of a strain in which this protein was missing.