母乳源乳双歧杆菌Probio-M8的遗传特征及基因组差异比较分析

【目的】母乳源乳双歧杆菌（Bifidobacterium animalis subsp. lactis） Probio-M8具有优良的益生特性,本文拟从全基因组水平解析Probio-M8的遗传特征,并与已有益生功效的乳双歧杆菌的基因组进行比较分析。【方法】本研究基于NCBI已公开的21株乳双歧杆菌和1株模式菌株DSM10140^T的基因组数据,构建了核心基因集与泛基因集,解析该群体的系统发育关系,比较分析Probio-M8的遗传特征及功能基因组。【结果】22株乳双歧杆菌的泛基因集包含1 618个基因,其中核心基因1 514个,占泛基因集的93.57%,表明乳双歧杆菌核心基因集高度保守。以1 514个核心基因构建系统发育树,发现22株乳双歧杆菌分为两个分支,AD011单独为一个分支,Probio-M8和其他菌株与模式菌株DSM10140^T聚在同一分支,且Probio-M8与V9、BB-12、Bi-07、HN019的遗传距离极为接近。进一步分析耐药基因和毒力基因,在Probio-M8与V9、BB-12、Bi-07、HN019基因组上均检测到DfrA...     

反刍动物产志贺毒素大肠杆菌的分离鉴定和致病潜力分析

产志贺毒素大肠杆菌(Shiga toxin-producing Escherichia coli,STEC)是重要的食源性病原,而STEC往往以正常菌群的形式存在于牛羊等反刍动物肠道.[目的]本研究对牛羊粪便样品中的STEC分离和鉴定并对分离株进行致病潜力分析.从江苏、云南和河北等地共分离到羊源STEC菌株11株,牛源...     

Adhesion properties of <Emphasis Type="Italic">Lactobacillus casei</Emphasis> strains to resected intestinal fragments and components of the extracellular matrix

Adhesion to intestinal epithelium is an outcome property for the selection of probiotic lactic acid bacteria strains. We have analyzed the adhesion properties of a collection of Lactobacillus casei strains from different origins, ranging from cheese isolates to commercial probiotics. Analysis of the surface characteristics of the strains by measuring adhesion to solvents (MATS test) showed that most of the strains have a basic and hydrophobic surface. The strains were able to bind ex vivo to human colon fragments at different levels and, in most cases, this adhesion correlated with the ability to in vitro binding of mucin. Attachment to this later substrate was not enhanced by growing the cells in the presence of mucin and was independent of proteinaceous factors. On the contrary, adhesion to other extracellular matrix components, such as collagen, fibronectin, or fibrinogen was partially or totally dependent on the presence of surface proteins. These results show that most of L. casei strains have in their surfaces factors that promote binding to intestinal epithelium, however, no clear correlation appears to exist between the origin of the strains and their adhesion capacities.     

仿刺参(Apostichopus japonicus)肠道源植物乳杆菌(Lactobacillus plantarum) HY21的生长及益生特性分析

【目的】在仿刺参(Apostichopus japonicus)健康养殖中为了寻找具有良好耐受性、益生性、安全性的益生菌,本研究从仿刺参肠道内容物中分离获得一株植物乳杆菌(Lactobacillus plantarum) HY21,对其益生潜能进行评价。【方法】利用摇瓶发酵培养、应用环境因子模拟试验、抗生素药敏测试等方法,分析L. plantarum HY21的生长特性、产酸性能、发酵液的抗氧化性质和对水产致病菌的抑制作用;检测菌体的疏水性、自聚性和共聚性、黏附力和药物敏感性,以及对应用环境因子的耐受性。【结果】L. plantarum HY21摇瓶发酵培养温度30℃、初始pH 8.0,于2-10 h为对数生长期,发酵18 h后pH达到最低3.6;发酵液对溶藻弧菌的抑菌直径达到(13.96±0.30) mm,对1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-trinitrophenylhydrazine, DPPH)自由基清除率达到95.45%±1.56%;L. plantarum HY21的疏水率为60.42%±2.78%、自聚率为22.69%±1.36%,与溶藻弧菌共聚率为27.98%±1.45%;对体表和肠道黏液蛋白的黏附量分别达到(1.66±0.01)×10⁶ CFU/mL和(1.23±0.15)×10⁶ CFU/mL;对13种常见的抗菌药物均表现敏感;菌体在模拟胃肠液(pH 3.0, pH 6.8)中孵育3 h存活率均可达99%以上,在10%鱼胆汁溶液中孵育3 h存活率达到68.11%±7.98%,在3%海盐溶液中孵育16 h存活率达到97.58%±7.14%。【结论】L. plantarum HY21具有良好的定殖作用、抑菌作用、抗氧化作用等益生特性,而且在应用环境中具有高存活率和高安全性,可以作为一株新的水产益生菌,为开发水产益生菌制剂提供科学理论依据。     

灌喂植物乳杆菌和干酪乳杆菌增加仔猪肠道菌群多样性及短链脂肪酸生成

【目的】研究断奶前给仔猪饲喂植物乳杆菌和干酪乳杆菌对断奶前、后肠道菌群组成、数量和短链脂肪酸(SCFA)浓度的影响,分析仔猪生长性能与肠道形态、微生物菌群及SCFAs的相关性,探讨测试菌株缓解仔猪断奶应激的可能机制。【方法】选取15窝7 d龄杜长大仔猪,随机分为3组,分别灌喂2 mL去离子水(对照组)、0.5×10~9 CFU/mL植物乳杆菌(LP组)或干酪乳杆菌(LC组)的菌液,每组以窝为单位5个重复,于21 d(断奶)、24 d和35 d屠宰,采集回肠和结肠食糜,分析菌群组成和数量的变化,测定SCFAs浓度。【结果】测试菌株均能显著提高断奶2周后回肠、结肠菌群多样性(P0.05),促进乳酸杆菌和双歧杆菌增殖;显著促进断奶前回肠和结肠中乙酸、丙酸、丁酸和总SCFA生成,促进断奶后乙酸和总SCFA产生;相关分析显示,测试菌株组仔猪腹泻率下降与SCFAs浓度上升、回肠绒毛高度增加和总菌数量上升显著相关,日增重提高与结肠乙酸和TSCFA浓度增加显著相关。【结论】测试菌株促进乳酸杆菌、双歧杆菌等有益菌增殖,增加肠道菌群多样性,促进肠道SCFAs生成。     

Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases

Summary We have cloned and sequenced the Escherichia coli K-12 ppsA gene. The ppsA gene codes for PEP synthase, which converts pyruvate into phosphoenolpyruvate (PEP), an essential step in gluconeogenesis when pyruvate or lactate are used as a carbon source. The open reading frame consists of 792 amino acids and shows homology with other phosphohistidine-containing enzymes that catalyze the conversion between pyruvate and PEP. These enzymes include pyruvate, orthophosphate dikinases from plants and Bacteroides symbiosus and Enzyme I of the bacterial PEP:carbohydrate phosphotransferase system.     

A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

Identification of two new genes,mukE andmukF, involved in chromosome partitioning inEscherichia coli

We have previously reported that the MukB protein is essential for chromosome partitioning inEscherichia coli and thatmukB mutants produce anucleate cells and are temperature-sensitive for colony formation. ThemukB gene maps at 21 min on theE. coli chromosome andsmtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report thatmukF andmukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in themukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for themukF, mukE, andmukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.     

混合菌群调节果蝇产卵偏嗜性

[目的]利用果蝇产卵偏嗜性作为评价指标,研究影响混合菌群平衡的因素及竞争机制,并揭示混合菌对果蝇后代存活率的影响.[方法]将植物乳杆菌(Lactobacillus planterum)、大肠杆菌(Escherichia coli)和二者混合菌发酵处理酪蛋白琼脂食物;利用双向选择产卵器检测成年雌性果蝇的产卵偏嗜性;平板计...     

The fadD gene of Escherichia coli K12 is located close to rnd at 39.6 min of the chromosomal -map and is a new member of the AMP-binding protein family

The fadD gene of Escherichia coli K12 was cloned and sequenced. The gene was identified by its ability to complement the corresponding mutant and by measuring the enzymatic activity after its expression in this mutant. The deduced polypeptide sequence exhibits similarity to other long chain acyl-CoA (coenzyme A) synthetases and a variety of other proteins, which together form a family of AMP-binding proteins. This family is extended by several new members and subdivided into four groups. fadD is assigned to a subgroup that does not include long chain acyl-CoA synthetases from eukaryotic organisms.     

Studies on the alteration of chromosome copy number and cell division potential in a dnaA mutant of Escherichia coli

Summary The dnaA167 mutant of Escherichia coli, N167, maintains, on the average, two replicating chromosomes per cell at the perimissive growth temperature of 30°C and only one per cell at the higher permissive growth temperature of 38°C. When the growth temperature of this mutant is changed from 30° to 38°C the cells rapidly readjust their chromosome copy number from two to one. I have examined the kinetics of this transition with reference to DNA replication and cell division. My results indicate that this mutant uncouples cell division from chromosome duplication to achieve the appropriate copy number, suggesting that the dnaA gene product may be involved in the coordination between these two cellular events.     

Identification and preliminary characterization of temperature-sensitive mutations affecting HlyB, the translocator required for the secretion of haemolysin (HlyA) from Escherichia coli

We have carried out a genetic analysis of Escherichia coli HlyB using in vitro(hydroxylamine) mutagenesis and regionally directed mutagenesis. From random mutagenesis, three mutants, temperature sensitive (Ts) for secretion, were isolated and the DNA sequenced: Glyl0Arg close to the N-terminus, Gly408Asp in a highly conserved small periplasmic loop region PIV, and Pro624Leu in another highly conserved region, within the ATP-binding region. Despite the Ts character of the Gly10 substitution, a derivative of HlyB, in which the first 25 amino acids were replaced by 21 amino acids of the Cro protein, was still active in secretion of HlyA. This indicates that this region of HlyB is dispensable for function. Interestingly, the Gly408Asp substitution was toxic at high temperature and this is the first reported example of a conditional lethal mutation in HlyB. We have isolated 4 additional mutations in PIV by directed mutagenesis, giving a total of 5 out of 12 residues substituted in this region, with 4 mutations rendering HlyB defective in secretion. The Pro624 mutation, close to the Walker B-site for ATP binding in the cytoplasmic domain is identical to a mutation in HisP that leads to uncoupling of ATP hydrolysis from the transport of histidine. The expression of a fully functional haemolysin translocation system comprising HlyC,A,B and D increases the sensitivity of E. coli to vancomycin 2.5-fold, compared with cells expressing HlyB and HlyD alone. Thus, active translocation of HlyA renders the cells hyperpermeable to the drug. Mutations in hlyB affecting secretion could be assigned to two classes: those that restore the level of vancomycin resistance to that of E. coli not secreting HlyA and those that still confer hypersensitivity to the drug in the presence of HlyA. We propose that mutations that promote vancomycin resistance will include mutations affecting initial recognition of the secretion signal and therefore activation of a functional transport channel. Mutations that do not alter HlyA-dependent vancomycin sensitivity may, in contrast, affect later steps in the transport process.     

kpsE和kpsD双基因缺失显著降低肠道外致病性大肠杆菌的毒力

【目的】探究荚膜对肠道外致病性大肠杆菌致病作用的影响。【方法】选取负责荚膜多糖转运的基因kpsE和kpsD,利用λRed重组系统构建APEC E058和UPEC U17荚膜缺失株E058ΔkpsED和U17ΔkpsED,并通过一系列的体内及体外试验对其生物学特性及致病性进行研究。【结果】双基因缺失株的生长速度较野生株没有明显差异,但缺失株抗血清补体杀菌能力和抗鸡巨噬细胞HD-11细胞吞噬能力显著下降。1日龄雏鸡LD50致病性试验结果显示,缺失株E058ΔkpsED和U17ΔkpsED对鸡失去致病力,而回复株毒力恢复至野生株水平;35日龄SPF鸡体内动态分布和竞争试验显示ΔkpsED缺失株在鸡体内定殖能力和竞争性生长能力显著下降,表明kpsED双基因的缺失能显著降低APEC E058和UPEC U17的致病力。【结论】荚膜与肠道外致病性大肠杆菌的致病性相关,是其重要的毒力因子。     

Norepinephrine-dependently released Dr fimbriae of diffusely adhering Escherichia coli strain IH11128 promotes a mitogen-activated protein kinase ERK1/2-dependent production of pro-inflammatory cytokine, IL-8 in human intestinal Caco-2/TC7 cells

The diffusely adhering Escherichia coli (Afa/Dr DAEC) are associated with recurrent urinary tract infections in adults as well as with diarrheal disease in infants. We previously demonstrated that in wild-type strain IH11128, the Dr fimbriae is released in the extracellular medium in response to multiple environmental signals such as temperature, low aeration and rich medium. A number of molecules of eukaryotic origin, such as catecholamines, have been reported to stimulate bacterial growth and virulence factor production. We show that norepinephrine affects the production and release of Dr fimbriae in Afa/Dr DAEC WT-IH11128 bacteria. The regulatory mechanism involved with norepinephrine-induced Dr fimbriae liberation was apparently due to a differential induction of genes draC, encoding the usher, and draE, encoding the major fimbrial subunit. In addition, we show that the released Dr fimbriae induces the phosphorylation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 (ERK1/2) and the production of the pro-inflammatory cytokine, IL-8 in fully differentiated cultured human intestinal Caco-2/TC7 cells.     

Genetic and molecular study of the inability of the yeast Kluyveromyces lactis var. drosophilarum to ferment lactose

The fermentation of lactose (Lac⁺) in the dairy yeast Kluyveromyces lactis var. lactis is controlled by the LAC4 (β-galactosidase) and LAC12 (lactose permease) genes. The complementation analysis of twelve Kl. lactis var. drosophilarum natural homothallic Lac^? strains of different origin was carried out using the genetic heterothallic lines of Kl. lactis var. lactis of the lac4LAC12 and LAC4lac12 genotypes. It was shown that the natural Lac^? strains did not possess the LAC4LAC12 gene cluster. Southern hybridization of chromosomal DNA with LAC4 and LAC12 probes, as well as recombination analysis, showed that Kl. lactis var. drosophilarum yeasts do not have even silent copies of these genes. As distinct from this yeast, natural Lac^? strains of the yeast Kl. marxianus are mutants impaired in the lactose permease gene (lac12 analogue), but possess an active β-galactosidase gene (LAC4 analogue). The origin of the LAC4LAC12 gene cluster of the dairy yeasts Kl. lactis is discussed.