青海湖裸鲤肠道乳酸菌多样性与抑菌活性

【目的】通过生理生化特性,结合16S r RNA基因序列分析研究青海湖裸鲤肠道乳酸菌分离株的多样性,并对这些代表株的抑菌活性进行初步探讨,以期筛选具有高效抑菌活性的鱼源益生菌。【方法】对分离的47株乳酸菌代表株进行p H、温度生长范围、耐盐性等生理生化特征检测,结合16S r RNA基因序列对已分离到的乳酸菌进行基因分型和菌种鉴定,采用牛津杯双层平板法检测乳酸菌代表株的抑菌活性。【结果】鉴定结果显示:23株为Lactobacillus fuchuensis(48.94%),12株为Lactobacillus curvatus(25.53%),3株为Leuconostoc fallax(6.38%),2株为Lactobacillus sakei(4.26%),2株为Weissella ceti(4.26%);2株为Lactococcus cremoris(4.26%),1株为Leuconostoc lactis(2.13%),1株为Weissella minor(2.13%),1株为Enterococcus devriesei(2.13%)。qz1217、qz1196、qz1220所在的A、B、C三组乳酸菌在5-50°C的温度范围内生长良好,qz1196、qz1220所在的B、C组在pH 3.0-10.0的范围内生长良好,几乎所有乳酸菌都具有耐6.5%盐浓度特性。13株乳酸菌菌株对6种病原菌都具有抑制作用。通过排除酸、过氧化氢实验,发现上清液仍然具有抑菌活性。对qz1251发酵液进行蛋白酶处理,抑菌活性消失,确定其抑菌物质属于蛋白类物质,是一种细菌素。【结论】青海湖裸鲤肠道附着乳酸菌的多样性为益生性乳酸菌的筛选提供优质资源及数据参考。     

连翘内生真菌的分离鉴定及其发酵液抑菌活性和HPLC测定

为获得能产生抑菌活性物质的连翘内生菌,从连翘新鲜组织中分离纯化内生真菌并进行形态学分析,牛津杯法测定发酵液的抑菌活性,对具有抑菌活性的菌株进行18S rRNA基因序列分析,并以连翘主要活性成分连翘苷和连翘酯苷A、B标准品为对照,HPLC测定发酵上清液中的连翘苷和连翘酯苷A、B含量。结果表明从连翘新鲜组织中获得了5株内生真菌,其中4株菌的发酵上清液对金黄色葡萄球菌和肠炎沙门氏菌有很好的抑菌活性,对这4株菌的分子生物学检测表明3株菌为枝胞菌,另1株菌为曲霉菌,HPLC结果显示这4株菌的发酵上清液中均含有一定浓度的连翘苷和连翘酯苷A、B,发酵液中发挥抑菌活性的成分是否与这三种物质有关需要进一步研究,但连翘内生真菌可以产生抑菌活性物质这一结果为后续利用微生物发酵生产抑菌物质提供了新的途径和新的菌种来源。     

北部湾近海珊瑚礁区系沉积物抗菌活性放线菌类群

通过对大肠埃希菌和枯草芽胞杆菌抗菌活性初步筛选,从北部湾近海珊瑚礁区5个沉积物样品中成功分离得到51株具有不同抗菌活性的放线菌,其中9株具有较强抗菌能力。根据这9株放线菌的菌落和孢子形态,可确定它们都属于链霉菌属。 RAPD-PCR分析表明这9株放线菌为6种不同类型,16S rDNA序列和系统发生树分析表明,9株放线菌可划分到4个大的类群6种不同类型,且结果显示RAPD-PCR聚类分析与16S rDNA序列聚类分析的结果具有较大的一致性。生理生化鉴定结果表明,分离株与亲缘关系最近的放线菌模式菌株的生理生化特征均存在差异,这说明分离株为放线菌新种的可能性比较大。这6种放线菌具有较为广谱的抑菌活性,并且抑菌活性均存在一定的差异,说明其可能分泌出多种结构功能不同的活性次生代谢产物。研究结果表明,广西北部湾近海珊瑚礁区系沉积物蕴藏着丰富的可供药物开发的放线菌资源。     

1株生防细菌的筛选、鉴定及其抑菌特性的初步研究

从鲜牛奶中筛选1株对番茄灰霉病菌具有拮抗作用的菌株A9,经16S rDNA序列分析,鉴定为枯草芽胞杆菌(Bacillus subtilis)。抑菌特性研究表明,该菌株代谢产物会造成番茄灰霉病菌菌丝畸形,同时抑制其孢子生长,使孢子细胞壁破裂;代谢粗提物经过DEAE-52离子交换层析及Sephadex-G50凝胶柱层析后,电泳检测到具有抑菌活性且分子量约为17ku的单一条带,经验证该抑菌物具有蛋白酶活性。     

一株抗G- 菌和酵母菌的乳酸乳球菌的分离鉴定与抗菌活性

以G+ 菌金黄色葡萄球菌(Staphylococcus aureus)作为指示菌, 通过抑菌筛选法从生牛奶中初筛得到具有抑菌活性的14株细菌菌株, 然后通过个体形态与培养特征观测、部分生理生化反应、G + C mol%测定、16S rDNA序列比对分析、PCR扩增特异性N-乙酰胞壁酸水解酶基因和序列对比分析等鉴定, 确定其中的一株具有较高抑菌活性的分离株为乳酸乳球菌乳酸亚种(Lactococcus lactis subsp. lactis)菌株, 命名为MB191。对多种G+ 细菌、G- 细菌、酵母菌和丝状真菌的对峙培养抗性测定结果表明, MB191除对供试G+ 细菌具有较高的抑菌活性以外, 还对丁香假单胞菌(Pseudomonas syringae)、荧光假单胞菌(P. fluorescens)等G- 细菌和汉逊德巴利酵母(Debaryomyces hansenii)等具有明显的抑菌活性。乳酸乳球菌的这一特性目前尚未见文献报道。     

土壤样品中粘细菌的分离鉴定及其生物活性检测

【目的】从土壤样品中分离粘细菌,对其进行纯化、归类与鉴定,以丰富粘细菌菌种资源;对纯化得到的粘细菌进行抑菌、杀虫及抗肿瘤生物活性的分析,为粘细菌次级代谢产物的开发奠定基础。【方法】采用灭活大肠杆菌和滤纸片诱导子实体法从土样中分离粘细菌,结合形态观察、生理生化特征和16S r DNA基因序列同源性分析,对分离到的各菌株进行鉴定并归类。通过平板扩散法、昆虫口服毒性测试、肿瘤细胞毒性实验等方法,检测粘细菌代谢产物生物活性。【结果】共分离得到35株粘细菌,初步分类为:粘球菌属(Myxococcus)9株;珊瑚球菌属(Corallococcus)9株;侏囊菌属(Nannocystis)11株;堆囊菌属(Sorangium)6株。从其中纯化得到8株粘细菌,对其进行了鉴定并命名。发现了具有高效且广谱的肿瘤细胞毒性效应菌株S22,S51和S55也具同样的细胞毒性;另外,还发现具有肿瘤细胞毒性的菌株S20和S22对枯草芽胞杆菌和白色念珠菌也具有较好的抑菌活性。【结论】土壤中存在丰富的粘细菌资源,发现了具有肿瘤细胞毒性的弱小珊瑚球菌与具有抑菌和强抗肿瘤活性的大孢珊瑚球菌,粘细菌是活性天然产物与新药开发的重要资源。     

沙棘根瘤内生细菌中抑菌促生菌株的筛选和鉴定

【背景】植物内生细菌可产生具有抑菌和促生活性的物质,既能抑制植物病原菌对寄主植物的侵染,也能促进植物的生长。沙棘根瘤内生细菌是根瘤内除共生固氮的弗兰克氏菌外,与沙棘共生的一大类微生物。研究具有抑菌和促生活性的植物内生菌,可为微生物菌肥的研究提供理论基础。【目的】筛选具有优良抑菌和促生活性的沙棘根瘤内生细菌,初步研究其抑菌和促生活性,并对菌株进行鉴定。【方法】采用双层琼脂法、琼脂扩散法、双层平板对峙法、牛津杯法进行沙棘根瘤拮抗性内生细菌的筛选。选取抑菌活性较高的内生细菌,分别采用Salkowski比色法、ChromeAzurolS(CAS)平板检测法和钼锑抗比色法进行产吲哚乙酸、铁载体及溶磷能力的测定。采用发酵液灌根法测定沙棘根瘤内生细菌SR308对黄瓜促生作用的盆栽效果。通过形态和培养特征、生理生化试验及16S rRNA基因序列分析法对菌株TT201和SR308进行鉴定。【结果】从131株沙棘根瘤内生细菌中筛选出9株具有较强抑菌活性的内生细菌,其中菌株TT201抑菌性最佳、抑菌谱广;菌株SR308的促生活性最好,其发酵液对黄瓜的生长具有较强的促进作用。对具有较强抑菌和促生活性的菌株TT201和SR308进行鉴定的结果表明,菌株TT201为侧孢短芽孢杆菌(Brevibacilluslaterosporus),菌株SR308为蕈状芽孢杆菌(Bacillusmycoides)。【结论】获得2株具有优良抑菌和促生活性的沙棘根瘤内生细菌,为进一步开发微生物农药及菌肥提供了资源。     

高抑菌活性乳酸杆菌的筛选及性质

从自酿酸奶中分离得到1株高抑菌活性菌株,经16S rDNA测序后鉴定为Lactobacillus sp.FSZ。以大肠杆菌、金黄色葡萄球菌为指示菌,取得良好抑菌效果。经组分分析及蛋白酶降解,抑菌活性物质确定为蛋白物质,推测其由一些高分子的蛋白类物质和低分子的多肽类物质组成。抑菌活性物质在酸性条件下显示出良好的抑菌活性,发酵液经60℃处理30 min后,活性基本没有下降,经100℃处理30 min仍保留83.9%的活性,表现出良好的热稳定性。     

美洲大蠊肠道内生细菌的分离及其初步抑菌活性研究

分离鉴定美洲大蠊肠道内生细菌,并初步研究了其抑菌活性。采用平板稀释法对美洲大蠊肠道内生细菌进行分离纯化,分离菌株通过形态学观察和16S rDNA基因序列BLAST比对进行种属鉴定。以金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌、肺炎克雷伯菌、大肠埃希氏菌4株细菌为对象,采用牛津杯法初步鉴定各分离菌株的抑菌活性。BLAST比对分析结果表明:从15只美洲大蠊肠道内分离鉴定出125株内生细菌,分属12科20属。初步抑菌活性显示:部分菌株对金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌、肺炎克雷伯菌、大肠埃希氏菌有明显抑菌活性。美洲大蠊肠道内生菌种类繁多,而且较多菌株具有抑菌活性。     

广西三种真红树植物可培养细菌多样性及其生物活性初筛

为了挖掘真红树植物潜在细菌新物种和生物活性物质,丰富红树林微生物多样性,为新型活性产物开发提供菌株资源。该文从秋茄、木榄和红海榄三种广西来源的真红树植物及其生境中,按根、茎、叶、花、果实和泥土分成22份样品,选用8种不同培养基分离可培养细菌,通过16S rRNA基因序列鉴定,分析其多样性,采用纸片法筛选细菌发酵粗提物的抑菌活性,点植法测试其酶活性。结果表明：(1)共分离获得可培养细菌35株,隶属于23个科28个属,芽孢杆菌属占细菌总数的14.3%,为优势菌属,同时发现11株潜在的新细菌资源。(2)活性筛选获得4株细菌具有抑菌活性,16株细菌具有酶活性,芽孢杆菌属是酶活性优势菌属。综上所述,广西真红树植物可培养细菌多样性丰富,部分细菌具有抑菌活性和酶活性,在新型抗生素和酶应用方面具有一定的开发潜力。     

Network Relationships of Bacteria in a Stable Mixed Culture

We investigated the network relationships of bacteria in a structurally stable mixed culture degrading cellulose. The mixed culture consists of four bacterial strains (a cellulose-degrading anaerobe [strain S], a saccharide-utilizing anaerobe [strain F], a peptide- and acetate-utilizing aerobe [strain 3] and a peptide-, glucose-, and ethanol-utilizing aerobe [strain 5]). Interspecies interactions were examined by analyzing the effects of culture filtrates on the growth of the other strains and by comprehensively analyzing population dynamics in the mixed-culture systems with all possible combinations of the four bacterial strains. The persistence of strain S depends on the effects of strain 5. However, strain 5 is a disadvantaged strain because strain 3 has bacteriocidal activity on strain 5. The extinction of strain 5 is indirectly prevented by strain F that suppresses the growth of strain 3. Although strain F directly has suppressive effects on the growth of strain S, strain F is essential for the persistence of strain S, considering the indirect effects (maintaining strain 5, which is essential for the survival of strain S, by inhibiting strain 3). These indirect relationships form a bacterial network in which all the relationships including suppressive effects were well balanced to maintain the structural stability. In addition to direct metabolite interactions, such kind of indirect relationships could have a great impact on microbial community structure in the natural environment.     

Binding of chloramphenicol and a fragment of aminoacyl-transfer ribonucleic acid to ribosomes and a ribosome precursor from a mutant of Escherichia coli.

During exponential growth, the mutatn strain Escherichia coli 15-28 accumulates 47S particles, which are unusual precursors to 50S ribosomal subunits. The 47S particles have little ability to bind chloramphenicol, but binding of a fragment of aminoacyl-tRNA is about half that by completed subunits. The 70S (and 50S) ribosomes of strain 15-28 and its parent (strain 15TP) do not differ in chloramphenicol binding. Although ribosomes from the mutant are less able than those from the parent to bind the fragment, this difference is not as marked as was found previously [Sims & Wild (1976) Biochem. J. 160, 721-726] for the binding of an analogue of peptidyl-tRNA and for peptidyltransferase activity. The altered activities may arise because strain 15-28 misassembles 50S subunits of altered conformation and because the few proteins that 47S patricles lack have vital functions in some of the partial reactions of protein synthesis.     

Recombinant Pseudomonas exoenzyme S and exoenzyme S from Pseudomonas aeruginosa DG1 share the ability to stimulate T lymphocyte proliferation.

Exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 have distinct characteristics, which has led to a controversy about their homology and their pathophysiologic consequences. We have been investigating the ability of exoenzyme S to activate T lymphocytes, and therefore performed studies to determine whether exoenzyme S from P. aeruginosa DG1 and recombinant exoenzyme S derived from strain 388 and expressed in Pseudomonas aeruginosa PA103 or in E. coli BL21(DE3), could induce T lymphocyte activation and proliferation. Both preparations were able to activate T cells and induce lymphocyte proliferation at similar levels as measured by flow cytometry of surface-activation markers and DNA synthesis, respectively. Further, a monoclonal antibody raised against exoenzyme S from strain DG1 partially neutralized T cell activation induced by recombinant exoenzyme S and bound to it in an immunoblot suggesting that the epitope responsible for T cell activation is shared by exoenzyme S from strain DG1 and recombinant exoenzyme S. These data suggest that the two different preparations of exoenzyme S, despite biochemical differences, share the characteristic that is responsible for T lymphocyte activation.     

Coexistence of antibiotic-producing and antibiotic-sensitive bacteria in biofilms is mediated by resistant bacteria

Antibiotic-sensitive bacteria have been found to coexist with antibiotic-producing bacteria in biofilms, but little is known about how the former develop in such an environment. Here we isolated pyocyanin-sensitive bacteria belonging to the genus Brevibacillus from a biofilm derived from soil extract and based on the preestablished biofilm of a pyocyanin producer, Pseudomonas aeruginosa strain P1. In addition, pyocyanin-resistant strains belonging to the genus Raoultella were isolated from the same biofilm. Microbial relationships within biofilms were examined by using three strains, strain P1, Brevibacillus strain S1, and Raoultella strain R1, each of which individually formed a biofilm within 2 days in a flow cell. Strain S1 did not fully develop on the preestablished biofilm of strain P1 during 4 days of cultivation, whereas a mutant of strain P1 which was deficient in pyocyanin production allowed strain S1 to cocolonize within a biofilm. On the other hand, strain R1 developed on the biofilm of strain P1 regardless of pyocyanin production. When mixed 1:1 inocula of strains S1 and R1 were introduced into the strain P1 biofilm, all three species were found in the 4-day biofilm. In the mixed biofilm, strain S1 was surrounded by the layer of strain R1 and seemed to be separated from strain P1 and the outflow solution. However, strain S1 did not survive in a three-species mixed culture under planktonic conditions. These results indicate that the survival of sensitive bacteria in biofilm with a pyocyanin producer is achieved by covering them with a layer of resistant bacteria. We also evaluated the influence of antibiotic production on the producer.     

Genome sequence of Brucella abortus vaccine strain S19 compared to virulent strains yields candidate virulence genes

The Brucella abortus strain S19, a spontaneously attenuated strain, has been used as a vaccine strain in vaccination of cattle against brucellosis for six decades. Despite many studies, the physiological and molecular mechanisms causing the attenuation are not known. We have applied pyrosequencing technology together with conventional sequencing to rapidly and comprehensively determine the complete genome sequence of the attenuated Brucella abortus vaccine strain S19. The main goal of this study is to identify candidate virulence genes by systematic comparative analysis of the attenuated strain with the published genome sequences of two virulent and closely related strains of B. abortus, 9-941 and 2308. The two S19 chromosomes are 2,122,487 and 1,161,449 bp in length. A total of 3062 genes were identified and annotated. Pairwise and reciprocal genome comparisons resulted in a total of 263 genes that were non-identical between the S19 genome and any of the two virulent strains. Amongst these, 45 genes were consistently different between the attenuated strain and the two virulent strains but were identical amongst the virulent strains, which included only two of the 236 genes that have been implicated as virulence factors in literature. The functional analyses of the differences have revealed a total of 24 genes that may be associated with the loss of virulence in S19. Of particular relevance are four genes with more than 60 bp consistent difference in S19 compared to both the virulent strains, which, in the virulent strains, encode an outer membrane protein and three proteins involved in erythritol uptake or metabolism.     

Concomitant Synthesis of Bacteriocin and Bacteriocin Inactivator from Serratia marcescens

We have found that Serratia marcescens strain P & S is bacteriocinogenic. However, the phenotypic expression of bacteriocin activity depends upon the temperature at which the cells are grown. When the organism is grown at 30 to 37 C, no bacteriocin activity can be demonstrated, whereas when it is grown at 39 C bacteriocin activity is readily observed. It appears that the P & S strain concomitantly synthesizes a bacteriocin and a substance which not only can inactivate the bacteriocin but also has a high activation energy for inactivation. This inactivator readily loses its activity when heated at 39 C for 1 hr. Two mutants were isolated from the P & S strain which can produce active bacteriocin when grown at temperatures from 30 to 39 C. It is significant that these mutants have considerably less bacteriocin inactivator. The data suggest that the inactivator is an extracellular protease. The ability of one of these mutants, JF58-12, to produce active bacteriocin at temperatures between 30 and 39 C is a stable property, whereas in the other mutant, JF48W, this property is unstable. JF48W was selected from the P & S strain in two steps: first a streptomycin-resistant variant (strain A-10) was isolated and from this mutant a strain (JF48W) was isolated which not only synthesized little of the inactivator but also did not synthesize the red pigmnet prodigiosin. This latter pleiotropic mutant appears to revert in one step to a phenotype similar to the P & S strain, since it is streptomycin-sensitive and produces prodigiosin and normal amounts of inactivator and the demonstration of bacteriocin activity is temperature-dependent.     

Histopathological study of rabbit intestinal mucosa infected with a hybrid strain of Shigella dysenteriae 1 carrying LPS biosynthesis genes of Salmonella enterica serovar typhimurium

The rfb gene cluster and the rfc gene of Salmonella enterica were introduced earlier into an invasive Shigella dysenteriae 1 strain by triparental cross. Antiserum was raised in rabbit against lipopolysaccharide isolated from the hybrid strain. Both the hybrid and the invasive S. dysenteriae 1 strain were found to have a titer of 1:2560 while for S. enterica, it was 1:640. Ligated ileal loops were prepared in rabbit, which were inoculated with 10(8) CFU ml(-1) each of the hybrid strain, and invasive S. dysenteriae 1 strain used as positive control. Escherichia coli K12 was also used as a negative control. After 18 h, the fluid accumulation ratios were 0.2 and 1.6 for hybrid and invasive strains of S. dysenteriae 1, respectively. Rabbit intestinal mucosa infected with hybrid S. dysenteriae 1 strain showed the presence of intact villus tips and unruptured intestinal mucosa whereas total necrosis of intestinal mucosa and villi was observed in the S. dysenteriae 1-infected region.     

Cloning, characterization and taxonomic significance of genes for the 5S ribosomal RNA of Leptonema illini strain 3055.

The genes encoding the 5S ribosomal RNA (rRNA) for Leptonema illini strain 3055 were isolated and sequenced. The 5S RNA molecule encoded was 117 nucleotides long. The genome of strain 3055 contained two genes for 5S rRNA that were located close together. The nucleotide sequences of the Leptonema illini genes exhibited less similarity to the rRNA gene of Leptospira interrogans strain Moulton and also to those of typical eubacterial genes than did the rRNA genes of other leptospires. However, the overall secondary structure of the 5S rRNA encoded exhibited a strong similarity to that of typical eubacterial 5S rRNA. Southern hybridization of the 5S rRNA gene probe with the genomic DNA of strain 965, which is currently classified as Leptospira biflexa, showed the latter to have close similarity to that of strain 3055. The physical map of strain 965 was quite similar to that of strain 3055 and was greatly different from that of any other strains of L. biflexa. In the organization of 5S rRNA genes, strain 965 is sufficiently different from other members of the genus Leptospira to be regarded as a member of the genus Leptonema.     

The significance of mating processes for the genetics and for the formation of resting eggs in monogonont rotifers

Crossbreeding experiments with three geographically distinct strains (E, S, and L) of the rotifer Brachionus plicatilis have been carried out in an attempt to elucidate the apparent male sterility of strain L, which is unable to produce resting eggs.The 9 crossing possibilities of the three strains have been investigated in 27 experiments. The results refute the concept of male sterility. L-males copulate successfully with mictic as well as with amictic females of strains E and S. Fertilized amictic E and S females produce defective resting eggs, which have only one thin shell and, which disintegrate after deposition. L-females cannot, as a rule, be fertilized. Moreover, crosses between strain E and S are succesful only in one direction; the reciprocal crosses failed.To explain the present results a hypothesis is suggested that the thickness of the body wall of newborn females differs in the three strains, and between mictic and amictic individuals. A sequence of gradually increasing body wall thickness of all types of females involved, together with a comparably increasing penetration ability of the males of strain E, S, and L explains the success or failure of all crosses, including the unilateral cross E × S.     

Cloning of a gene involved in rRNA precursor processing and 23S rRNA cleavage in Rhodobacter capsulatus.

In Rhodobacter capsulatus wild-type strains, the 23S rRNA is cleaved into [16S] and [14S] rRNA molecules. Our data show that a region predicted to form a hairpin-loop structure is removed from the 23S rRNA during this processing step. We have analyzed the processing of rRNA in the wild type and in the mutant strain Fm65, which does not cleave the 23S rRNA. In addition to the lack of 23S rRNA processing, strain Fm65 shows impeded processing of a larger 5.6-kb rRNA precursor and slow maturation of 23S and 16S rRNAs from pre-23S and pre-16S rRNA species. Similar effects have also been described previously for Escherichia coli RNase III mutants. Processing of the 5.6-kb precursor was independent of protein synthesis, while the cleavage of 23S rRNA to generate 16S and 14S rRNA required protein synthesis. We identified a DNA fragment of the wild-type R. capsulatus chromosome that conferred normal processing of 5.6-kb rRNA and 23S rRNA when it was expressed in strain Fm65.