一株ganghwense发光杆菌的分类鉴定及相关特性的研究

对从海鲜食品(虾蛄)中分离到的1株细菌"M-1"进行系统分类鉴定.采用常规方法[1]进行分离培养,以形态学特征、培养特性、生理生化特征及分子生物学等方法对其进行分类鉴定.该菌株为革兰阴性杆状细菌,细菌宽度＞1 μm;16S rDNA序列测定与photobacterium ganghwense相似为99%(1 455/1 467),生理生化特征与发光杆菌属相近.菌株"M-1"是1株发光杆菌.     

一株多功能芽孢杆菌的鉴定

【摘 要】 目的 从土壤筛选所得1株芽孢杆菌并对该菌进行鉴定.方法 通过表型特征、生理生化特性和16S rDNA序列同源性分析（Gen Bank登录号为: JN609382）。结果 形态学观察：菌落呈圆形,白色微黄,中央凸起,半透明,长时间培养容易形成褶皱;革兰染色为阳性;芽孢中生,周生鞭毛,细长且多。生理生化特征：参照《常见细菌手册》和《伯杰氏细菌鉴定手册》,其中接触酶反应、V-P测定、卵磷脂以及革兰染色呈阳性;能够利用D-葡萄糖、D-木糖、D-甘露醇产酸,并能使淀粉水解;不能利用丙酸盐,不能形成吲哚,不能水解酪氨酸,生理生化试验结果显示该芽孢杆菌与枯草芽孢杆菌的特征一致。分子生物学鉴定：在Gen Bank上经过BLAT分析,其与枯草芽孢杆菌的相似性最高（99％）。结论 鉴定该菌为枯草芽孢杆菌。     

一株产生糖脂菌种的初步鉴定及发酵条件的考察

对从炼油厂附近的油污土样中分离纯化出的 1株杆菌进行了鉴定。该细菌为革兰氏阳性 ,有芽孢 ,可运动、不抗酸 ,能发酵豆油、色拉油形成稳定的乳化液。其细胞形态、培养特征和生理生化特性 ,基本上与凝结芽孢杆菌一致。研究了该菌株生产糖脂的摇瓶发酵工艺条件 ,进行了 10L罐发酵实验 ,糖脂产量达到 7.0 73g/L。     

新疆棉花黄萎病菌拮抗细菌的分离、筛选与鉴定

【背景】棉花黄萎病是由大丽轮枝菌(Verticillium dahliae Kleb.)引起的一种世界性病害,近年来对该病害的生物防治因具有环境友好和人畜安全的特性而倍受关注。【目的】筛选棉花黄萎病高效拮抗细菌并对其进行鉴定,为棉花黄萎病的生物防治扩充菌种资源。【方法】采用稀释涂布平板法分离细菌,并进行拮抗细菌的初筛和复筛,通过形态特征、生理生化特征和16S rRNA基因序列分析对筛选到的细菌进行鉴定,确定其分类地位。【结果】初筛分离到535株对病原菌具有拮抗作用的细菌,并选取了108株拮抗细菌进行复筛,最终筛选到了4株优势拮抗细菌。通过形态观察、生理生化特征和16SrRNA基因序列分析,将菌株BHZ-29、SHT-15、SHZ-24和SMT-24分别鉴定为贝莱斯芽孢杆菌(Bacillusvelezensis)、枯草芽孢杆菌斯皮兹仁亚种(Bacillus subtilis subsp. spizizenii)、萎缩芽孢杆菌(Bacillus atrophaeus)和香草芽孢杆菌(Bacillus vanillea)。【结论】获得了4株高效拮抗细菌,并且首次报道了香草芽孢杆菌对棉花黄萎病菌具有抑制作用。     

一株引起马来甜龙竹组培污染内生菌的分离与鉴定

【目的】对一株引起马来甜龙竹组培污染内生菌的分离与鉴定。【方法】采用改良的NA培养基分离纯化菌株,并通过菌体的形态结构观察、生理生化试验及其16SrDNA序列同源性分析对其进行鉴定。【结果】菌株SWFU01的形态特征及生理生化试验结果与解淀粉芽孢杆菌[Bacillus amyloliquefaciens(Fukumoto)Priest et al.]的描述基本相同;16S rDNA序列分析表明,该菌株与解淀粉芽孢杆菌JS在同一系统发育分支,其同源性为99.28%。【结论】综合形态学特征、生理生化特征以及16S rDNA序列分析的研究结果,菌株SWFU01被鉴定为解淀粉芽孢杆菌。     

细菌群体感应抑制活性微生物Zou 03的筛选与鉴定

从近海区生态环境中分离纯化98株海洋菌株,以根癌农杆菌WCF47为敏感检测菌株,筛选出1株具细菌群体感应抑制活性的菌株Zou03,对其进行形态、生理生化特征鉴定和16S rDNA分子鉴定。结果显示,Zou03具枯草芽胞杆菌(Bacillus subtilis)的典型特征,其16S rDNA序列通过对比分析,与GenBank中枯草芽胞杆菌16SrDNA的部分序列同源性为100%。综合形态、生化特征及16S rDNA序列对比分分析,鉴定菌株Zou03为枯草芽胞杆菌。表明近海区生态环境中存在具有抑制细菌群体感应活性的微生物,有利于海洋微生物资源开发,为以致病菌群体感应系统为靶点的新型疗法提供新技术。     

一株来自蚯蚓的产纤溶酶蜡状芽孢杆菌Bacillus cereus的筛选及鉴定

对来自4个不同省份的5条蚯蚓的肠道及体表细菌进行分离,共获得122株细菌。通过脱脂奶粉平板法初筛,纤维蛋白平板法复筛,以透明圈为筛选标记,共筛选出产纤溶酶菌株12株,其中菌株SC-3-W-3的纤溶酶活力较高,达到了538.64 U/mL(相当于尿激酶的活力单位)。通过对其形态、培养、生理生化特征进行研究,发现其与蜡状芽孢杆菌Bacillus cereus Frankland的特征很相符。进一步对SC-3-W-3的16S rDNA序列及系统发育分析表明,该菌株与蜡状芽孢杆菌的同源性高达100%。综合生理生化及16S rDNA序列比对结果,将SC-3-W-3菌株鉴定为蜡状芽孢杆菌。     

细菌纤维素生产菌株的分离和菌种初步鉴定

从长膜的醋醅中分离出一株发酵生产细菌纤维素产量较高且稳定的醋酸菌M12。根据《常见细菌系统鉴定手册》和《伯杰氏细菌鉴定手册》第九版,对醋酸菌M12进行了形态和生理生化特征的分析、测定了G＋Cmol％含量,初步鉴定该菌为醋化醋杆菌木质亚种（Acetobacter xylinum subsp.xylinum,又称木醋杆菌）。     

贵州半夏块茎腐烂病病原菌的分离与鉴定

【目的】对贵州省半夏块茎腐烂病病原菌进行分离和鉴定,为开发有效的防治技术体系提供依据。【方法】采用组织分离法进行病原菌分离,根据柯氏法则进行致病性测定,结合形态学、生理生化特性和分子生物学方法进行鉴定。【结果】从贵州3个半夏产地患病样品中分离出12株病原细菌和5株病原真菌。经形态学、生理生化特性和分子生物学方法鉴定,12株病原细菌为胡萝卜软腐果胶杆菌胡萝卜软腐亚种;ZJ、Z3病原真菌为尖孢镰刀菌,D3、D5、H1为茄病镰刀菌。【结论】贵州省半夏块茎腐烂病的病原菌有病原细菌和病原真菌。     

一株吡虫啉杀虫剂降解菌BB-1的分离鉴定

目的:从福建安溪茶园土壤分离能降解吡虫啉杀虫剂的细菌菌株,对其进行分类鉴定.方法:采用室内培养试验方法,通过富集驯化、平板划线分离得到1株优势细菌BB-1,采用形态、生理生化特征和16S rDNA序列分析对菌株BB-1进行鉴定.结果:菌株BB-1 与多株苍白杆菌(Ochrobactrum)的亲缘关系最近,该菌株鉴定为苍白杆菌属(Ochrobactrum sp.).BB-1在吡虫啉浓度为400mg/L的LB培养基中培养0h～5h为生长延迟期,4h～20h为对数生长期,20h～42h为稳定期,42h以后为衰亡期.菌株BB-1对多种抗生素敏感,可耐受的NaCl浓度超过10%.结论:BB-1能降解吡虫啉杀虫剂,分类上应属于Ochrobactrum sp..     

Cloning and expression of genes of the luminescence system in Photobacterium leiognathi

The genes of Photobacterium leiognathi luminescence system were cloned in plasmid pUC18. Escherichia coli cells harboring a recombinant plasmid pPHL1 are luminescent. pPHL1 contains luciferase genes and genes responsible for aldehyde biosynthesis. The luminescence of Escherichia coli is subject to autoinductor regulation similar to the one existing in luminescent bacteria. The 2.7 kb fragment of Photobacterium leiognathi DNA containing the genes for alpha- and beta-luciferase subunits were cloned in pUC19.     

The lux genes of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish. Nucleotide sequence, difference in gene organization, and high expression in mutant Escherichia coli

The lux genes required for light expression in the luminescent bacterium Photobacterium leiognathi (ATCC 25521) have been cloned and expressed in Escherichia coli and their organization and nucleotide sequence determined. Transformation of a recombinant 9.5-kbp chromosomal DNA fragment of P. leiognathi into an E. coli mutant (43R) gave luminescent colonies that were as bright as those of the parental strain. Moreover, expression of the lux genes in the mutant E. coli was strong enough so that not only were high levels of luciferase detected in crude extracts, but the fatty-acid reductase activity responsible for synthesis of the aldehyde substrate for the luminescent reaction could readily be measured. Determination of the 7.3-kbp nucleotide sequence of P. leiognathi DNA, including the genes for luciferase (luxAB) and fatty-acid reductase (luxCDE) as well as a new lux gene (luxG) found recently in luminescent Vibrio species, showed that the order of the lux genes was luxCDABEG. Moreover, luxF, a gene homologous to luxB and located between luxB and luxE in Photobacterium but not Vibrio strains, was absent. In spite of this different lux gene organization, an intergenic stem-loop structure between luxB and luxE was discovered to be highly conserved in other Photobacterium species after luxF.     

Conditions that influence bacterial luminescence in the presence of blood serum

Conditions that influence the luminescence of natural and recombinant luminescent bacteria in the presence of blood serum were studied. In general, blood serum quenched the luminescence of the marine Photobacterium phosphoreum and the recombinant Escherichia coli strains harboring the luminescent system genes of Photobacterium leiognathi, but enhanced the luminescence of the soil bacterium Photorhabdus luminescens Zm1 and the recombinant E. coli strain harboring the lux operon of P. luminescens Zm1. The quenching effect of blood serum increased with its concentration and the time and temperature of incubation. The components of blood serum that determine the degree and specificity of its action on bacterial luminescence were identified.     

Heterogeneity of the populations of marine luminescent bacteria Photobacterium leiognathi under different conditions of cultivation

Manifestation of pleiotropic effects in the isogenic variants of luminescent bacteria Photobacterium leiognathi 54 was investigated. The decrease or increase of the expression level of bioluminescence was caused by changes in lux operon regulation. The dynamics of the bioluminescence of dark and dim variants did not differ from the dynamics of the initial luminescent variant, but dependence of the level of luminescence intensity on the exogenous autoinductor of the lux operon was revealed. The investigated variants of P. leiognathi 54 inherited fairly stable morphological characteristics, colony architectonics, level of luminescence, and activity of some enzymes; variants with reduced bioluminescence formed colonies of the S type. Stable bright variants with S- and R-type colonies appeared both in the initial strain population and in the dark variant population, but with smaller frequency. Populations of the bright variant with R-type colonies were most heterogeneous; this can be determined by the lack of glucose repression of the bioluminescence in contrast to other investigated variants of P. leiognathi.     

Phylogenetic diversity and cosymbiosis in the bioluminescent symbioses of "Photobacterium mandapamensis"

"Photobacterium mandapamensis" (proposed name) and Photobacterium leiognathi are closely related, phenotypically similar marine bacteria that form bioluminescent symbioses with marine animals. Despite their similarity, however, these bacteria can be distinguished phylogenetically by sequence divergence of their luminescence genes, luxCDAB(F)E, by the presence (P. mandapamensis) or the absence (P. leiognathi) of luxF and, as shown here, by the sequence divergence of genes involved in the synthesis of riboflavin, ribBHA. To gain insight into the possibility that P. mandapamensis and P. leiognathi are ecologically distinct, we used these phylogenetic criteria to determine the incidence of P. mandapamensis as a bioluminescent symbiont of marine animals. Five fish species, Acropoma japonicum (Perciformes, Acropomatidae), Photopectoralis panayensis and Photopectoralis bindus (Perciformes, Leiognathidae), Siphamia versicolor (Perciformes, Apogonidae), and Gadella jordani (Gadiformes, Moridae), were found to harbor P. mandapamensis in their light organs. Specimens of A. japonicus, P. panayensis, and P. bindus harbored P. mandapamensis and P. leiognathi together as cosymbionts of the same light organ. Regardless of cosymbiosis, P. mandapamensis was the predominant symbiont of A. japonicum, and it was the apparently exclusive symbiont of S. versicolor and G. jordani. In contrast, P. leiognathi was found to be the predominant symbiont of P. panayensis and P. bindus, and it appears to be the exclusive symbiont of other leiognathid fishes and a loliginid squid. A phylogenetic test for cospeciation revealed no evidence of codivergence between P. mandapamensis and its host fishes, indicating that coevolution apparently is not the basis for this bacterium's host preferences. These results, which are the first report of bacterial cosymbiosis in fish light organs and the first demonstration that P. leiognathi is not the exclusive light organ symbiont of leiognathid fishes, demonstrate that the host species ranges of P. mandapamensis and P. leiognathi are substantially distinct. The host range difference underscores possible differences in the environmental distributions and physiologies of these two bacterial species.     

Genomic polymorphism in symbiotic populations of Photobacterium leiognathi

Photobacterium leiognathi forms a bioluminescent symbiosis with leiognathid fishes, colonizing the internal light organ of the fish and providing its host with light used in bioluminescence displays. Strains symbiotic with different species of the fish exhibit substantial phenotypic differences in symbiosis and in culture, including differences in 2-D PAGE protein patterns and profiles of indigenous plasmids. To determine if such differences might reflect a genetically based symbiont-strain/host-species specificity, we profiled the genomes of P. leiognathi strains from leiognathid fishes using PFGE. Individual strains from 10 species of leiognathid fishes exhibited substantial genomic polymorphism, with no obvious similarity among strains; these strains were nonetheless identified as P. leiognathi by 16S rDNA sequence analysis. Profiling of multiple strains from individual host specimens revealed an oligoclonal structure to the symbiont populations; typically one or two genomotypes dominated each population. However, analysis of multiple strains from multiple specimens of the same host species, to determine if the same strain types consistently colonize a host species, demonstrated substantial heterogeneity, with the same genomotype only rarely observed among the symbiont populations of different specimens of the same host species. Colonization of the leiognathid light organ to initiate the symbiosis therefore is likely to be oliogoclonal, and specificity of the P. leiognathi/leiognathid fish symbiosis apparently is maintained at the bacterial species level rather than at the level of individual, genomotypically defined strain types.     

Phylogenetic analysis of the incidence of lux gene horizontal transfer in Vibrionaceae

Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT in Vibrionaceae and its possible impact on speciation, we assessed the incidence of interspecies transfer of the lux genes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11 Aliivibrio, Photobacterium, and Vibrio species, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrB or pyrH) and a representative lux gene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB, recA, and pyrH) and multiple lux genes (luxCDABEG). In nearly all cases, housekeeping gene and lux gene phylogenies were congruent, and there was no instance in which the lux genes of one luminous species had replaced the lux genes of another luminous species. Therefore, the lux genes are predominantly vertically inherited in Vibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) the lux genes of the only known luminous strain of Vibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to the lux genes of Vibrio harveyi; (ii) the lux genes of two luminous strains of Vibrio chagasii, 21N-12 and SB-52, were closely related to those of V. harveyi and Vibrio splendidus, respectively; (iii) the lux genes of a luminous strain of Photobacterium damselae, BT-6, were closely related to the lux genes of the lux-rib(2) operon of Photobacterium leiognathi; and (iv) a strain of the luminous bacterium Photobacterium mandapamensis was found to be merodiploid for the lux genes, and the second set of lux genes was closely related to the lux genes of the lux-rib(2) operon of P. leiognathi. In none of these cases of apparent HGT, however, did acquisition of the lux genes correlate with phylogenetic divergence of the recipient strain from other members of its species. The results indicate that horizontal transfer of the lux genes in nature is rare and that horizontal acquisition of the lux genes apparently has not contributed to speciation in recipient taxa.     

Phylogeny, genomics, and symbiosis of Photobacterium

Photobacterium comprises several species in Vibrionaceae, a large family of Gram-negative, facultatively aerobic, bacteria that commonly associate with marine animals. Members of the genus are widely distributed in the marine environment and occur in seawater, surfaces, and intestines of marine animals, marine sediments and saline lake water, and light organs of fish. Seven Photobacterium species are luminous via the activity of the lux genes, luxCDABEG. Much recent progress has been made on the phylogeny, genomics, and symbiosis of Photobacterium. Phylogenetic analysis demonstrates a robust separation between Photobacterium and its close relatives, Aliivibrio and Vibrio, and reveals the presence of two well-supported clades. Clade 1 contains luminous and symbiotic species and one species with no luminous members, and Clade 2 contains mostly nonluminous species. The genomes of Photobacterium are similar in size, structure, and organization to other members of Vibrionaceae, with two chromosomes of unequal size and multiple rrn operons. Many species of marine fish form bioluminescent symbioses with three Photobacterium species: Photobacterium kishitanii, Photobacterium leiognathi, and Photobacterium mandapamensis. These associations are highly, but not strictly species specific, and they do not exhibit symbiont-host codivergence. Environmental congruence instead of host selection might explain the patterns of symbiont-host affiliation observed from nature.     

Growth and luminescence of luminous bacteria promoted by agents of microbial origin

The examination of four species of luminous bacteria Photobacterium leiognathi, Photobacterium phosphoreum, Vibrio fischeri and Vibrio harveyi has enabled us to reveal some nutrient medium components effecting growth, luminescence intensity and luciferase synthesis. These agents are nucleic components (nucleotides, nucleosides and amine bases), amino acids and vitamins, which are part of hydrolysates from the biomass of various lithotrophic microorganisms, hydrogen-oxidizing, ironoxidizing and carboxydobacteria. The effect of promoting agents essentially alters the physiological state and ultrastructure of the cells of luminous bacteria and increases luciferase biosynthesis two- to three-fold compared to a control.