溶藻弧菌溶血活性检测及溶血素基因、启动子区功能

[目的]研究溶藻弧菌的溶血现象,溶血素基因vah的分布及vah基因、vah启动子区对溶藻弧菌溶血活性的贡献.[方法]对46株分离自华南沿海水生动物体内和海水的溶藻弧菌环境株及溶藻弧菌标准株1.1587进行溶血实验;比较具有溶血活性的溶藻弧菌野生株ZJ051、vah基因大肠杆菌BL21重组表达株、vah缺失突变株和基因回补株间溶血能力的差异;检测vah基因在溶藻弧菌中的分布,比较溶血株与非溶血株vah基因及上游启动子区的序列差异.[结果]47.8％的溶藻弧菌菌株产生溶血活性,因此溶血现象普遍存在于溶藻弧菌环境株中;vah基因的表达产物具有溶血活性,vah基因缺失突变株不具有溶血活性,而vah基因回补株恢复溶血活性.vah基因普遍存在于溶藻弧菌中,且基因序列非常相似,氨基酸序列完全相同,然而不同菌株的启动子区第188-190碱基位点存在差异.[结论]溶藻弧菌vah基因是造成溶藻弧菌溶血的直接原因,但溶藻弧菌溶血能力的差异并非是由vah基因本身差异决定,极有可能与启动子区第188-190碱基位点相关.     

高产铁载体假单胞菌的筛选及其对铁氧化物的利用

筛选高产铁载体的微生物,研究铁载体的抑菌作用和对不溶性未定型铁氧化物(poorly crystalline iron hydroxides,PCIH)的利用。CAS法筛选高产铁载体菌株,采用琼脂扩散法和生长抑制测定铁载体的抑菌作用,利用16S r RNA基因序列比对鉴定分离菌株,并根据分离菌株的生长情况,确定铁载体对不溶性PCIH的利用。从土壤样品中共筛选到172株产铁载体的菌株,高产铁载体菌株13株,其中仅有菌株Z158的发酵液对金黄色葡萄球菌、藤黄微球菌、普通变形杆菌和副溶血性弧菌具有抑菌作用,抑菌率分别为51.3%、50.2%、37.1%和28.0%。比对该菌株的16S r RNA基因序列,确定Z158属于Pseudomonas aeruginosa。当不溶性的PCIH作为唯一可利用的铁源时,菌株Z158培养24 h的生物量比无铁条件下提高了46.1%。铜绿假单胞菌Z158分泌的铁载体能够抑制病原菌的生长,同时还能获取不溶性未定型铁氧化物PCIH中的铁元素。     

乌梅提取物对溶藻弧菌的抑制效应与机理

[背景] 溶藻弧菌（Vibrio alginolyticus）能够感染鱼、虾、贝等海洋经济动物,给海水养殖业带来了严重的经济损失,对公众健康及食品安全也构成较大威胁。[目的] 通过研究乌梅（Fructus mume）对溶藻弧菌的抑菌效应及其机理,为乌梅在水产养殖中的进一步开发利用提供理论依据。[方法] 采用试管二倍稀释法测定乌梅提取物（Fructus mume Extract,FME）对溶藻弧菌的最小抑菌浓度（Minimal Inhibitory Concentration,MIC）和最小杀菌浓度（Minimum Bactericidal Concentration,MBC）,电导率仪检测溶藻弧菌的相对电导率,分光光度法分析溶藻弧菌的核酸泄露和呼吸链脱氢酶活性及生物膜抑制率,蛋白质电泳检测溶藻弧菌的蛋白质合成情况,扫描电子显微镜观察溶藻弧菌的亚显微结构。[结果] 乌梅提取物对溶藻弧菌的MIC和MBC分别为1.953 mg/mL和3.906 mg/mL;乌梅提取物显著增加了溶藻弧菌的相对电导率和核酸泄露,明显降低了溶藻弧菌蛋白质的合成能力,对于弧菌的亚显微形态产生了较大影响。同时乌梅提取物显著抑制了溶藻弧菌生物膜的形成和呼吸链脱氢酶的活性。[结论] 乌梅提取物通过增加溶藻弧菌细胞膜通透性及显著抑制生物膜的生成、呼吸链脱氢酶活性及蛋白质的合成,实现抑制及杀灭溶藻弧菌的目的。     

Pseudomonas pseudoalcaligenes B50铁载体合成相关基因pyrD的克隆与功能分析

从棉花根际分离的一株细菌B50在低铁条件下可以产生铁载体。通过形态学特征、生理生化特征及其16SrDNA序列分析,将其鉴定为Pseudomonas pseudoalcaligenes。采用三亲本杂交的方法将转座子Tn5-1063a(含luxAB)导入B50中,进行转座子插入诱变,获得突变株。用TAIL-PCR方法得到与铁吸收有关的pyrD基因序列。通过互补实验验证pyrD与铁载体的合成有关。Pseudomonas pseudoalcaligenes能够产生铁载体属于首次报道。     

海洋微生物铁载体的研究进展

铁是影响初级生产力的主要限制性因子之一,其在海洋环境中的分布具有空间异质性。由于铁在海洋中主要以溶解度较低、易沉降的三价态（Fe³⁺）形式存在,因此溶解铁对海洋生物而言是一种稀缺资源。为了获得生命代谢所需的铁,微生物进化出多种铁摄取的策略来满足需求,其中铁载体（siderophores）是最典型的代表。铁载体作为重要的代谢辅因子,除了铁循环以外,也强烈影响着其他元素的循环。基于铁载体的重要性,深入理解它的合成、转运和调控机制是系统认识海洋铁循环和生命过程的重要环节之一。本文以近20年的研究为重点,总结了铁载体的最新进展,包括其类型、合成/运输系统、获取途径、调控机制以及铁载体的功能与应用,旨在更好地认识铁载体在海洋微生物生态学过程中的作用,加深对海洋铁循环动力学机制的理解。     

假单胞菌株JKD—2分泌铁载体抑制稻瘟病菌

利用铬奥醇（CAS）分析法测定了假单胞菌（Pseudomonassp．）JKD－2分泌铁载体的特征。在无铁环境下JKD-2菌能分泌高亲和力的铁载体;在低铁条件下,铁载体的分泌量减少;在富铁环境下,则不能分泌。结果还显示菌株JKD-2在无铁条件下分泌的铁载体,能在低铁条件下抑制稻瘟病菌（Piriculariaoryzae）的生长。     

假单胞菌株JKD—2分泌铁载体抑制稻瘟病菌*

利用铬奥醇（CAS）分析法测定了假单胞菌（Pseudomonassp．）JKD－2分泌铁载体的特征。在无铁环境下JKD-2菌能分泌高亲和力的铁载体;在低铁条件下,铁载体的分泌量减少;在富铁环境下,则不能分泌。结果还显示菌株JKD-2在无铁条件下分泌的铁载体,能在低铁条件下抑制稻瘟病菌（Piriculariaoryzae）的生长。     

江苏省水产品中溶藻弧菌分布情况

随机采集江苏省各地区的超市和农贸市场鲜活水产样品252份,参考国家标准中副溶血性弧菌的检验方法,采用形态特征观察、生化特性测定并结合特异性基因测定的方法对溶藻弧菌进行分离鉴定。结果显示,252份样品中共有198份分离鉴定出溶藻弧菌,其分离率为79%。其中,淡水产品的检出率为76%,海水产品的检出率为81%。淡水鱼中的溶藻弧菌检出率低于淡水虾贝类产品和海水产品;沿江地区的又高于沿海地区和内陆地区,农贸市场的高于超市。结果表明,海水产品与淡水产品中溶藻弧菌的携带情况非常普遍。     

铁对产铁载体的沼泽红假单胞菌光合色素与铁载体合成的影响

【目的】探求铁对1株能产生铁载体的不产氧光合细菌(Anoxygenic Phototrophic Bacteria,APB)光合色素和铁载体合成的影响。【方法】通用CAS法检测铁载体产生,Arnow、Csaky和Shenker法检测铁载体类型;吸收光谱法和HPLC法分析光合色素的组分和含量。【结果】Rhodopseudomonas palustris CQV97能够产生异羟肟酸型铁载体,未添加FeCl3时,铁载体含量最高,铁载体的产生与生长并非关联型。随FeCl3浓度升高,菌体生长潜伏期缩短,生长速率、最终生物量以及细菌叶绿素(Bacteriochlorophyll,BChl)a和类胡萝卜素(Carotenoid,Car)含量均提高,而检测到的游离铁载体含量降低;菌体积累的BChl a的组成和相对含量未见明显变化,但主要的Car组分由Spirilloxanthin转化为Rhodopin,菌体中积累Car组分的平均共轭体系降低,Car组成的改变与色素提取液的Car特征性光谱蓝移现象相吻合。【结论】首次报道APB能够产生铁载体,CQV97菌株能够产生异羟肟酸型铁载体。阐明了铁对CQV97生长、铁载体产生和光合色素合成的影响规律,这些研究结果为深入开展APB铁载体分离纯化以及生物功能研究奠定了基础。     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾（Litopenaeus vannamei）幼体中分离到两株病原菌zouA和zouB,常规形态和生理生化试验表明均为弧菌属菌种,弧菌编码鉴定系统分别鉴定为溶藻弧菌（Vibrioatginolyticus）和副溶血弧菌（V.parahaemolyticus）。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98％,相互间不能区分;HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98％以上,而与所有其它弧菌的相似性不到92％。结合表型和分子特征的鉴定结果,菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

Effect of exogenous siderophores on iron uptake activity of marine bacteria under iron-limited conditions

More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM. The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore, N,N'-bis (2,3-dihydroxybenzoyl)-O-serylserine from a Vibrio sp. Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria. Radioisotope studies using (59)Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores. Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols. An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores. This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores. Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.     

Iron-binding proteins and heme compounds as iron sources forVibrio anguillarum

Utilization of several iron sources available from the host was investigated in different strains ofVibrio anguillarum. We tested the ability to use transferrins, heme, hemoglobin, and haptoglobin-hemoglobin as iron sources in strains ofV. anguillarum possessing different iron uptake systems mediated by siderophores. Only the wild-type pathogenic strains with an intact siderophore-mediated iron transport system were able to obtain iron from transferrins. None of the low-virulence derivatives lacking siderophore production could grow in the presence of transferrins. However, all strains, wild-type and iron-deficient derivatives, could utilize heme, hemoglobin, and haptoglobin-hemoglobin as iron sources when added to iron-deficient media. The ability to grow in fish serum was also evaluated. Although only wild-type strains could grow in fresh serum, derivative strains lacking siderophore production also were able to grow when serum was heat inactivated or when a utilizable siderophore was present in serum. The results indicate that besides the siderophore-mediated mechanism,V. anguillarum can also obtain iron from other sources presumably available from the host, although its importance for growth in vivo is so far unknown.     

Effect of Exogenous Siderophores on Iron Uptake Activity of Marine Bacteria under Iron-Limited Conditions

More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM. The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore, N,N′-bis (2,3-dihydroxybenzoyl)-O-serylserine from a Vibrio sp. Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria. Radioisotope studies using ⁵⁹Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores. Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols. An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores. This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores. Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.     

Synthesis of siderophores by pathogenicVibrio species

PathogenicVibrio species were assayed for the production and utilization of siderophores. Although some similarities were observed in the types of siderophores secreted, chemical and biological assays indicated that several different iron transport systems are expressed by members of this genus. Bioassays indicated that each species tested produced compounds that stimulated their growth in low-iron media. Phenolate compounds were detected by chemical assay of culture supernatants of iron-starvedV. cholerae, V. fluvialis, V. vulnificus, andV. anguillarum, but notV. parahaemolyticus orV. alginolyticus. Vibrio vulnificus synthesizes an additional, relatively species-specific compound. This unique siderophore has greater biological activity than theV. vulnificus phenolate.     

Bacterial growth stimulation with exogenous siderophore and synthetic N-acyl homoserine lactone autoinducers under iron-limited and low-nutrient conditions

The growth of marine bacteria under iron-limited conditions was investigated. Neither siderophore production nor bacterial growth was detected for Pelagiobacter sp. strain V0110 when Fe(III) was present in the culture medium at a concentration of <1.0 microM. However, the growth of V0110 was strongly stimulated by the presence of trace amounts of exogenous siderophore from an alpha proteobacterium, V0902, and 1 nM N-acyl-octanoylhomoserine lactone (C(8)-HSL), which is known as a quorum-sensing chemical signal. Even though the iron-binding functionality of a hydroxamate siderophore was undetected in the supernatant of V0902, a hydroxamate siderophore was detected in the supernatant of V0110 under the above conditions. These results indicated that hydroxamate siderophore biosynthesis by V0110 began in response to the exogenous siderophore from V0902 when in the presence of C(8)-HSL; however, C(8)-HSL production by V0110 and V0902 was not detected. Direct interaction between V0902 and V0110 through siderophore from V0902 was observed in the dialyzing culture. Similar stimulated growth by exogenous siderophore and HSL was also observed in other non-siderophore-producing bacteria isolated from marine sponges and seawater. The requirement of an exogenous siderophore and an HSL for heterologous siderophore production indicated the possibility that cell-cell communication between different species was occurring.     

铁限制条件下东海原甲藻分泌铁载体

在铁限制条件下,进行东海原甲藻分泌铁载体的动态研究。对藻类在富铁与缺铁条件下生长状况、生长过程中分泌铁载体的情况以及海藻接种量对铁载体分泌的影响进行了连续观测,结果表明:东海原甲藻在缺铁条件下生长状况远不如在富铁条件下;随着藻类的生长,分泌铁载体不断增多,达指数生长期时,其分泌量也达到了最大值,之后藻类的生长和铁载体分泌都呈现下降趋势;高接种量东海原甲藻能分泌较多的铁载体,并在较短时间到达峰值。     

Influence of iron on growth and extracellular products of Acinetobacter baumannii

Iron is an important nutrient required by bacteria for optimal growth. Acquisition of iron from the host where iron is restricted is an important mediator of bacterial pathogenesis. In iron deplete chemically defined medium (CDM-Fe) growth of Acinetobacter baumannii was restricted as compared to iron replete medium (CDM + Fe). Bacteria developed four high molecular weight outer membrane proteins (OMPs) of 88, 84, 80 and 77 kDa in CDM-Fe medium which were absent in CDM + Fe medium, and are known iron regulated outer membrane proteins (IROMPs). A. baumannii secreted siderophores extracellularly into the medium which act as iron chelators which had been demonstrated in the supernatants of CDM-Fe media. The siderophore was of catechol type. This shows that A. baumannii under iron restricted conditions express IROMPs along with production of catechol type siderophore in order to acquire iron from the external milieu.     

Bacterial Growth Stimulation with Exogenous Siderophore and Synthetic N-Acyl Homoserine Lactone Autoinducers under Iron-Limited and Low-Nutrient Conditions

The growth of marine bacteria under iron-limited conditions was investigated. Neither siderophore production nor bacterial growth was detected for Pelagiobacter sp. strain V0110 when Fe(III) was present in the culture medium at a concentration of <1.0 μM. However, the growth of V0110 was strongly stimulated by the presence of trace amounts of exogenous siderophore from an alpha proteobacterium, V0902, and 1 nM N-acyl-octanoylhomoserine lactone (C₈-HSL), which is known as a quorum-sensing chemical signal. Even though the iron-binding functionality of a hydroxamate siderophore was undetected in the supernatant of V0902, a hydroxamate siderophore was detected in the supernatant of V0110 under the above conditions. These results indicated that hydroxamate siderophore biosynthesis by V0110 began in response to the exogenous siderophore from V0902 when in the presence of C₈-HSL; however, C₈-HSL production by V0110 and V0902 was not detected. Direct interaction between V0902 and V0110 through siderophore from V0902 was observed in the dialyzing culture. Similar stimulated growth by exogenous siderophore and HSL was also observed in other non-siderophore-producing bacteria isolated from marine sponges and seawater. The requirement of an exogenous siderophore and an HSL for heterologous siderophore production indicated the possibility that cell-cell communication between different species was occurring.     

Siderophore protection against colicins M, B, V, and Ia in Escherichia coli.

A variety of natural and synthetic siderophores capable of supporting the growth of Escherichia coli K-12 on iron-limited media also protect strain RW193+ (tonA+ ent-) from the killing action of colicins B, V, and Ia. Protective activity falls into two categories. The first, characteristic of enterobactin protection against colicin B and ferrichrome protection against colicin M, has properties of a specific receptor competition between the siderophore and the colicin. Thus, enterobactin specifically protects against colicin B in fes- mutants (able to accumulate but unable to utilize enterobactin) as predicted by our proposal that the colicin B receptor functions in the specific binding for uptake of enterobactin (Wayne and Neilands, 1975). Similarly ferrichrome specifically protects against colicin M in SidA mutants (defective in hydroxamate siderophore utilization). The second category of protective response, characteristic of the more general siderophore inhibition of colicins B, V, and Ia, requires the availability or metabolism of siderophore iron. Thus, enterobactin protects against colicins V and Ia, but only when the colicin indicator strain is fes+, and hydroxamate siderophores inhibit colicins B, V, and Ia, but only when the colicin indicator strain is SidA+. Moreover, ferrichrome inhibits colicins B, V, and Ia, yet chromium (III) deferriferrichrome is inactive, and ferrichrome itself does not prevent adsorption of colicin Ia receptor material in vitro. Although the nonspecific protection against colicins B, V, and Ia requires iron, the availability of siderophore iron for cell growth is not sufficient to bring about protection. None of the siderophores tested protect cells against the killing action of colicin E1 or K, or against the energy poisons azide, 2, 4-dinitrophenol, and carbonylcyanide m-chlorophenylhydrazone. We suggest that nonspecific siderophore protection against colicins B, V, and Ia may be due either to an induction of membrane alterations in response to siderophore iron metabolism or to a direct interference by siderophore iron with some unknown step in colicin action subsequent to adsorption.     

Regulation of internal solute concentrations of marine Vibrio alginolyticus in response to external NaCl concentration.

Slightly halophilic marine Vibrio alginolyticus grown in the range of NaCl from 0.2 to 1.5 M maintained the total internal solute concentration always higher than the external medium by about 0.25 osM. The concentrations of macromolecules such as DNA, RNA, and protein were little affected by the increase in medium NaCl. The internal K+ concentration was kept to about 400 mM in the range of medium NaCl from 0.4 to 0.8 M; it rose to 510 mM when the bacterium was grown in 1.5 M NaCl, indicating that K+ increased only slightly in response to the large increase in medium NaCl. Thus, in contrast to the case of nonhalophilic and extremely halophilic bacteria, K+ was unlikely to act as a major component to regulate the internal solute concentration of marine V. alginolyticus. The internal Na+ and Cl- concentrations were maintained always lower than those in the growth medium, but they increased in response to the increase in medium NaCl. The concentration of internal Na+ was close to that of K+ at the concentration of medium NaCl that supports the optimal growth of this organism. The total amino acid content of V. alginolyticus increased from 76 to 413 mM by the increase in medium NaCl from 0.2 to 1.5 M. The concentrations of glutamic acid and prolined were 254 and 72 mM, respectively, when grown in 1.5 M NaCl. These results indicated that Na+, Cl- and amino acids, especially glutamic acid and proline, contributed to the regulation of internal solute concentration of V. alginolyticus in response to the increased external NaCl.