Correlation between pigmentation and antifouling compounds produced by Pseudoalteromonas tunicata

Pseudoalteromonas tunicata is a marine bacterium with the ability to prevent biofouling by the production of at least four target-specific compounds. In addition to these antifouling compounds, P. tunicata produces at least two pigments. These include a yellow and a purple pigment which, when combined, give the bacterium a dark green appearance. Transposon mutagenesis was used in this study to investigate the correlation between pigment production and the expression of specific antifouling phenotypes in P. tunicata. Four different categories of pigmentation mutants were isolated including yellow, dark-purple, light-purple and white mutants. The mutants were tested for their ability to inhibit the settlement of invertebrate larvae, algal spore germination, fungal growth and bacterial growth. The results showed that the yellow-pigmented mutants retained full antifouling activity, whereas the purple and white mutant strains had lost some, or all, of their ability to inhibit target organisms. This demonstrates that the loss of antifouling capabilities correlates with the loss of yellow pigment and not purple pigment. Sequencing and analysis of the genes disrupted by the transposons in these mutants identified a number of potential biosynthetic enzymes and transport systems involved in the synthesis and regulation of pigmentation and fouling inhibitors in this organism.     

Effect of biofilm formation by Pseudoalteromonas spongiae on induction of larval settlement of the polychaete Hydroides elegans

The effects of culture conditions and chloramphenicol treatment on the induction of the marine bacterium Pseudoalteromonas spongiae to larval settlement of Hydroides elegans were investigated. The results showed that P. spongiae cells grown in the medium containing both yeast extract and peptone (YP-grown P. spongiae) was highly inductive to larval settlement, whereas P. spongiae cells grown in the medium containing only peptone (P-grown P. spongiae) or YP-grown P. spongiae cells treated with chloramphenicol at the onset of biofilm development (YPC-grown P. spongiae) did not induce larval settlement. Analysis of biofilm formation, biofilm structure, and the surface protein profile indicated that only the induction-capable YP-grown P. spongiae formed a well-developed biofilm, while the P-grown P. spongiae and the YPC-grown P. spongiae did not. We report here for the first time that bacterial biofilm formation was associated with its induction of larval settlement.     

Sulphide as a larval settlement cue forCapitella sp I

Pioneering marine benthic invertebrates are capable of locating and colonizing newly created and recently disturbed mud bottoms within a few days. The results of this study demonstrate that sulphides — naturally occurring products of anaerobic organic matter decomposition — promote the larval settlement of the pioneering polychaeteCapitella sp I in both laboratory and semi-natural conditions.Settlement was enhanced both in sediments enriched with sulphides and in sulphidec, sediment-free conditions when compared with controls. A sulphide concentration ranging between 0.1 mM and 1.0 mM elicited optimal settlement with subsequent metamorphosis and survival of the settled worms.This is the first time a geochemically-mediated larval settlement response has been demonstrated.     

Substrate selection and larval settlement by Cupelopagis vorax

Cupelopagis vorax was sampled for one year with a glass slide sampler. Slides were collected every two weeks and the numbers and locations of settled individuals noted. Reproductive condition of the collected rotifers was recorded. The population appeared suddenly and rapidly attained peak numbers. C. vorax shows a distinct preference for the underside of horizontally-oriented surfaces. Sexual reproduction occurred when the number of settling individuals was maximum.     

Extracellular secretion of Pseudoalteromonas sp. cold-adapted esterase in Escherichia coli in the presence of Pseudoalteromonas sp. components of ABC transport system

Recently we described identification and characterization of GDSL esterase EstA from psychrotrophic bacterium Pseudoalteromonas sp. 643A. Attempts to obtain heterologous overexpression of this enzyme in Escherichia coli system were not satisfactory. The EstA protein was expressed as inclusion bodies, most of that were inactive after purification step, and the recovery of esterolytic activity was very low after refolding. Based on the sequence analysis we found that the esterase EstA gene is clustered with three genes encoding components of ABC transport system. These genes, designated abc1, abc2, and abc3 encode an ATP-binding protein (ABC1) and two permease proteins (ABC2 and ABC3). In present study, to obtain larger amounts of the active cold-adapted EstA esterase from Pseudoalteromonas sp. 643A, we designed a two-plasmid E. coli expression system where the gene encoding EstA enzyme was cloned into pET30b(+) expression vector and three genes encoding components of ABC transport system were cloned into pACYC-pBAD vector. It was shown that the created expression system was useful for extracellular production of active EstA enzyme which was purified from the culture medium. In the presence of all the three transporter proteins the secretion of EstA was at the highest level. When one or two of these components were missing, EstA secretion was also possible, but not so effective. It indicates that ABC2 and ABC3 proteins of Pseudoalteromonas sp. 643A could be replaced with their homologous proteins of E. coli.     

Metalloid Reducing Bacteria Isolated from Deep Ocean Hydrothermal Vents of the Juan de Fuca Ridge, Pseudoalteromonas telluritireducens sp. nov. and Pseudoalteromonas spiralis sp. nov

Five strains of Gram-negative, rod, curved rod and spiral-shaped bacteria were isolated from the vicinity of deep ocean hydrothermal vents along the Main Endeavour Segment of the Juan de Fuca Ridge in the Pacific Ocean. All strains showed remarkable resistance to high levels of toxic metalloid oxyanions, and were capable of reducing the oxyanions tellurite and selenite to their less toxic elemental forms. Phylogenetic analysis of four strains identified these isolates as close relatives of the genus Pseudoalteromonas within the class Gammaproteobacteria. Pseudoalteromonas agarivorans was the closest relative of strains Te-1-1 and Se-1-2-red^T, with, respectively, 99.5 and 99.8% 16S rDNA sequence similarity. Strain Te-2-2^T was most closely related to Pseudoalteromonas paragorgicola, with 99.8% 16S rDNA sequence similarity. The DNA G+C base composition was 39.6 to 41.8 mol%, in agreement with other members of the genus Pseudoalteromonas. However, the isolates showed important morphological and physiological differences from previously described species of this genus, with one group forming rod-shaped bacteria typical of Pseudoalteromonas and the other forming vibrioid- to spiral-shaped cells. Based on these differences, and on phylogenetic data, we propose the creation of the new species Pseudoalteromonas telluritireducens sp. nov., with strain Se-1-2-red^T (DSMZ=16098^T=VKM B-2382^T) as the type strain, and Pseudoalteromonas spiralis sp. nov., with strain Te-2-2^T (DSMZ=16099^T=VKM B-2383^T) as the type strain. The EMBL accession numbers for the 16S rDNA sequences are: Te-1-1, AJ314843; Te-2-2^T, AJ314842; Se-1-2-or, AJ314844; Se-1-2-red^T, AJ314845.     

极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶发酵条件的优化

对极地适冷菌Pseudoalteromonas sp. QI-1产适冷蛋白酶的发酵条件进行优化。结果表明:菌株QI-1的最适生长和产酶温度均为5℃;最佳接种量为1%;发酵培养基的最适初始pH和最佳装样量分别为5和10%;盐度为2%时对菌株的生长和产酶最为有利;麸皮和醋酸钠分别为最佳N源和C源;添加0.75%酪蛋白时菌株QI-1胞外蛋白酶的活性最高;10 mmol/L Mg2+和0.5%Tween-80有利于产酶。正交试验结果表明:菌株Pseudoalteromonassp. QI-1产蛋白酶较佳培养基配方(g/L)为麸皮5,酵母粉2.5,酪蛋白3,MgCl2.6H2O 3,KCl 1.5;发酵液比酶活为166.20 U/mL,较优化前提高了约56%。     

Bio-removal of cadmium by growing deep-sea bacterium Pseudoalteromonas sp. SCSE709-6

Effective bio-removal of heavy metals is important for water treatment. Although a number of microorganism species demonstrated the ability of living cells to remove cadmium, most of them were tested at fixed concentration of metals, salinity, and temperature. This paper reported a research on the screening and performance of a newly developed deep-sea bacterium, Pseudoalteromonas sp. SCSE709-6, for Cd(II) removal by growing cells under a range of experimental conditions: 0–50 mg/L of Cd(II), 15–30 °C of incubation temperatures, 6.5–8.0 of initial pH, and 1.5–5.0 % of salinity. Study results revealed that Pseudoalteromonas sp. SCSE709-6 could remove more than 96 % of Cd(II) on growth. The Cd(II) bioremoval was in correlation but not in accordance with biomass. As cadmium concentrations increased, the Cd(II) removal by cell adsorption played an increasingly important role compared with that of intracellular accumulation. For the removal mechanism, Fourier transform infrared spectroscopy revealed that carboxyl, amido and hydroxyl of saccharides, and proteins in the extracellular polymeric substances are the most active groups for Cd(II) absorption. The bacterium reported in this study offers a new microbe strain for Cd(II) bioremediation.     

Promoting larval settlement of coral Pocillopora damicornis by calcium

Larval settlement is a critical bottleneck in the process of coral sexual propagation. Promoting coral larval settlement by inducers is a promising strategy in coral reef restoration engineering. In this study, the settlement-promoting effect of Ca²⁺ on larvae of the brooding coral Pocillopora damicornis was investigated for the first time. Treatment with 40 mM CaCl₂ for 24 h effectively promoted coral larval settlement (~ 80%). Moreover, CaCl₂ is comparable with the natural inducer, crustose coralline algae (CCA), in both promoting coral larval settlement and post-settlement growth. CaCl₂ showed toxic effects on larval survival and growth at high concentrations, and this could be minimized by optimizing CaCl₂ concentration and shortening the exposure period. Our study suggests that applying Ca²⁺ to effectively and efficiently induce coral larval settlement is viable for laboratory research and small-scale aquaculture systems, and it might become a useful tool in future coral reef restoration engineering.

     

Structure of glycerophosphate-containing O-specific polysaccharide from Pseudoalteromonas sp. KMM 639

On the basis of acid hydrolysis, dephosphorylation, methylation, and 13C NMR spectroscopy data, the O-specific polysaccharide of Pseudoalteromonas sp. KMM 639 was shown to be a glycerophosphate-containing polymer built of repeating disaccharide units of the following structure: [formula: see text]     

假交替单胞菌LJ1菌株产褐藻胶裂解酶的培养条件优化及酶学性质

[目的]为了优化Lj1菌株的培养条件使之产生高活性的胞外褐藻胶裂解酶.[方法]通过富集培养技术从海带筛选到一株褐藻胶裂解酶产生菌Lj1,依据表型特征、脂肪酸组成分析及16S rRNA基因序列分析对该菌株进行鉴定.通过单因子和正交试验对Lj1菌株产胞外褐藻胶裂解酶的培养条件进行了优化.[结果]Lj1菌株属于假交替单胞菌属(Pseudoalteromonas).该菌株产酶的最佳培养基组成为:褐藻胶3g/L、(NH4)2SO43 g/L、NaCl 20 g/L、KH2PO4 0.1 g/L、CaCl2 0.1 g/L;最佳培养条件为:250 mL三角烧瓶中装液量25 mL、接种量3%、摇瓶转速150 r/min、pH7.5、培养温度为28℃、培养时间为24 h.LJl菌株所产褐藻胶裂解酶的最适温度为40℃,最适pH7.6,最适NaCl浓度为0.3 mol/L.1 mol/1.金属离子Mg2+对酶活力有明显的促进作用,而C02+和Zn2+对酶活力有较强的抑制作用.[结论]LJ1菌株是Pseudoalteromonas新的胞外褐藻胶裂解酶产生菌,在最佳培养条件下,该菌株的酶活力提高了66%.     

适冷海洋细菌交替假单胞菌(Pseudoalteromonas sp.)MB-1内切葡聚糖酶基因的克隆和表达

从黄海深海海底淤泥中筛选出一株产纤维素酶的适冷革兰氏阴性杆菌MB 1,克隆和分析了MB 1的 16SrDNA序列 (GenBank接受号 :AY5 5 132 1) ,经鉴定为交替假单胞菌 (Pseudoalteromonas) ,命名为Pseudoalteromonassp .MB 1。克隆了该菌适冷内切葡聚糖酶基因celA(GenBank接受号 :AY5 5 132 2 ) ,并在大肠杆菌 (Escherichiacoli)BL2 1中进行了表达。重组E .coli菌体破碎后 ,获取上清液 ,其中融合蛋白GST CelA浓度约为 78 5mg L。分析了融合酶GST CelA的性质 ,其最适反应温度为 35℃ ,最适反应pH值为 7 2 ,为中性适冷酶。实验结果为交替假单胞菌低温纤维素酶的基础理论和应用研究奠定了基础     

交替假单胞菌(Pseudoalteromonas sp.)DY3菌株产内切葡聚糖酶的性质研究及基因克隆

从深海样品ESO109中分离到一株具有高内切葡聚糖酶活力的细菌DY3,16SrDNA序列分析表明该菌与交替假单胞菌属(Pseudoalteromonas sp．)的Pseudoalteromonas citrea和Pseudoalteromonas elyakovii的同源性为99％。PCR扩增DY3的内切葡聚糖酶基因celX全长1479bp,编码一个492AA的蛋白质。酶的氨基酸序列分析表明CelX与Rseudoalteromonas haloplanktis的内切葡聚糖酶CelG有95％的相似性,包括一个糖基水解酶家族5的催化结构域,一个连接序列和位于C端的的CBM5结构域。对酶性质的初步研究发现,CelX的最适温度为40℃,酶的最适pH在6～7之间。     

海洋细菌Pseudoalteromonas sp. NJ631中NRPS基因簇及核心模件的发掘

[目的]运用基因组信息发掘技术(Genome Mining)对海洋细菌Pseudoalteromonas sp.NJ631基因组中的非核糖体肽合成酶(Nonribosomal peptides synthetases,NRPSs)基因簇资源及其核心模件进行发掘分析,旨在为Pseudoalteromonas属中非核糖体肽(Nonribosomal peptides,NRPs)的发现提供理论依据和数据支持.[方法]依托第二代测序技术获得的Pseudoalteromonas sp.NJ631基因组序列草图,在分析其次生代谢产物编码基因的基础上,利用NRPS-PKS knowledgebase在线预测软件鉴定潜在的NRPSs基因簇,并对其基因组中的NRPSs核心模件腺苷酰化(Adenylation,A)结构域编码基因信息进行发掘.[结果]在NJ631基因组序列草图中发现3个典型结构组成的NRPSs基因簇,命名为NGC1、NGC2和NGC3,分别位于scaffold6,9和11.进一步的结构域预测分析表明,3个NRPSs基因簇均含有3个ORFs,其中NGC1编码7个NRPSs模块 ;NGC2和NGC3均编码6个NRPSs模块.对A结构域的信息发掘显示NJ631基因组中含有38个A结构域编码基因,特异性选择18种氨基酸底物.[结论]通过运用基因组信息发掘技术对海洋细菌Pseudoalteromonas sp.N J631全基因组信息进行NRPSs基因簇及核心模件A结构域的发掘分析,结果提示,通常只在放线菌或真菌中发现的NRPSs基因资源也在Pseudoalteromonas属中大量存在.研究结果也为今后Pseudoalteromonas属中非核糖体肽(Nonribosomal peptides,NRPs)的发现提供理论依据.     

Antibiofilm Activity of the Marine Bacterium Pseudoalteromonas sp. Strain 3J6

Biofilm formation results in medical threats or economic losses and is therefore a major concern in a variety of domains. In two-species biofilms of marine bacteria grown under dynamic conditions, Pseudoalteromonas sp. strain 3J6 formed mixed biofilms with Bacillus sp. strain 4J6 but was largely predominant over Paracoccus sp. strain 4M6 and Vibrio sp. strain D01. The supernatant of Pseudoalteromonas sp. 3J6 liquid culture (SN_3J6) was devoid of antibacterial activity against free-living Paracoccus sp. 4M6 and Vibrio sp. D01 cells, but it impaired their ability to grow as single-species biofilms and led to higher percentages of nonviable cells in 48-h biofilms. Antibiofilm molecules of SN_3J6 were able to coat the glass surfaces used to grow biofilms and reduced bacterial attachment about 2-fold, which might partly explain the biofilm formation defect but not the loss of cell viability. SN_3J6 had a wide spectrum of activity since it affected all Gram-negative marine strains tested except other Pseudoalteromonas strains. Biofilm biovolumes of the sensitive strains were reduced 3- to 530-fold, and the percentages of nonviable cells were increased 3- to 225-fold. Interestingly, SN_3J6 also impaired biofilm formation by three strains belonging to the human-pathogenic species Pseudomonas aeruginosa, Salmonella enterica, and Escherichia coli. Such an antibiofilm activity is original and opens up a variety of applications for Pseudoalteromonas sp. 3J6 and/or its active exoproducts in biofilm prevention strategies.Biofilms are defined as microbial communities of cells that are irreversibly attached to a substratum, to an interface, or to each other and are embedded into a matrix of extracellular polymeric substances that they have produced (8). It is now considered that most (if not all) bacteria are capable of forming biofilms and that this is their predominant bacterial life-style. Biofilm formation is a complex biological phenomenon and has been generally described as a temporal process involving a succession of distinct stages: a reversible and then irreversible attachment of planktonic bacteria onto a surface, the formation of microcolonies either by the clonal growth of attached cells or by the active translocation of cells across the surface, the coalescence of growing microcolonies to form a macrocolony, and cell dispersal. It should, however, be noted that this developmental model still requires further experimental validation, especially concerning the possibility of a hierarchical order of genetic pathways (26). Furthermore, Karatan and Watnick (17) pointed out that there are as many different types of biofilms as there are bacteria and that a single bacterium may even make several different types of biofilms under different environmental conditions. Biofilm formation is associated with the virulence of pathogenic bacteria, and cells included within a biofilm are generally more resistant (up to 1,000-fold) to antibiotics and disinfectants than free-living bacteria (8, 26). Biofilms are therefore a major concern in medicine and in medical environments but also in all domains where their growth constitutes a source of contamination for humans or animals (food industry, cooling towers, and water pipes, etc.) or leads to economical losses (biofouling of boats and immersed structures and material biocorrosion, etc.). The development of antibiofilm strategies is therefore of major interest and currently constitutes an important field of investigation in which environmentally friendly antibiofilm molecules or organisms are highly valuable (5, 7, 9).Marine bacteria belonging to the genus Pseudoalteromonas of the class Gammaproteobacteria are often found in association with marine eukaryotes, and their ability to produce a variety of biological activities has attracted particular attention (2, 11, 13, 15, 28). We previously isolated marine bacteria attached to solid surfaces (glass in most cases) immersed for 3 or 6 h in the Morbihan Gulf or in the Bay of Brest, France (10, 20, 21, 27). Out of the three Pseudoalteromonas strains isolated, we were able to tag strain 3J6 with a green fluorescent protein (GFP)-encoding plasmid. This allowed us to investigate whether Pseudoalteromonas sp. strain 3J6 affected the biofilm growth of other marine bacterial isolates. Here, we report that strain 3J6 predominated in two-species biofilms over Paracoccus sp. strain 4M6 and Vibrio sp. strain D01. Although devoid of antibacterial activity against planktonic cells, Pseudoalteromonas sp. 3J6 exoproducts impaired biofilm formation by Paracoccus sp. 4M6 and Vibrio sp. D01. We characterized the effects of these exoproducts on the latter strains and on other bacteria.     

假交替单胞菌JIV-49产抗真菌活性物质的发酵条件研究

目的:对假交替单胞菌JIV-49进行了发酵条件的研究。方法:采用单因素实验法对JIV-49产抗真菌活性物质的发酵培养基及主要的影响因子如初始pH值、温度、培养时间、接种量、转速、摇瓶装液量等进行了考察。结果:确定了最佳培养基为:牛肉膏为2.5%、KBr为0.01%、盐浓度为6%;最佳培养条件为:初始pH值为7.5、温度为30℃、培养时间为96h、接种量为7%,摇床转速为180r/min,摇瓶装液量为75ml/500ml。结论:初步确定了JIV-49发酵的条件,为工业化生产抗真菌活性物质提供了科学依据。