Isolation and characterization of algicidal bacteria from <Emphasis Type="Italic">Cochlodinium polykrikoides</Emphasis> culture

In this study, we analyzed a bacterial community closely associated with Cochlodinium polykrikoides that caused harmful algal blooming in the sea. Filtration using a plankton mesh and percoll gradient centrifugation were performed to eliminate free-living bacteria. Attached bacteria were analyzed by culture-dependent and culture-independent methods. Five culturable bacterial strains were isolated and identified from the C. polykrikoides mixed bacterial community. The isolates belonged to α-Proteobacteria (Nautella sp., Sagittula sp., and Thalassobius sp.) and γ-Proteobacteria (Alteromonas sp. and Pseudoalteromonas sp.). All of the 5 isolates showed algicidal activity against C. polykrikoides and produced extracellular compounds responsible for algicidal properties after entering the stationary phase. The algicidal compounds produced by the 5 isolates were heat-stable and had molecular masses of less than 10,000 Da. Furthermore, the algicidal compounds were relatively specific for C. polykrikoides in terms of their algicidal activities. Culture-independent analysis of the bacterial community in association with C. polykrikoides was performed using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). On the basis of the PCR-DGGE profile, Sagittula sp. was identified as a dominant species in the bacterial community of C. polykrikoides.     

青藏高原察尔汗盐湖地区可培养中度嗜盐菌的群落结构与多样性

【目的】研究青海察尔汗盐湖地区的可培养中度嗜盐菌的群落结构及多样性。【方法】采用多种选择性培养基进行中度嗜盐菌的分离、培养;通过16S r RNA基因序列扩增、测定,根据序列信息,进行系统进化树构建、群落结构组成分析及多样性指数计算。【结果】从察尔汗盐湖卤水及湖泥中分离到中度嗜盐菌421株,合并重复菌株后共83株中度嗜盐菌。菌株16S rRNA基因序列信息显示,4株中度嗜盐菌为潜在的新分类单元。83株嗜盐细菌分布于3个门的6个科16个属。其中,Bacillus属、Oceanobacillus属和Halomonas属为优势属。多样性结果显示,水样中的菌株多样性高于泥样,而泥样中的菌株优势度高于水样。【结论】察尔汗盐湖中度嗜盐菌具有丰富的遗传多样性,种群种类丰富,优势菌群集中,该盐湖地区存在可分离培养的中度嗜盐菌的疑似新物种。     

茅尾海红树根际土壤可培养细菌多样性及抑菌活性研究

为挖掘茅尾海红树植物根际土壤可培养细菌资源,研究其抑菌活性,该文使用培养基对茅尾海5种红树植物(红海榄、黄槿、无瓣海桑、桐花树、阔苞菊)的7份根际土壤进行富集培养,选用6种不同分离培养基对富集样品进行可培养细菌的分离纯化,基于菌株16S rRNA基因序列信息进行物种多样性分析,并采用纸片法筛选具有抑制表皮葡萄球菌、耐甲氧西林金黄色葡萄球菌、铜绿假单胞菌活性的菌株。结果表明:(1)从7份红树植物根际土壤样品中分离到120种可培养细菌,隶属于35科47属,其中链霉菌属(Streptomyces sp.)占菌种总数的 14.2%,同时发现5种潜在新菌株。(2)通过抑菌活性初筛,发现9种细菌的发酵粗提物对至少一种致病菌具有抑菌活性。综上表明,茅尾海红树植物根际土壤可培养细菌多样性丰富,并且部分菌株具有抑制人类致病菌活性。该研究结果为新型抗生素的开发与利用提供了菌种资源。     

Interactions of Botryococcus braunii Cultures with Bacterial Biofilms

Unicellular microalgae generally grow in the presence of bacteria, particularly when they are farmed massively. This study analyzes the bacteria associated with mass culture of Botryococcus braunii: both the planktonic bacteria in the water column and those forming biofilms adhered to the surface of the microalgal cells (∼10⁷–10⁸ culturable cells per gram microalgae). Furthermore, we identified the culturable bacteria forming a biofilm in the microalgal cells by 16S rDNA sequencing. At least eight different culturable species of bacteria were detected in the biofilm and were evaluated for the presence of quorum-sensing signals in these bacteria. Few studies have considered the implications of this phenomenon as regards the interaction between bacteria and microalgae. Production of C4-AHL and C6-AHL were detected in two species, Pseudomonas sp. and Rhizobium sp., which are present in the bacterial biofilm associated with B. braunii. This type of signal was not detected in the planktonic bacteria isolated from the water. We also noted that the bacterium, Rhizobium sp., acted as a probiotic bacterium and significantly encouraged the growth of B. braunii. A direct application of these beneficial bacteria associated with B. braunii could be, to use them like inoculants for large-scale microalgal cultures. They could optimize biomass production by enhancing growth, particularly in this microalga that has a low growth rate.     

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。     

Characterization and removal of biofouling from reverse osmosis membranes (ROMs) from a desalination plant in Northern Chile,using Alteromonas sp. Ni1-LEM supernatant

Abstract

Biofouling control in reverse osmosis membranes (ROMs) is challenging due to the high cost of treatments, and reduction in the life of ROMs. This study characterizes the biofouling in the ROMs from a desalination plant and reports its effective removal using the supernatant obtained from Alteromonas sp. strain Ni1-LEM. The characterization of the bacterial community revealed that the most abundant taxa in ROMs were the genera Fulvivirga and Pseudoalteromonas, and unclassified species of the families Flavobacteriaceae and Sphingomonadaceae. This bacterial community significantly decreased upon treatment with the supernatant from Alteromonas sp. Ni1-LEM, resulting in the prevalence of the genus Pseudoalteromonas. Furthermore, this bacterial supernatant significantly inhibited cell adhesion of seven benthic microalgae isolated from ROMs as well as promoting cell detachment of the existing microbial biofilms. The study showed that the extracellular supernatant modified the conformation of extracellular polymeric substances (EPS) in the biofouling of ROMs without any biocidal effects.     

Bacteria Associated with the Bleached and Cave Coral Oculina patagonica

The relative abundance of bacteria in the mucus and tissues of Oculina patagonica taken from bleached and cave (azooxanthellae) corals was determined by analyses of the 16S rRNA genes from cloned libraries of extracted DNA and from isolated colonies. The results were compared to previously published data on healthy O. patagonica. The bacterial community of bleached, cave, and healthy corals were completely different from each other. A tight cluster (>99.5% identity) of bacteria, showing 100% identity to Acinetobacter species, dominated bleached corals, comprising 25% of the 316 clones sequenced. The dominant bacterial cluster found in cave corals, representing 29% of the 97 clones sequenced, showed 98% identity to an uncultured bacterium from the Great Barrier Reef. Vibrio splendidus was the most dominant species in healthy O. patagonica. The culturable bacteria represented 0.1–1.0% of the total bacteria (SYBR Gold staining) of the corals. The most abundant culturable bacteria in bleached, cave, and healthy corals were clusters that most closely matched Microbulbifer sp., an α-proteobacterium previously isolated from healthy corals and an α-protobacterium (AB026194), respectively. Three generalizations emerge from this study on O. patagonica: (1) More bacteria are associated with coral tissue than mucus; (2) tissue and mucus populations are different; (3) bacterial populations associated with corals change dramatically when corals lack their symbiotic zooxanthellae, either as a result of the bleaching disease or when growing in the absence of light.     

Effect of temperature on the growth rates of halotolerant and halophilic bacteria isolated from Antarctic saline lakes

Summary The effect of temperature on the growth rates of Halomonas subglaciescola and a Halobacterium sp., halotolerant and halophilic bacteria isolated from Antarctic saline lakes, was predicted accurately by models which propose a linear relationship between temperature and the square root of the reciprocal of the growth rate. The cardinal temperatures of the strains examined were in general 5–10°C lower than those of taxonomic counterparts isolated from temperate and tropical environments. Temperature dependence data for strains of the Halobacterium sp. and annual temperature profiles of Deep Lake allowed estimation of the in situ potential for growth.     

不同寡营养培养条件下南海水体细菌群落结构及其对碳源的利用特征

【背景】绝大多数海洋微生物不可培养,为挖掘海洋生态系统中可培养的微生物资源,研究者尝试寡营养培养等方法。【目的】比较不同寡营养培养条件下南海水体细菌数量、群落结构及其对碳源的利用特征差异。【方法】采用原2216E培养液(Y)、稀释10倍(Y-10)和稀释50倍(Y-50)的2216E培养液培养南海海水样品,用荧光定量PCR法和16S rRNA基因检测细菌数量和菌群结构;利用平板计数法计数异养细菌的数量,纯化鉴定可培养细菌;采用Biolog EcoPlateTM微板法分析不同培养基中细菌群落对碳源的利用特征。【结果】Y组细菌总数高于Y-10组和Y-50组,差异不显著(P>0.05),但异养细菌数量显著高于Y-10组和Y-50组(P<0.05)。16S rRNA基因测序结果表明,不同稀释倍数下的细菌群落结构差异明显,Y组检测出10门193属,优势类群为Proteobacteria(56.44%)和Bacteroides (37.27%);Y-10组检测出15门220属,优势类群为Proteobacteria (40.30%)、Bacteroides(36.91%)和Firmic...     

Seasonal changes in numbers and the location of a particular bacterial strain of Alteromonas sp. in seagrass sediments

Many variables must be considered in seeking to describe differences in population sizes for native aquatic bacterial populations. In this study of seagrass- and nearby plant-free sediments, seasonal effects on total bacterial counts were found to be highly significant, outweighing the significance of factors such as geographic variability, but on populations of a chosen Alteromonas sp., they were not significant at the 5% level. Summer counts for both populations were higher than those for winter; this result is likely to reflect the higher productivity of the host Zostera capricomi in summer months, resulting in the exudation of increased amounts of organic nutrients. The Alteromonas sp. occurred in greatest abundance (1.8% of the total population) at the seagrass sediment site from which it was originally isolated and formed up to 1.5% of the population in adjacent plant-free sediments. In fluorescent microscopy studies with labeled antibodies, the Alteromonas sp. was found to be ubiquitous in seagrass and plant-free sediments but was found closely associated in much higher numbers with seagrass root-rhizome tissue, suggesting a possible nutritional relationship between plant and bacterium. In associated trials of sediment preservation techniques, bacterial counts of replicate sediments preserved with glutaraldehyde (3% v/v) were higher than those obtained using Lugol's iodine or freezing. Correspondence to: P.W. Glazebrook     

小花老鼠簕可培养细菌多样性及其生物学活性研究

小花老鼠簕(Acanthus ebracteatus)是一种生长在红树生态系统的珍稀真红树植物,具有较高的药用价值。为研究小花老鼠簕内生及根际可培养细菌多样性,挖掘其潜在新物种及具有特殊生物学活性的菌株,该文利用7种不同培养基,通过传统稀释涂布法对小花老鼠簕各植物组织及根际土壤可培养细菌进行分离,基于16S rRNA基因序列解析其内生及根际细菌群落结构和多样性特征,应用植物病原菌平板对峙实验和平铺捕食活性测试分析其可培养细菌的抗菌活性。结果表明：(1)基于16S rRNA基因序列分析,发现从小花老鼠簕的根、茎、叶、花及根际土壤中分离得到144株可培养细菌,这些细菌隶属于18目26科37属66种,芽孢杆菌属(Bacillus)和链霉菌属(Streptomyces)为优势菌属,分别占细菌种数的15.1%和13.6%;(2)拮抗多种植物病原菌试验结果显示,获得29株具有拮抗植物病原菌活性的细菌,10株具有广谱抑菌活性,其中链霉菌属菌株拮抗作用最强且菌株Y129为潜在新物种。(3)捕食活性测试结果显示,有5株细菌对金黄色葡萄球菌(Staphylococcus aureus)、耐甲氧西林金黄色葡...     

Rapid screening and dereplication of bacterial isolates from marine sponges of the Sula Ridge by Intact-Cell-MALDI-TOF mass spectrometry (ICM-MS)

Rapid grouping of bacterial isolates is critical in comprehensive microbial studies of environmental samples or screening programmes e.g. in unknown marine environments where large numbers of strains have to be isolated on different growth media. Sets of bacteria have been cultured from the marine sponges Isops phlegraei, Haliclona sp. 1, Phakellia ventilabrum and Plakortis sp. growing at a depth of about 300 m on the Sula Ridge close to the Norwegian coast. We employed Intact-Cell MALDI-TOF (ICM) mass spectrometry to achieve a rapid proteometric clustering of a subset of the strain collection including 456 isolates. Cluster analysis of mass spectra resolved the strains into 11 groups corresponding to species of Alteromonas (15), Bacillus (3), Colwellia (31), Erythrobacter (19), Marinobacter (14), Marinococcus (6), Pseudoalteromonas (297), Pseudomonas (56), Roseobacter (3), Sphingomonas (2) and Vibrio (10) as verified by 16 S rDNA analysis. A further discrimination into subgroups was demonstrated for different isolates from the genus Pseudoalteromonas. The approach described here permits the rapid identification of isolates for dereplication, and the selection of strains representing rare species for subsequent characterization.     

Mercury resistance and volatilization by oil utilizing haloarchaea under hypersaline conditions

The hydrocarbon utilizing haloarchaea, Haloferax (two strains), Halobacterium and Halococcus from a hypersaline coastal area of the Arabian Gulf, had the potential for resistance and volatilization of Hg²⁺. Individual haloarchaea resisted up to between 100 and 200 ppm HgCl₂ in hydrocarbon free media with salinities between 1 and 4 M NaCl, but only up to between 20 and 30 ppm in a mineral medium containing 3 M NaCl, with 0.5% (w/v) crude oil, as a sole source of carbon and energy. Halococcus and Halobacterium volatilized more mercury than Haloferax. The individual haloarchaea consumed more crude oil in the presence of 3 M NaCl than in the presence of 2 M NaCl. At both salinities, increasing the HgCl₂ concentration in the medium from 0 to 20 ppm resulted in decreasing the oil consumption values by the individual haloarchaea. However, satisfactory oil consumption still occurred in the presence of 10 ppm HgCl₂. It was concluded that haloarchaea with the combined potential for mercury resistance and volatilization and hydrocarbon consumption could be useful in removing toxic mercury forms effectively from oil free, mercury contaminated, hypersaline environments, and mercury and oil, albeit less effectively, from oily hypersaline environments.     

16S Ribosomal DNA-Based Analysis of Bacterial Diversity in Purified Water Used in Pharmaceutical Manufacturing Processes by PCR and Denaturing Gradient Gel Electrophoresis

The bacterial community in partially purified water, which is prepared by ion exchange from tap water and is used in pharmaceutical manufacturing processes, was analyzed by denaturing gradient gel electrophoresis (DGGE). 16S ribosomal DNA fragments, including V6, -7, and -8 regions, were amplified with universal primers and analyzed by DGGE. The bacterial diversity in purified water determined by PCR-DGGE banding patterns was significantly lower than that of other aquatic environments. The bacterial populations with esterase activity sorted by flow cytometry and isolated on soybean casein digest (SCD) and R2A media were also analyzed by DGGE. The dominant bacterium in purified water possessed esterase activity but could not be detected on SCD or R2A media. DNA sequence analysis of the main bands on the DGGE gel revealed that culturable bacteria on these media were Bradyrhizobium sp., Xanthomonas sp., and Stenotrophomonas sp., while the dominant bacterium was not closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods of quality control for pharmaceutical water.     

Predominant culturable crude oil-degrading bacteria in the coast of Kuwait

Total of 272 crude oil-degrading bacteria were isolated from seven locations along the coast of Kuwait. The analysis of the 16S rDNA sequences of isolated bacteria revealed the predominance of six bacterial genera: Pseudomonas, Bacillus, Staphylococcus, Acinetobacter, Kocuria and Micrococcus. Investigation of the factors associated with bacterial predominance revealed that, dominant culturable crude oil-degrading bacteria were better crude oil utilizers than the less frequently occurring isolates. Bacterial predominance was also influenced by the ability of bacteria to adapt to the level of organic content available. Predominant culturable bacteria constituted 89.7–54.2% of the total crude oil-degrading bacterial communities. Using 16S-RFLP analyses to assess the diversity of the dominant crude oil-degrading bacterial genera, four phylotypes of Pseudomonas sp. and seven phylotypes of Bacillus sp. were determined. This suggested high degree of diversity of crude oil-degrading bacterial population at the strain level, but low diversity at the genus level.     

Macroalgal Morphogenesis Induced by Waterborne Compounds and Bacteria in Coastal Seawater

Axenic gametes of the marine green macroalga Ulva mutabilis Føyn (Ria Formosa, locus typicus) exhibit abnormal development into slow-growing callus-like colonies with aberrant cell walls. Under laboratory conditions, it was previously demonstrated that all defects in growth and thallus development can be completely abolished when axenic gametes are inoculated with a combination of two specific bacterial strains originally identified as Roseobacter sp. strain MS2 and Cytophaga sp. strain MS6. These bacteria release diffusible morphogenetic compounds (= morphogens), which act similar to cytokinin and auxin. To investigate the ecological relevance of the waterborne bacterial morphogens, seawater samples were collected in the Ria Formosa lagoon (Algarve, Southern Portugal) at 20 sampling sites and tidal pools to assess their morphogenetic effects on the axenic gametes of U. mutabilis. Specifically the survey revealed that sterile-filtered seawater samples can completely recover growth and morphogenesis of U. mutabilis under axenic conditions. Morphogenetic activities of free-living and epiphytic bacteria isolated from the locally very abundant Ulva species (i.e., U. rigida) were screened using a multiwell-based testing system. The most represented genera isolated from U. rigida were Alteromonas, Pseudoalteromonas and Sulfitobacter followed by Psychrobacter and Polaribacter. Several naturally occurring bacterial species could emulate MS2 activity (= induction of cell divisions) regardless of taxonomic affiliation, whereas the MS6 activity (= induction of cell differentiation and cell wall formation) was species-specific and is probably a feature of difficult-to-culture bacteria. Interestingly, isolated bacteroidetes such as Algoriphagus sp. and Polaribacter sp. could individually trigger complete Ulva morphogenesis and thus provide a novel mode of action for bacterial-induced algal development. This study also highlights that the accumulation of algal growth factors in a shallow water body separated from the open ocean by barrier islands might have strong implications to, for example, the wide usage of natural coastal seawater in algal (land based) aquacultures of Ulva.     

长春花内生细菌多样性与柑橘黄龙病菌的相关性

【目的】分析感柑橘黄龙病长春花植株与健康长春花植株不同部位内生细菌菌群结构变化,为柑橘黄龙病菌与长春花内生细菌的相关性研究提供理论基础。【方法】本研究利用兼性厌氧可培养技术、16S rDNA限制性片段长度多态性分析(Restriction fragment length polymorphism,RFLP)以及16S rDNA序列分析相结合的方法。【结果】分别从感病和健康长春花叶、茎、根的组织中分离获得67株内生细菌,与GenBank中29种细菌的相似性达到97%-100%。其中短小杆菌属(Curtobacterium sp.)、欧文氏菌属(Erwinia sp.)、蜡样芽胞杆菌(Bacillus cereus)为感病长春花内生细菌的优势菌群,鞘胺醇单胞菌属(Brevundimonas sp.)、芽胞杆菌属(Bacillus sp.)为健康长春花内生细菌的优势菌群;马胃葡萄球菌(Staphylococcus equorum)为两者的共同优势菌群。通过RFLP方法分析,感病株得到16个、健株得到23个操作分类单元(Operational TaxonomicUnits,OTUs),感病植株中除柑橘黄龙病菌Candidatus Liberibacter asiaticus外,还有丰富的CandidatusLiberibacter sp.存在。【结论】感病与健康长春花植株中均含有丰富的内生细菌,黄龙病菌的存在改变了长春花原有内生细菌的菌群结构,且菌群多样性下降。可见长春花内生细菌在一定程度上受到柑橘黄龙病菌的抑制。     

不同培养基组合提高土壤细菌可培养性的研究

为选择性采用多培养基组合以提高土壤细菌可培养性,利用变性梯度凝胶电泳(DGGE)技术研究了贫营养、富营养和自然营养培养基在3种培养方式下获得细菌种群的差异。结果表明:平板培养条件下,细菌在贫营养培养基上生长较慢,菌落连续稳定形成。培养5d后,富营养的LB培养基和贫营养的R2A培养基获得菌落数最多,分别是贫营养的0.1×LB培养基获得菌落数的5.1倍和5.3倍。7种培养基中,LB培养基获得细菌种群数目最多,营养成分适当稀释后,培养物中有新的种群出现。贫营养培养基和富营养培养基培养物DGGE图谱相似性低,条带互补性强。三角瓶静置培养时,R2A和LB培养基获得细菌种群数目较多,其它几种培养基获得的细菌类群都能在这2种培养基中找到。试管静置培养条件下,LB培养基获得细菌种群数目最多,某些种群也只出现在R2A培养基和TSB培养基上,R2A及LB培养基与TSB培养基获得的细菌种群差异较为明显。研究结果为特殊培养基设计及选用合适培养基分离土壤细菌提供参考。     

Tributyltin-resistant marine bacteria: a summary of recent work

Summary A tributyltin chloride (TBTCl)-resistant bacterium,Alteromonas sp. M-1, was isolated from coastal seawater. This bacterium grew in medium containing 125 M TBTCl. TBTCl added to the medium was taken up by this bacterium, however, the amount of TBTCl in the cellular fraction was low after the logarithmic phase, suggesting the existence of a TBTCl-efflux system. A genetic library was constructed using plasmid vector pUC 19. Three positive clones were obtained, by whichE. coli was transformed to TBTCl resistance. Of the three clones, the shortest fragment fromHindIII-library was analyzed. This fragment was 1.8 kb long and contained one complete open reading frame. The predicted amino acid sequence of this open reading frame had a homologous domain to transglycosylases of bacteriophage andE. coli. TBTCl-tolerant marine bacteria other thanAlteromonas sp. M-1 were obtained from natural seawater to which TBTCl was added. DNA-DNA hybridization was performed between the three cloned fragments fromAlteromonas sp. M-1 and chromosomal DNA of the TBTCl-tolerant bacteria. Some strains hybridized with the fragments and some did not, suggesting that several genes are responsible for TBTCl tolerance.