Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus.

The growth, toxicity, and associated bacterial flora of 10 clonal cultures of the toxic benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus isolated from the coastal waters of southwest Puerto Rico have been examined. Clonal cultures of O. lenticularis grew more rapidly and at broader temperature ranges than those of G. toxicus. All five Ostreopsis clones were toxic, while only one of the five Gambierdiscus clones was poisonous. The degree of toxicity among poisonous clones was highly variable. The number of associated bacterial genera and their frequency of occurrence were quite variable among clones of both dinoflagellate genera. Bacterial isolates represented six genera (Nocardia, Pseudomonas, Vibrio, Aeromonas, Flavobacterium, and Moraxella) in addition to coryneform bacteria. Extracts of dinoflagellate-associated bacteria grown in pure culture were not toxic. Gambierdiscus clones were characterized by the frequent presence of Pseudomonas spp. (four of five clones) and the absence of coryneforms. In O. lenticularis, only one of five clones showed the presence of Pseudomonas spp., and Moraxella sp. was absent altogether. Detailed analyses of toxicity and associated microflora in a selected Ostreopsis clone, repeatedly cultivated (four times) over a period of 160 days, showed that peak cell toxicities developed in the late static and early negative culture growth phases. Peak Ostreopsis cell toxicities in the stationary phase of culture growth were correlated with significant increases in the percent total bacteria directly associated with these cells. Changes in the quantity of bacteria directly associated with microalgal cell surfaces and extracellular matrices during culture growth may be related to variability and degree of toxicity in these laboratory-cultured benthic dinoflagellates.     

Associated bacterial flora, growth, and toxicity of cultured benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus

The growth, toxicity, and associated bacterial flora of 10 clonal cultures of the toxic benthic dinoflagellates Ostreopsis lenticularis and Gambierdiscus toxicus isolated from the coastal waters of southwest Puerto Rico have been examined. Clonal cultures of O. lenticularis grew more rapidly and at broader temperature ranges than those of G. toxicus. All five Ostreopsis clones were toxic, while only one of the five Gambierdiscus clones was poisonous. The degree of toxicity among poisonous clones was highly variable. The number of associated bacterial genera and their frequency of occurrence were quite variable among clones of both dinoflagellate genera. Bacterial isolates represented six genera (Nocardia, Pseudomonas, Vibrio, Aeromonas, Flavobacterium, and Moraxella) in addition to coryneform bacteria. Extracts of dinoflagellate-associated bacteria grown in pure culture were not toxic. Gambierdiscus clones were characterized by the frequent presence of Pseudomonas spp. (four of five clones) and the absence of coryneforms. In O. lenticularis, only one of five clones showed the presence of Pseudomonas spp., and Moraxella sp. was absent altogether. Detailed analyses of toxicity and associated microflora in a selected Ostreopsis clone, repeatedly cultivated (four times) over a period of 160 days, showed that peak cell toxicities developed in the late static and early negative culture growth phases. Peak Ostreopsis cell toxicities in the stationary phase of culture growth were correlated with significant increases in the percent total bacteria directly associated with these cells. Changes in the quantity of bacteria directly associated with microalgal cell surfaces and extracellular matrices during culture growth may be related to variability and degree of toxicity in these laboratory-cultured benthic dinoflagellates.     

南美白对虾肠道微生物群落的分子分析

采用分子生物学手段16S rDNA克隆文库方法对实验室养殖条件下的南美白对虾肠道细菌进行了多样性研究。用限制性片段长度多态性(RFLP)方法从文库中筛选出可能不同细菌来源的克隆子12个,测定其16S rDNA片段核甘酸序列,将所获得的序列与GenBank数据库进行BLAST比对,结果表明:南美白对虾肠道的16S rDNA克隆文库中126个克隆子分属2个不同的细菌类群:变形细菌(Proteobacteria)和厚壁细菌(Firmicutes),其中厚壁细菌为优势菌群占到75.4%,且与最相似序列同源性均低于94%;变形细菌占到24.6%,与最相似序列同源性均高于98%,分别为希瓦氏菌属(Shewanella),泛菌属(Pantoea),Aranicola属,假单胞菌属(Pseudomonas)和弧菌属(Vibrio)。     

Microbial diversity in Maras salterns, a hypersaline environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 x 10(6) to 3 x 10(6) cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4',6'-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon "Haloquadra walsbyi," although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the gamma-proteobacterium "Pseudomonas halophila" DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the "P. halophila" cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

High-diversity biofilm for the oxidation of sulfide-containing effluents

In the present work, we describe for the first time the utilization of a complex microbial biofilm for the treatment of sulfide-containing effluents. A non-aerated packed-column reactor was inoculated with anoxic lake sediment and exposed to light. A biofilm developed in the column and showed a stable oxidation performance for several weeks. Microbial species composition was analyzed by microscopy, pigment analysis and a bacterial 16S rRNA gene clone library. Colorless sulfur bacteria, green algae and purple sulfur bacteria were observed microscopically. Pigment composition confirmed the presence of algae and purple sulfur bacteria. The clone library was dominated by alpha-Proteobacteria (mostly Rhodobacter group), followed by gamma-Proteobacteria (Chromatiaceae-like and Thiothrix-like aerobic sulfur oxidizers) and the Cytophaga-Flavobacterium-Bacteroides group. Plastid signatures from algae were also present and a few clones belonged to both the beta- (Rhodoferax sp., Thiobacillus sp.) and delta-Proteobacteria (Desulfocapsa sp.) and to the low G+C Gram-positive bacteria (Firmicutes group). The coexistence of aerobic, anaerobic, phototrophic and chemotrophic microorganisms in the biofilm, the species richness found within these metabolic groups (42 operational taxonomic units) and the microdiversity observed within some species could be very important for the long-term functioning and versatility of the reactor.     

Development of microsatellite markers for Pythium helicoides

Pseudomonas sp. strain WBC-3 utilizes methyl parathion ( O,O -dimethyl O - p -nitrophenol phosphorothioate) or para -nitrophenol as the sole source of carbon, nitrogen and energy. A gene encoding methyl parathion hydrolase (MPH) had been characterized previously and found to be located on a typical class I composite transposon that comprised IS 6100 (Tn mph ). In this study, the transposability of this transposon was confirmed by transposition assays in two distinct mating-out systems. Tn mph was demonstrated to transpose efficiently in a random manner in Pseudomonas putida PaW340 by Southern blot and in Ralstonia sp. U2 by sequence analysis of the Tn mph insertion sites, both exhibiting MPH activity. The linkage of the mph -like gene with IS 6100 , together with the transposability of Tn mph , as well as its capability to transpose in other phylogenetically divergent bacterial species, suggest that Tn mph may contribute to the wide distribution of mph -like genes and the adaptation of bacteria to organophosphorus compounds.     

Application of broad-range 16S rRNA PCR amplification and DGGE fingerprinting for detection of tick-infecting bacteria

Ticks play an important role in the transmission of arthropod-borne diseases of viral, protozoal and bacterial origin. The present article describes a molecular-biological based method, which facilitated the broad-range analyses of bacterial communities in ixodid ticks (Ixodes ricinus). DNA was extracted both from single ticks and pooled adult ticks. Eubacterial 16S rRNA gene fragments (16S rDNA) were amplified by polymerase chain reaction (PCR) with broad-range ribosomal primers. Sequences spanning the hypervariable V3 region of the 16S rDNA and representing individual bacterial taxons were separated by denaturing gradient gel electrophoresis (DGGE). For phylogenetic identification, DGGE bands were exised, cloned and sequenced. In addition, we set up a 16S rDNA clone library which was screened by DGGE. Sequences were compared with sequences of known bacteria listed in the GenBank database. A number of bacteria were affiliated with the genera Rickettsia, Bartonella, and Borrelia, which are known to be pathogenic and transmitted by ticks. Two sequences were related to the yet to be cultivated Haemobartonella. To our knowledge, Haemobartonella has never been directly detected in I. ricinus. In addition, members of the genera Staphylococcus, Rhodococcus, Pseudomonas, and Moraxella were detected, which have not been identified in ticks so far. Two bacteria were most closely related to a rickettsial endosymbiont of an Acanthamoeba sp., and to an endosymbiont (Legionellaceae, Coxiella group) of the microarthropod Folsomia candida. The results prove that 16S rDNA genotyping in combination with DGGE analysis is a promising approach for the detection and identification of bacteria infecting ticks, regardless of whether these bacteria are fastidious, obligate intracellular or noncultivable.     

Bacterial diversity in the bacterioneuston (sea surface microlayer): the bacterioneuston through the looking glass

The bacterioneuston is defined as the community of bacteria present within the neuston or sea surface microlayer. Bacteria within this layer were sampled using a membrane filter technique and bacterial diversity was compared with that in the underlying pelagic coastal seawater using molecular ecological techniques. 16S rRNA gene libraries of approximately 500 clones were constructed from both bacterioneuston and the pelagic water samples and representative clones from each library were sequenced for comparison of bacterial diversity. The bacterioneuston was found to have a significantly lower bacterial diversity than the pelagic seawater, with only nine clone types (ecotaxa) as opposed to 46 ecotaxa in the pelagic seawater library. Surprisingly, the bacterioneuston clone library was dominated by 16S rRNA gene sequences affiliated to two groups of organisms, Vibrio spp. which accounted for over 68% of clones and Pseudoalteromonas spp. accounting for 21% of the library. The dominance of these two 16S rRNA gene sequence types within the bacterioneuston clone library was confirmed in a subsequent gene probing experiment. 16S rRNA gene probes specific for these groups of bacteria were designed and used to probe new libraries of 1000 clones from both the bacterioneuston and pelagic seawater DNA samples. This revealed that 57% of clones from the bacterioneuston library hybridized to a Vibrio sp.-specific 16S rRNA gene probe and 32% hybridized to a Pseudoalteromonas sp.-specific 16S rRNA gene probe. In contrast, the pelagic seawater library resulted in only 13% and 8% of 16S rRNA gene clones hybridizing to the Vibrio sp. and Pseudoalteromonas sp. probes respectively. Results from this study suggest that the bacterioneuston contains a distinct population of bacteria and warrants further detailed study at the molecular level.     

Microbial diversity within early-stage cultured Panulirus ornatus phyllosomas

A thorough understanding of the microorganisms and pathogens associated with the larval stage of the tropical ornate rock lobster, Panulirus ornatus, is required to overcome disease outbreaks that currently block aquaculture attempts. This study used microscopy in addition to culture and molecularly based microbiological techniques to characterize the bacterial community associated with cultured, developmental stage PI to PII P. ornatus phyllosomas. Scanning electron microscopy demonstrated colonization of phyllosomas by filamentous, rod-shaped, and coccus-shaped bacteria. A clone library constructed from dead phyllosomas sampled from the larval rearing tank on day 10 was dominated by Thiothrix-affiliated sequences (56% of clones). A comparable library from live phyllosomas also contained Thiothrix-affiliated sequences, though these only represented 19% of clones within the library. Fluorescent in situ hybridization (FISH) confirmed identification of the filamentous bacteria as Thiothrix sp., being present on dead phyllosomas. FISH also identified Leucothrix sp. and Vibrio sp., as well as a range of other rod- and coccus-shaped bacteria, colonizing both live and dead phyllosomas. The development of the microbial community associated with phyllosomas was monitored through a standard larval rearing run using denaturing gradient gel electrophoresis (DGGE). Vibrio sp.-affiliated bands dominated the profiles of live animals through the rearing period and dead phyllosomas sampled on selected days. The population of Vibrio sp. associated with phyllosomas was monitored with culture-based analysis on selective media and demonstrated to increase significantly on day 7, coinciding with the beginning of the larval molt. An isolated Vibrio harveyi strain demonstrated an identical 16S rRNA sequence with retrieved DGGE and clone library sequences. Colonization of phyllosomas with filamentous bacterial species potentially hinders the ability of the animals to molt and, combined with the added stress of the molt process, likely results in reduced immune function, allowing opportunistic pathogenic Vibrio sp. to cause larval mortalities.     

Algal growth enhancement by bacteria: Is consumption of photosynthetic oxygen involved?

Abstract: Pseudomonas diminuta and P. vesicularis , two obligate aerobes isolated from laboratory algal cultures, stimulated the growth of the green microalgae Scenedesmus bicellularis and Chlorella sp., without releasing any growth promoting substance. An intimate contact between both microorganisms was necessary for significant algal growth enhancement. The possibility of algal growth stimulation by bacterial attenuation of photosynthetic oxygen tension was indirectly examined by simulating the effect of bacteria through a physical removal of oxygen (air suction). Vacuum-treated cultures showed an increase in growth rate and photosynthetic activity as compared to the control, a result which cannot be explained by differences in CO₂/HCO₃⁻ pump activity. In the presence of P. diminuta , the photosynthetic activity of S. bicellularis was more strongly stimulated under a limited concentration of inorganic carbon. It is suggested that, apart from a CO₂ supply, aerobic bacteria can promote algal growth by reducing the photosynthetic oxygen tension within the microenvironment of the algal cells, thereby creating more favorable conditions for optimal photosynthetic algal growth.     

Molecular and culture-dependent analyses revealed similarities in the endophytic bacterial community composition of leaves from three rice (Oryza sativa) varieties

The endophytic bacterial communities of the three most important rice varieties cultivated in Uruguay were compared by a multiphasic approach. Leaves of mature plants grown in field experiments for two consecutive crop seasons were studied. No significant differences were found in the heterotrophic bacterial density for the three varieties. Pantoea ananatis and Pseudomonas syringae constituted 51% of the total of the isolates. These species were always present regardless of the variety or the season. Molecular analysis based on the 16S rRNA gene was performed by terminal restriction fragment length polymorphism (T-RFLP) and cloning. T-RFLP analysis revealed that bacterial communities grouped according to the variety, although the three varieties presented communities that showed 74% or higher similarities. Brevundimonas, the dominant genus in the clone library (18% of the clones), which might be present in all varieties according to T-RFLP profiles, was not recovered by cultivation. Conversely, bacteria from the genus Pseudomonas were not detected in the clone library. These results indicate that communities established in leaves of physiologically different rice varieties were highly similar and composed by a reduced group of strongly associated and persistent bacteria that were partially recovered by cultivation.     

Impact of cultivation on characterisation of species composition of soil bacterial communities

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere. The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.     

胜利油田单12高温油藏非培养和富集培养样品的细菌组成特性

[目的]通过比较分析油藏样品的微生物群落结构特点,认识油藏微生物的生态功能.[方法]利用3种油藏微生物研究中常用的富集培养方法,对胜利油田单12区块S12-4油井产出水样品进行了选择性富集培养,运用构建16S rRNA基因文库的方法分析了富集样品和非培养样品的细菌多样性.[结果]通过16S rRNA基因序列比对发现,非培养样品、异养菌富集样品、烃降解菌富集样品和硫酸盐还原菌富集样品中的优势菌分别为Pseudomonas属,Thermotoga属,Thermaerobacter属和Thermotoga属的成员.多样性分析结果表明,非培养样品的微生物多样性最丰富,同时非培养样品和富集样品的微生物群落结构存在很大的差异,富集样品中的微生物包括优势菌在油藏原位环境中含量很低.[结论]细菌组成差异的比较结果,对油藏微生物的生态功能研究和微生物驱油潜力评估具有重要意义.     

Microbial Diversity in Maras Salterns, a Hypersaline Environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 × 10⁶ to 3 × 10⁶ cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon “Haloquadra walsbyi,” although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the γ-proteobacterium “Pseudomonas halophila” DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the “P. halophila” cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta

Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.     

Using a green fluorescent protein gene-labeled p-nitrophenol-degrading Moraxella strain to examine the protective effect of alginate encapsulation against protozoan grazing

A gfp-labeled p-nitrophenol-degrading Moraxella strain G21 was used to study grazing of a Tetrahymena thermophila strain in liquid medium. This allowed visualization of the feeding process. Under an epifluorescent microscope, individual G21 fluorescent cells could be seen in vacuoles within the protozoans. Most of the G21 cells appeared to be lysed by T. thermophila and green fluorescent protein released from the bacteria yielded brightly fluorescent food vacuoles inside the protozoans, Examination of population dynamics of a mixed culture of T. thermophila and Moraxella sp. G21 showed that the protozoan reduced the bacterial density from 7.6 to 5.8 log CFU/ml in 2 days. Encapsulating the bacteria in alginate prevented grazing by the protozoans and the density of G21 cells in the beads increased steadily from about 8.3 to 8.9 log CFU/ml in 15 days regardless of the presence of the protozoans.     

Identification of a plasmid-borne parathion hydrolase gene from Flavobacterium sp. by southern hybridization with opd from Pseudomonas diminuta.

Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.     

螺旋粉虱成虫体内细菌群落多样性的PCR-DGGE 和16S rRNA文库序列分析

为了解螺旋粉虱Aleurodicus dispersus体内细菌多样性和主要优势菌群结构, 用PCR-DGGE和16S rRNA文库对采自于海南省番石榴上螺旋粉虱雌、 雄成虫体内的细菌群落进行了分析。用PCR扩增体内细菌16S rRNA基因, 构建雌、 雄虫克隆文库; 再用限制性片段长度多态性（restriction fragment length polymorphism, RFLP）方法从文库中筛选不同16S rRNA基因图谱, 根据图谱对克隆子进行分型。从螺旋粉虱雌、 雄两个样品中共获得10 种分类操作单元(operational taxonomic unit, OTUs)。以16S rRNA基因为基础构建系统发育树, 系统发育分析表明, 螺旋粉虱雌、 雄成虫体内优势菌群主要为发酵菌属Zymobacter, 杀雄菌属Arsenophonus, 泛菌属Pantoea和假单胞菌属Pseudomonas。Candidatus Portiera aleyrodidarum和Arsenophonus sp.可能为其体内共生菌群, 在所有样品中均可稳定地检测到。这些微生物可能对螺旋粉虱生长发育、 繁殖和性比调控起到重要的协同作用。     

Role of dissolution rate and solubility in biodegradation of aromatic compounds

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.     

Role of dissolution rate and solubility in biodegradation of aromatic compounds.

Strains of Moraxella sp., Pseudomonas sp., and Flavobacterium sp. able to grow on biphenyl were isolated from sewage. The bacteria produced 2.3 to 4.5 g of protein per mol of biphenyl carbon, and similar protein yields were obtained when the isolates were grown on succinate. Mineralization of biphenyl was exponential during the phase of exponential growth of Moraxella sp. and Pseudomonas sp. In biphenyl-supplemented media, Flavobacterium sp. had one exponential phase of growth apparently at the expense of contaminating dissolved carbon in the solution and a second exponential phase during which it mineralized the hydrocarbon. Phase-contrast microscopy did not show significant numbers of cells of these three species on the surface of the solid substrate as it underwent decomposition. Pseudomonas sp. did not form products that affected the solubility of biphenyl, although its excretions did increase the dissolution rate. It was calculated that Pseudomonas sp. consumed 29 nmol of biphenyl per ml in the 1 h after the end of the exponential phase of growth, but 32 nmol of substrate per ml went into solution in that period when the growth rate had declined. In a medium with anthracene as the sole added carbon source, Flavobacterium sp. converted 90% of the substrate to water-soluble products, and a slow mineralization was detected when the cell numbers were not increasing. Flavobacterium sp. and Beijerinckia sp. initially grew exponentially and then arithmetically in media with phenanthrene as the sole carbon source. Calculations based on the growth rates of these bacteria and the rates of dissolution of phenanthrene suggest that the dissolution rate of the hydrocarbon may limit the rate of its biodegradation.