Raceway cultivation of Spirulina platensis using underground water

The semi-outdoor cultivation of Spirulina platensis was attempted using an underground-water-based medium. Occurrence of contaminant organisms such as Chlorella sp. and Chlamydomonas sp. was not found from a microscopic observation and bacteria were not detected from denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA during the cultivation, owing to pH control and the high quality of the underground water. The mean productivity was high at 10.5 g/m2/d with a range of 4.2-12.3 g/m2/d despite the unfavorable weather conditions of the rainy season. The cultivated S. platensis included a normal protein content of 58.9%. Consequently, the underground water improved the biomass productivity and the biomass quality because of an abundant supplementation of natural minerals and through a contaminant-free culture.     

豆豉纤溶酶产生菌分离和鉴定

从全国各地收集豆豉样品,采用不同的培养基进行富集培养,并利用纤维蛋白平板法高效获得了13株形态差异较大的产纤溶酶菌株。通过传统方法、化学方法以及16S rRNA序列分析对这13株菌进行分类鉴定,它们分属于芽孢杆菌属、链霉菌属、假单胞菌属以及节杆菌属,包括9种细菌,丰富了豆豉纤溶酶产生菌菌种资源。     

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.     

Evaluation of candidate probiotic strains for gilthead sea bream larvae (Sparus aurata) using an in vivo approach

AIMS: The aim of this study was to evaluate the effect of six bacterial strains on gilthead sea bream larvae (Sparus aurata). METHODS AND RESULTS: Six bacterial strains isolated from well-performing live food cultures were identified by sequencing fragments of their 16s rDNA genome to the genus level as Cytophaga sp., Roseobacter sp., Ruergeria sp., Paracoccus sp., Aeromonas sp. and Shewanella sp. Survival rates of gilthead sea bream larvae transferred to seawater added these bacterial strains at concentrations of 6 +/- 0.3 x 10(5) bacteria ml(-1) were similar to those of larvae transferred to sterilized seawater and showed an average of 86% at 9 days after hatching, whereas, survival rates of larvae transferred to filtered seawater were lower (P < 0.05), and showed an average of 39%, 9 days after hatching. CONCLUSION: Several bacterial strains isolated from well-performing live food cultures showed a positive effect for sea bream larvae when compared with filtered seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach used in this study could be applied as an in vivo evaluation method of candidate probiotic strains used in the rearing of marine fish larvae.     

Phylogeny and diversity of Bradyrhizobium strains isolated from the root nodules of peanut (Arachis hypogaea) in Sichuan, China.

Twenty-two rhizobial strains isolated from the root nodules of two Chinese peanut cultivars (Arachis hypogaea L. Tianfu no. 3 and a local cultivar) growing at four different sites in the Sichuan province, Southwest China, were characterized by growth rate, rep-PCR, PCR-RFLP of 16S rDNA, partial sequencing of ribosomal genes, and fatty acid-methyl ester analysis (FAME), and compared with strains representing Bradyrhizobium japanicum, B. elkanii and other unclassified Bradyrhizobium sp. All peanut isolates from Sichuan were bradyrhizobia. Dendrograms constructed using the rep-PCR fingerprints grouped the strains mainly according to their geographic and cultivar origin. Based on PCR-RFLP and partial sequence analysis of 16S rDNA it appears that peanut bradyrhizobial strains from Sichuan are similar to peanut strains from Africa and Israel, and closely related to B. japonicum. In contrast, analysis of FAME data using two-dimensional principal component analysis indicated that Bradyrhizobium sp. (Arachis) were similar to, but slightly different from other bradyrhizobia. The presence and level of fatty acid 16:1 w5c was the distinguishing feature. The results of PCR-RFLP of the 16S rRNA gene, the partial sequence analysis of 16S rDNA, and FAME were in good agreement.     

Differences Between Bacterial Communities Associated with the Surface or Tissue of Mediterranean Sponge Species

Bacterial communities associated with the surfaces of several Mediterranean sponge species (Agelas oroides, Chondrosia reniformis, Petrosia ficiformis, Geodia sp., Tethya sp., Axinella polypoides, Dysidea avara, and Oscarella lobularis) were compared to those associated with the mesohyl of sponges and other animate or inanimate reference surfaces as well as with those from bulk seawater. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified bacterial 16S ribosomal RNA genes obtained from the surfaces and tissues of these sponges demonstrated that the bacterial communities were generally different from each other. The bacterial communities from sponges were different from those on reference surfaces or from bulk seawater. Additionally, clear distinctions in 16S rDNA fingerprint patterns between the bacterial communities from mesohyl samples of "high-microbial abundance (HMA) sponges" and "low-microbial abundance sponges" were revealed by DGGE and cluster analysis. A dominant occurrence of particularly GC-rich 16S ribosomal DNA (rDNA) fragments was found only in the DGGE banding pattern obtained from the mesohyl of HMA sponges. Furthermore, sequencing analysis of 16S rDNA fragments obtained from mesohyl samples of HMA sponges revealed a dominant occurrence of sponge-associated bacteria. The bacterial communities within the mesohyl of HMA sponges showed a close relationship to each other and seem to be sponge-specific.     

抗菌和细胞毒活性海洋细菌的筛选及其次生代谢基因证据

从不同海域的海水、海泥和海洋生物中分离海洋细菌,利用琼脂扩散法和MTT法对细菌培养液的乙酸乙酯提取物进行了抗菌和细胞毒活性筛选,并对具有细胞毒活性的细菌菌株进行了16SrRNA系统发生学分析和多聚酮合酶(PKSⅠ型)、非核糖体肽合成酶(NRPS)的筛选。结果显示,在分离到的346株海洋细菌中,42株细菌具有抗菌活性,12株具有细胞毒活性。对12株具有细胞毒活性的细菌菌株进行了16SrRNA系统发生学分析,它们分别属于Agrobacterium,Pseudoalteromons,Bacillus,Paracoccus,Rheinheimera,Aerococcus,Exiguobacterium和Alteromonas8个属。对这12株具有细胞毒活性的细菌菌株进行进一步的PKS和NRPS筛选,得到了4株含有PKSⅠ型的KS结构域或NPRS的A结构域的海洋细菌,为从聚酮和非核糖体肽等生物合成途径去深入研究其次生代谢产物提供了基因学的证据。     

Phylogenetic analysis of aerobic freshwater and marine enrichment cultures efficient in hydrocarbon degradation: effect of profiling method

Aerobically grown enrichment cultures derived from hydrocarbon-contaminated seawater and freshwater sediments were generated by growth on crude oil as sole carbon source. Both cultures displayed a high rate of degradation for a wide range of hydrocarbon compounds. The bacterial species composition of these cultures was investigated by PCR of the 16S rDNA gene using multiple primer combinations. Near full-length 16S rDNA clone libraries were generated and screened by restriction analysis prior to sequence analysis. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out using two other PCR primer sets targeting either the V3 or V6-V8 regions, and sequences derived from prominent DGGE bands were compared to sequences obtained via cloning. All data sets suggested that the seawater culture was dominated by alpha-subgroup proteobacteria, whereas the freshwater culture was dominated by members of the beta- and gamma-proteobacteria. However, the V6-V8 primer pair was deficient in the recovery of Sphingomonas-like 16S rDNA due to a 3' terminal mismatch with the reverse primer. Most 16S rDNA sequences recovered from the marine enrichment were not closely related to genera containing known oil-degrading organisms, although some were detected. All methods suggested that the freshwater enrichment was dominated by genera containing known hydrocarbon-degrading species.     

Isolation of marine bacteria with antimicrobial activities from cultured and field-collected soft corals

Bacteria associated with eight field-collected and five cultured soft corals of Briareum sp., Sinularia sp., Sarcophyton sp., Nephtheidae sp., and Lobophytum sp. were screened for their abilities in producing antimicrobial metabolites. Field-collected coral samples were collected from Nanwan Bay in southern Taiwan. Cultured corals were collected from the cultivating tank at National Museum of Marine Biology and Aquarium. A total of 1,526 and 1,138 culturable, heterotrophic bacteria were isolated from wild and cultured corals, respectively; seawater requirement and antimicrobial activity were then assessed. There is no significant difference between the ratio of seawater-requiring bacteria on the wild and cultured corals. The ratio of antibiotic-producing bacteria within the seawater-requiring bacteria did not differ between the corals. Nineteen bacterial strains that showed high antimicrobial activity were selected for 16S rDNA sequencing. Three strains could be assigned at the family level (Rhodobacteraceae). The remaining 16 strains belong to eight genera: Marinobacterium (2 strains), Pseudoalteromonas (1), Vibrio (5), Enterovibrio (1), Tateyamaria (1), Labrenzia (2), and Pseudovibrio (4). The crude extract from bacteria strains CGH2XX was found to have high cytotoxicity against the cancer cell line HL-60 (IC₅₀?=?0.94???g/ml) and CCRF-CEM (IC₅₀?=?1.19???g/ml). Our results demonstrate that the marine bacteria from corals have great potential in the discovery of useful medical molecules.     

A molecular biological protocol to distinguish potentially human pathogenic Stenotrophomonas maltophilia from plant-associated Stenotrophomonas rhizophila

In recent years, the importance of the Gram-negative bacterium Stenotrophomonas as an opportunistic pathogen as well as in biotechnology has increased. The aim of the present study was to develop new methods for distinguishing between strains closely related to the potentially human pathogenic Stenotrophomonas maltophilia and those closely related to the plant-associated Stenotrophomonas rhizophila. To accomplish this, 58 strains were characterized by 16S rDNA sequencing and amplified ribosomal DNA restriction analysis (ARDRA), and the occurrence of specific functional genes. Based on 16S rDNA sequences, an ARDRA protocol was developed which allowed differentiation between strains of the S. maltophilia and the S. rhizophila group. As it was known that only salt-treated cells of S. rhizophila were able to synthesize the compatible solute glucosylglycerol (GG), the ggpS gene responsible for GG synthesis was used for differentiation between both species and it was confirmed that it only occurred in S. rhizophila strains. As a further genetic marker the smeD gene, which is part of the genes coding for the multidrug efflux pump SmeDEF from S. maltophilia, was used. Based on the results we propose a combination of fingerprinting techniques using the 16S rDNA and the functional genes ggpS and smeD to distinguish both Stenotrophomonas species.     

Isolation and molecular characterization of thiosulfate-oxidizing bacteria from an Italian rice field soil

In rice paddy soils an active cycling of sulfur compounds takes place. To elucidate the diversity of thiosulfate-oxidizing bacteria these organisms were enriched from bulk soil and rice roots by the most probable number method in liquid medium. From the MPN enrichment cultures 21 bacterial strains were isolated on solid mineral medium, and could be further shown to produce sulfate from thiosulfate. These strains were characterized by 16S rDNA analyses. The isolates were affiliated to seven different phylogenetic groups within the alpha- and beta-subclass of Proteobacteria. Two of these phylotypes were already described as S-oxidizers in this environment (Xanthobacter sp. and Bosea sp. related strains), but five groups represented new S-oxidizers in rice field soil. These isolates were closely related to Mesorhizobium loti, to Hydrogenophaga sp., to Delftia sp., to Pandoraea sp. or showed sequence similarity to a strain of Achromobacter sp.     

The phylogenetic relationships of cyanobacteria inferred from 16S rRNA,gyrB, rpoC1 and rpoD1 gene sequences

Phylogenetic analysis of cyanobacteria was carried out using the small subunit rRNA (16S rRNA), DNA gyrase subunit B (gyrB), DNA-dependent RNA polymerase gamma subunit (rpoC1) and a principal sigma factor of E. coli sigma(70) type for DNA-dependent RNA polymerase (rpoD1) gene sequences of 24 strains which contained 5 subgroups of cyanobacteria-3 strains of the Chroococcales, 5 strains of the Pluerocapsales, 7 strains of the Oscillatoriales, 7 strains of the Nostocales and 2 strains of the Stigonematales. Degenerated PCR primers of gyrB, rpoC1 and rpoD1 genes were designed using consensus amino acid sequences registered in GenBank. The phylogenetic positions of cyanobacteria were resolved through phylogenetic analysis based on 16S rDNA, gyrB, rpoC1 and rpoD1 gene sequences. Phylogenies of gyrB, rpoC1 and rpoD1 support 16S rRNA-based classification of cyanobacteria. Interestingly, phylogenies from amino acid sequences deduced from gyrB and combined amino acid sequences deduced from rpoC1 and rpoD1 genes strongly support that of 16S rRNA, but the branching pattens of the trees based on 16S rDNA, GyrB, rpoC1, rpoD1 and combined amino acid sequences deduced from rpoC1 and rpoD1 were not congruent. In this study, we showed the correlation among phylogenetic relationships of 16S rDNA, gyrB, rpoC1 and rpoD1 genes. The phylogenetic trees based on the sequences of 16S rDNA, GyrB, rpoC1, rpoD1 and the combined amino acid sequences deduced from rpoC1 and rpoD1 showed that the lateral gene transfer of rRNA might be suspected for Synechocystis sp. PCC 6803.     

Disruption of a gene essential for sulfoquinovosyldiacylglycerol biosynthesis in Sinorhizobium meliloti has no detectable effect on root nodule symbiosis

The sulfolipid sulfoquinovosyldiacylglycerol is commonly found in the thylakoid membranes of photosynthetic bacteria and plants. While there is a good correlation between the occurrence of sulfolipid and photosynthesis, a number of exceptions are known. Most recently, sulfoquinovosyldiacylglycerol was discovered in the non-photosynthetic, root nodule-forming bacterium Sinorhizobium meliloti. This discovery raised the questions of the phylogenetic origin of genes essential for the biosynthesis of this lipid in S. meliloti and of a function of sulfolipid in root nodule symbiosis. To begin to answer these questions, we isolated and inactivated the sqdB gene of S. meliloti. This gene and two other genes located directly 3' of sqdB are highly similar to the sqdB, sqdC, and sqdD genes known to be essential for sulfolipid biosynthesis in the photosynthetic, purple bacterium Rhodobacter sphaeroides. This observation confirms the close phylogenetic kinship between these two species. Furthermore, the reduced similarity of sqdB to the plant ortholog SQD1 of Arabidopsis thaliana does not support a previous sqd gene transfer from the plant as a consequence of close symbiosis. A sulfolipid-deficient mutant of S. meliloti disrupted in sqdB is capable of inducing functional nodules and does not show an obvious disadvantage under different laboratory culture conditions. Thus far, no specific function can be assigned to bacterial sulfolipid, in either nodule-associated or free-living cells. S. meliloti contains a rich set of polar membrane lipids some of which, including sulfolipid, may become critical only under growth conditions that still need to be discovered.     

Molecular characterization of the genes encoding acetohydroxy acid synthase in the cyanobacterium Spirulina platensis.

The enzyme acetohydroxy acid synthase (AHS), which catalyses the first common step in the biosynthesis of isoleucine, leucine and valine, has been demonstrated to be present in Spirulina platensis in two isoenzymic forms. The complete nucleotide sequences of the genes ilvX and ilvW encoding these two enzymes have been determined. Sequence analysis revealed the presence of two open reading frames, of 1836 and 1737 nucleotides for ilvX and ilvW, respectively. The predicted amino acid sequences of the two isoenzymes, compared with the Synechococcus PCC 7942 AHS enzyme and the large subunits of the Escherichia coli AHSI, II, III isoenzymes, revealed a notable degree of similarity. A small subunit has not been identified for either of the S. platensis AHS isoenzymes. Analysis by Northern blot hybridization demonstrated that the ilvX and ilvW genes are transcribed to give mRNA species of approximately 2.15 kb and 1.95 kb, respectively.     

金沙江干热河谷区田菁根瘤菌多样性与系统发育

[目的]揭示金沙江干热河谷区这一特殊地理环境下田菁根瘤菌的多样性和系统发育地位. [方法]采用了数值分类、16S rDNA PCR-RFLP、16S rDNA和GSⅡ序列分析方法.[结果]数值分类结果表明:在93%的水平上,待测菌株分布于6个群,其中4个群分别与R.tropici、 R.etli、 S.saheli、A.rubi的参比菌株聚在一起,两个群没有参比菌株与之聚群.16S rDNA PCR-RFLP结果与数值分类基本一致,只有两个独立群有所差异.16S rDNA序列分析表明:两独立群中心菌株SCAU176 和SCAU144与R.huautlense聚在一起,与该种同源性分别为100%和98.9%.GSⅡ序列分析中SCAU176 和SCAU144单独聚在一起,与最近的参比菌株R.tropici的同源性系数在90%以下.[结论]金沙江干热河谷区田菁根瘤菌具有较为丰富的多样性,在系统发育地位上分布于Sinorhizobium、Agrobacterium和Rhizobium三个属.     

香蕉炭疽病拮抗细菌的分离和初步鉴定

对36株从药用植物蛇足石杉中获得内生细菌进行香蕉炭疽病拮抗细菌筛选。采用对峙培养筛选法、碱基序列比对及同源性分析进行研究。结果获得2株对香蕉炭疽病有拮抗作用的菌株,其中JXS1-6和AHL1-1菌株的抑菌带宽达9 mm及4 mm。通过16S rDNA碱基序列比对及同源性分析,JXS1-6和AHL1-1均鉴定为伯克霍尔德属(Burkholderia),属于伯克霍尔德科(Burkholderiaceae),伯克霍尔德目(Burkholderiales),β-变形菌纲(Betaproteobacteria)。     

微量元素锗对四种微藻光合色素的影响

采用溶液培养方法,以锗作为胁迫因子,研究了锗对４种微藻“钝顶螺旋藻（Ｓｐｉｒｕｌｉｎａ ｐｌａｔｅｎｓｉｓ）、盐生杜氏藻（Ｄｕｎａｌｉｅｌｌａ ｓａｌｉｎａ）、淇江叉鞭金藻（Ｄｉｃｒａｔｅｒｉａ ｚｈａｎｊｉａｎｇｅｎｓｉｓ）和微绿藻（Ｎａｎｎｏｃｈｌｏｒｏｐｓｉｓ．ｓｐ）光合色素的影响。实验结果表明,经１０ｍｇ／ｄｍ＾３锗处理后,四种藻类细胞中的光合色素都发生了较大变化,有些色素发生明显的增加或     

Phenotypic and genotypic diversity of rhizobia nodulating Pterocarpus erinaceus and P. lucens in Senegal

A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus ennaceus and Pterocarpus lucens respectively native of sudanean and sahelian regions of Senegal were characterized. These isolates were compared to representative strains of known rhizobial species. Twenty-two new isolates were slow growers and twenty-eight were fast growers. A polyphasic approach was performed including comparative total protein sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profile analysis; 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) sequence analysis. By SDS-PAGE the slow growing isolates grouped in one major cluster containing reference strains of Bradyrhizobium sp. including strains isolated in Africa, in Brazil and in New Zealand. Most of the fast-growing rhizobia grouped in four different clusters or were separate strains related to Rhizobium and Mesorhizobium strains. The 16S rDNA and 16S-23S rDNA IGS sequences analysis showed accurately the differentiation of fast growing rhizobia among the Rhizobium and Mesorbizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrbizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrbizobium sp. (Lupinus) and Blastobacter denitrificans. This position of ORS 199 was not confirmed by IGS sequence divergence. We found no clear relation between the diversity of strains, the host plants and the ecogeographical origins.     

Occurrence of tetracycline resistance genes tet(M) and tet(S) in bacteria from marine aquaculture sites

Occurrence of tetracycline resistance genes encoding ribosomal protection proteins was examined in 151 tetracycline-resistant bacterial isolates from fish and seawater at coastal aquaculture sites in Japan and Korea. The tet(M) gene was detected in 34 Japanese and Korean isolates, which included Vibrio sp., Lactococcus garvieae, Photobacterium damsela subsp. piscicida, and unidentified Gram-positive bacteria. The majority of these bacterial isolates displayed high-level resistance with a minimum inhibitory concentrations (MICs) equal to or greater than 250 microg/ml of oxytetracycline and only four isolates had MICs less than 31.3 microg/ml. 16S rDNA RFLP typing of tet(M)-positive Vibrio isolates suggests that these are clonal populations of the same phylotype specific to a particular location. One Vibrio clone (phylotype III), however, is widely disseminated, being detected during different sampling years, at different locations, and in different fish species in both Japan and Korea. The tet(S) gene was detected in L. garvieae from yellowtail in Japan and in Vibrio sp. from seawater in Korea. This is the first report of tet(S) occurrence in Gram-negative facultative anaerobes. These results suggest that tet(M) and tet(S) genes are present in fish intestinal and seawater bacteria at aquaculture sites and could be an important reservoir of tetracycline resistance genes in the marine environment.