Qiliang Lai

A pyrene-degrading consortium from deep-sea sediment of the West Pacific and its key member Cycloclasticus sp. P1

A pyrene-degrading bacterial consortium was obtained from deep-sea sediments of the Pacific Ocean. The consortium degraded many kinds of polycyclic aromatic hydrocarbons (PAHs), including naphthalene, phenanthrene, pyrene, acenaphthene, fluorene, anthracene, fluoranthene, 2-methylnaphthalene and 2,6-dimethylnaphthalene, but it did not grow with chrysene and benzo[alpha]pyrene. With methods of plate cultivation and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), 72 bacteria belonging to 22 genera were detected from this consortium. Among the detected bacteria, the following genera frequently occurred: Flavobacterium, Cycloclasticus, Novosphingobium, Halomonas, Achromobacter, Roseovarius and Alcanivorax. The first two genera showed the strongest bands in denaturing gradient gel electrophoresis (DGGE) profiles and appeared in all PAH treatments. By now, only one isolate designated P1 was confirmed to be a pyrene degrader. It was identified to be Cycloclasticus spirillensus (100%). Although P1 can degrade pyrene independently, other bacteria, such as Novosphingobium sp. (Band 14), Halomonas sp. (Band 16) and an unidentified bacterium (Band 35), were involved in pyrene degradation in some way; they persist in the consortium in the test of dilution to extinction if only the consortium was motivated with pyrene. However, the secondary most important member Flavobacterium sp. evaded from the community at high dilutions. As a key member of the consortium, P1 distinguished itself by both cell morphology and carbon source range among the isolates of this genus. Based on intermediate analyses of pyrene degradation, P1 was supposed to take an upper pathway different from that previously reported. Together with the results of obtained genes from P1 homology with those responsible for naphthalene degradation, its degradation to pyrene is supposed to adopt another set of genes unique to presently detected. Summarily, an efficient pyrene-degrading consortium was obtained from the Pacific Ocean sediment, in which Cycloclasticus bacterium played a key role. This is the first report to exploit the diversity of pyrene-degrading bacteria in oceanic environments.

Biodiversity of polycyclic aromatic hydrocarbon-degrading bacteria from deep sea sediments of the Middle Atlantic Ridge

The bacteria involved in the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in deep sea subsurface environments are largely unknown. In order to reveal their biodiversity, sediments from 2.2 m under the bottom surface at a water depth of 3542 m were sampled on the Middle Atlantic Ridge with a gravity column sampler. The sediments were promptly enriched with either crude oil or a mixture of PAHs (naphthalene, phenanthrene and pyrene) as the sole carbon source, and further enriched with the PAH mixture mentioned above in the lab. The resulting consortia were named C2CO and C2PPN respectively. Their bacterial composition was analysed with plate cultivation, PCR-DGGE and 16S rDNA library analysis. On plates, isolates belonging to Pseudoalteromonas, Halomonas, Marinobacter, Thalassospira and Tistrella dominated the culturable populations. With PCR-DGGE, five major bands closely related to Cycloclasticus, Alteromonas, Thalassospira, Alcanivorax and Rhodospirillaceae were detected in consortium C2CO, while only one major band of Cycloclasticus was detected in consortium C2PPN. In addition, the dynamics of community structure in response to aromatic substrate alterations were examined. As a result, three ribotypes of Cycloclasticus were detected by 16S rDNA library analysis, one which played a key role in phenanthrene degradation; two Alteromonas bacteria dominated the naphthalene reselected consortium. Although bacteria of the two genera grew as the main members of the communities, none of them were isolated, probably owing to their poor cultivability. These results confirm that bacteria of Cycloclasticus are important obligate PAH degraders in marine environments, and coexist with other degrading bacteria that inhabit the deep subsurface sediment of the Atlantic. This supports the view that PAH accumulation and bioattenuation occur in remote areas consistently and continuously.

Gene diversity of CYP153A and AlkB alkane hydroxylases in oil‐degrading bacteria isolated from the Atlantic Ocean

Alkane hydroxylases, including the integral-membrane non-haem iron monooxygenase (AlkB) and cytochrome P450 CYP153 family, are key enzymes in bacterial alkane oxidation. Although both genes have been detected in a number of bacteria and environments, knowledge about the diversity of these genes in marine alkane-degrading bacteria is still limited, especially in pelagic areas. In this report, 177 bacterial isolates, comprising 43 genera, were obtained from 18 oil-degrading consortia enriched from surface seawater samples collected from the Atlantic Ocean. Many isolates were confirmed to be the first oil-degraders in their affiliated genera including Brachybacterium, Idiomarina, Leifsonia, Martelella, Kordiimonas, Parvibaculum and Tistrella. Using degenerate PCR primers, alkB and CYP153A P450 genes were surveyed in these bacteria. In total, 82 P450 and 52 alkB gene fragments were obtained from 80 of the isolates. These isolates mainly belonged to Alcanivorax, Bacillus, Erythrobacter, Martelella, Parvibaculum and Salinisphaera, some of which were reported, for the first time, to encode alkane hydroxylases. Phylogenetic analysis showed that both genes were quite diverse and formed several clusters, most of which were generated from various Alcanivorax bacteria. Noticeably, some sequences, such as those from the Salinisphaera genus, were grouped into a distantly related novel cluster. Inspection of the linkage between gene and host revealed that alkB and P450 tend to coexist in Alcanivorax and Salinisphaera, while in all isolates of Parvibaculum, only P450 genes were found, but of multiple homologues. Multiple homologues of alkB mostly cooccurred in Alcanivorax isolates. Conversely, distantly related isolates contained similar or even identical sequences. In summary, various oil-degrading bacteria, which harboured diverse P450 and alkB genes, were found in the surface water of Atlantic Ocean. Our results help to show the diversity of P450 and alkB genes in prokaryotes, and to portray the geographic distribution of oil-degrading bacteria in marine environments.

Genomic insights into the taxonomic status of the Bacillus cereus group.

The identification and phylogenetic relationships of bacteria within the Bacillus cereus group are controversial. This study aimed at determining the taxonomic affiliations of these strains using the whole-genome sequence-based Genome BLAST Distance Phylogeny (GBDP) approach. The GBDP analysis clearly separated 224 strains into 30 clusters, representing eleven known, partially merged species and accordingly 19–20 putative novel species. Additionally, 16S rRNA gene analysis, a novel variant of multi-locus sequence analysis (nMLSA) and screening of virulence genes were performed. The 16S rRNA gene sequence was not sufficient to differentiate the bacteria within this group due to its high conservation. The nMLSA results were consistent with GBDP. Moreover, a fast typing method was proposed using the pycA gene, and where necessary, the ccpA gene. The pXO plasmids and cry genes were widely distributed, suggesting little correlation with the phylogenetic positions of the host bacteria. This might explain why classifications based on virulence characteristics proved unsatisfactory in the past. In summary, this is the first large-scale and systematic study of the taxonomic status of the bacteria within the B. cereus group using whole-genome sequences, and is likely to contribute to further insights into their pathogenicity, phylogeny and adaptation to diverse environments.

Multiple alkane hydroxylase systems in a marine alkane degrader, Alcanivorax dieselolei B-5

Alcanivorax dieselolei strain B-5 is a marine bacterium that can utilize a broad range of n-alkanes (C(5) -C(36) ) as sole carbon source. However, the mechanisms responsible for this trait remain to be established. Here we report on the characterization of four alkane hydroxylases from A. dieselolei, including two homologues of AlkB (AlkB1 and AlkB2), a CYP153 homologue (P450), as well as an AlmA-like (AlmA) alkane hydroxylase. Heterologous expression of alkB1, alkB2, p450 and almA in Pseudomonas putida GPo12 (pGEc47ΔB) or P. fluorescens KOB2Δ1 verified their functions in alkane oxidation. Quantitative real-time RT-PCR analysis showed that these genes could be induced by alkanes ranging from C(8) to C(36) . Notably, the expression of the p450 and almA genes was only upregulated in the presence of medium-chain (C(8) -C(16) ) or long-chain (C(22) -C(36) ) n-alkanes, respectively; while alkB1 and alkB2 responded to both medium- and long-chain n-alkanes (C(12) -C(26) ). Moreover, branched alkanes (pristane and phytane) significantly elevated alkB1 and almA expression levels. Our findings demonstrate that the multiple alkane hydroxylase systems ensure the utilization of substrates of a broad chain length range.

Phylogenetic Diversity of the Bacillus pumilus Group and the Marine Ecotype Revealed by Multilocus Sequence Analysis

Bacteria closely related to Bacillus pumilus cannot be distinguished from such other species as B. safensis, B. stratosphericus, B. altitudinis and B. aerophilus simply by 16S rRNA gene sequence. In this report, 76 marine strains were subjected to phylogenetic analysis based on 7 housekeeping genes to understand the phylogeny and biogeography in comparison with other origins. A phylogenetic tree based on the 7 housekeeping genes concatenated in the order of gyrB-rpoB-pycA-pyrE-mutL-aroE-trpB was constructed and compared with trees based on the single genes. All these trees exhibited a similar topology structure with small variations. Our 79 strains were divided into 6 groups from A to F; Group A was the largest and contained 49 strains close to B. altitudinis. Additional two large groups were presented by B. safensis and B. pumilus respectively. Among the housekeeping genes, gyrB and pyrE showed comparatively better resolution power and may serve as molecular markers to distinguish these closely related strains. Furthermore, a recombinant phylogenetic tree based on the gyrB gene and containing 73 terrestrial and our isolates was constructed to detect the relationship between marine and other sources. The tree clearly showed that the bacteria of marine origin were clustered together in all the large groups. In contrast, the cluster belonging to B. safensis was mainly composed of bacteria of terrestrial origin. Interestingly, nearly all the marine isolates were at the top of the tree, indicating the possibility of the recent divergence of this bacterial group in marine environments. We conclude that B. altitudinis bacteria are the most widely spread of the B. pumilus group in marine environments. In summary, this report provides the first evidence regarding the systematic evolution of this bacterial group, and knowledge of their phylogenetic diversity will help in the understanding of their ecological role and distribution in marine environments.

Oceanibaculum indicum gen. nov., sp. nov., isolated from deep seawater of the Indian Ocean.

A taxonomic study was carried out on strain P24(T), which was isolated from a polycyclic aromatic hydrocarbon-degrading consortium, enriched from a deep-seawater sample collected from the Indian Ocean. The isolate was Gram-negative, rod-shaped, motile by means of a polar flagellum, moderately halophilic and capable of reducing nitrate to nitrite. Growth was observed at salinities of 0-9 % and at temperatures of 10-42 degrees C. The strain was unable to degrade Tween 80 or gelatin. The dominant fatty acids were C(16 : 0) (15.2 % of the total), C(18 : 0) (10.3 %), C(18 : 1)omega7c (52.0 %), C(18 : 1) 2-OH (4.7 %) and C(19 : 0)omega8c cyclo (4.7 %). The G+C content of the chromosomal DNA was 64.8 mol%. 16S rRNA gene sequence comparisons showed that strain P24(T) was related most closely to Thalassobaculum litoreum CL-GR58(T) (92.7 % similarity); levels of similarity between strain P24(T) and type strains of recognized species in the family Rhodospirillaceae were all less than 90.8 %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain P24(T) formed a distinct evolutionary lineage within the family Rhodospirillaceae. Strain P24(T) could be distinguished from phylogenetically related genera based on differences in several phenotypic properties. On the basis of the phenotypic and phylogenetic data presented, strain P24(T) is considered to represent a novel species of a new genus, for which the name Oceanibaculum indicum gen. nov., sp. nov. is proposed. The type strain is P24(T) (=CCTCC AB 208226(T)=LMG 24626(T)=MCCC 1A02083(T)).

Novosphingobium indicum sp nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from a deep-sea environment

A novel polycyclic aromatic hydrocarbon (PAH)-degrading bacterium, strain H25T, which was isolated from deep-sea water of the Indian Ocean, was studied phenotypically, genotypically and phylogenetically. Strain H25T can utilize several PAHs including phenanthrene and fluoranthene as sole carbon sources. The 16S rRNA gene sequence of strain H25T showed the highest similarity with that of Novosphingobium naphthalenivorans TUT562T (96.3%), and showed lower similarities (92.1-96.0%) with other members of the genus Novosphingobium. The major fatty acids of strain H25T were C14:0 2-OH (3.2%), C16:0 (13.6%), C16:1omega7c (5.2%), C18:0 (13.4%) and C18:1omega7c (57.0%), which accounted for 92.3% of the total fatty acids. It had ubiquinone 10 as the major respiratory quinone and spermidine as the major polyamine. All these characteristics were consistent with those of recognized Novosphingobium species. Results of DNA-DNA hybridization experiments and BOX-PCR fingerprint comparisons also indicate that strain H25T represents a novel Novosphingobium species, for which the name Novosphingobium indicum sp. nov. is proposed. The type strain is H25T (=MCCC 1A01080T=CGMCC 1.6784T=LMG 24713T).

Marispirillum indicum gen. nov., sp. nov., isolated from a deep-sea environment

A taxonomic study was carried out on strain B142(T), which was isolated from a crude-oil-degrading microbial consortium via enrichment with deep water from the Indian Ocean. Cells of the isolate were Gram-negative, oxidase-negative, catalase-positive, helical in shape, motile by means of polar flagella (three per cell) and moderately halophilic. Growth was observed at salinities of 0.5-12 % and at temperatures of 10-41 degrees C. The micro-organism was capable of denitrification, but was unable to degrade Tween 80 or gelatin. The predominant fatty acids were C(16 : 1)omega7c and/or iso-C(15 :0 )2-OH (6.4 %), C(16 : 0) (15.7 %), C(18 : 1)omega7c (45 %), C(18 : 0) (6.8 %) and C(19 : 0)omega8c cyclo (6.7 %). The G+C content of the chromosomal DNA was 67.3 mol%. Comparisons of 16S rRNA gene sequences showed that strain B142(T) was most closely related to the type strains of two Insolitispirillum peregrinum subspecies (93.0-93.1 % sequence similarity), two Novispirillum itersonii subspecies (92.8-92.9 %) and Caenispirillum bisanense (91.7 %); sequence similarities with respect to other taxa were below 90.5 %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain B142(T) formed a distinct evolutionary lineage within the family Rhodospirillaceae. Strain B142(T) was distinguishable from phylogenetically related genera with regard to several phenotypic properties. On the basis of phenotypic and phylogenetic data, therefore, strain B142(T) represents a novel genus and species, for which the name Marispirillum indicum gen. nov., sp. nov. is proposed. The type strain is B142(T) (=CCTCC AB 208225(T)=LMG 24627(T)=MCCC 1A01235(T)).

Flavobacterium beibuense sp. nov., isolated from marine sediment

A taxonomic study was carried out on strain F44-8(T), which was isolated from a crude-oil-degrading consortium, enriched from marine sediment of the Beibu Gulf, PR China. The 16S rRNA gene sequence of strain F44-8(T) showed highest similarities to those of Flavobacterium frigoris LMG 21922(T) (93.3 %), Flavobacterium terrae R2A1-13(T) (93.3 %) and Flavobacterium gelidilacus LMG 21477(T) (93.1 %). Sequence similarities to other members of the genus Flavobacterium were <93.0 %. The dominant fatty acids of strain F44-8(T) were iso-C(15 : 0), summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c), iso-C(15 : 1) G and iso-C(17 : 0) 3-OH. The DNA G+C content of strain F44-8(T) was 38.6 mol%. These results are consistent with characteristics of members of the genus Flavobacterium. Strain F44-8(T) could, however, be readily distinguished from all known Flavobacterium species by a number of phenotypic features. Therefore, according to the phenotypic and 16S rRNA gene sequence data, strain F44-8(T) represents a novel species in the genus Flavobacterium, for which the name Flavobacterium beibuense sp. nov. is proposed (type strain F44-8(T) =CCTCC AB 209067(T) =LMG 25233(T) =MCCC 1A02877(T)).