Diversity and distribution of microbial long-chain fatty acid biosynthetic genes in the marine environment

Bacterial production of long-chain fatty acids via a polyketide synthase-related mechanism has thus far only been investigated in isolate-based studies. Here, the genetic capacity for production of long-chain fatty acids was investigated using a culture-independent approach. PCR primers targeting the keto-acyl synthase (KS) domain of the pfaA gene involved in omega-3 polyunsaturated fatty acid (PUFA) biosynthesis were used to construct clone libraries to investigate KS sequence diversity in disparate marine habitats. Of the 446 sequences recovered, 123 (27.6%) clustered with KS sequences involved in the synthesis of eicosapentaenoic acid (EPA, C20:5n-3), docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6). The remaining 72.4% of clones formed environmental-only groups or grouped with the KS domains of pfaA homologues from organisms producing unidentified products. In total, 17 groups were recovered - four known and 13 newly identified. A query of metagenomic data sets revealed sequences related to EPA KS domains, as well as sequences related to four environmental-only groups discovered in the clone libraries. The phylogenetic affiliation and end product of these environmental-only KS clusters is unknown. These findings reveal a widespread capacity for long-chain fatty acid production in marine microorganisms, including biosynthetic pathways not yet characterized.     

Diversity and detection of nitrate assimilation genes in marine bacteria.

A PCR approach was used to construct a database of nasA genes (called narB genes in cyanobacteria) and to detect the genetic potential for heterotrophic bacterial nitrate utilization in marine environments. A nasA-specific PCR primer set that could be used to selectively amplify the nasA gene from heterotrophic bacteria was designed. Using seawater DNA extracts obtained from microbial communities in the South Atlantic Bight, the Barents Sea, and the North Pacific Gyre, we PCR amplified and sequenced nasA genes. Our results indicate that several groups of heterotrophic bacterial nasA genes are common and widely distributed in oceanic environments.     

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria

Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the beta- and the gamma-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2-3) nearly identical copies in the beta-subdivision representatives but in single copies in the gamma-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the beta-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.     

Diversity of ammonia monooxygenase (amoA) genes across environmental gradients in Chesapeake Bay sediments

Chesapeake Bay, the largest estuary in North America, encompasses a wide range of nutrient loading and trophic levels from the rivers and upper Bay to the sea, providing an ideal natural environment in which to explore relationships between functional diversity, physical/chemical complexity and ecosystem function (e.g. nitrification). In this study, amoA gene fragments (encoding subunit A of the key nitrification enzyme, ammonia monooxygenase) were PCR‐amplified from DNA extracted from sediment cores collected at five stations spanning gradients of salinity, ammonium, nitrate, oxygen and organic carbon along the Bay and Choptank River, a subestuary of the Bay. Phylogenetic analysis of ～30 amoA clones from each station revealed extensive diversity within the β‐Proteobacteria group of ammonia‐oxidizing bacteria (AOB), with the vast majority of sequences falling into coherent phylogenetic clusters distinct from sequences of cultivated AOB. Over 70% of the clones fell into two major phylogenetic clusters that appear to represent novel groups of Nitrosomonas‐like and Nitrosospira‐like amoA sequences that may be specific to estuarine and marine environments. Rarefaction analysis, estimators of genetic variation and dissimilarity indices all revealed differences in the relative amoA‐based diversity and/or richness among most of the stations, with the highest diversity at the North Bay station and the lowest at the mesohaline stations. Although salinity appears to play a role, no single physical or chemical parameter entirely explains the pattern of diversity along the estuary, suggesting that a complex combination of environmental factors may shape the overall level of AOB diversity in this dynamic environment.     

Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes

Eighteen 4-t-octylphenol-degrading bacteria were isolated and screened for the presence of degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from multicomponent phenol hydroxylase, single component monooxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Seventeen of the 18 isolates exhibited the presence of a 232 bp amplicon that shared 61-92% identity to known multicomponent phenol hydroxylase gene sequences from short and/or medium-chain alkylphenol-degrading strains. Twelve of the 18 isolates were positive for a 324 bp region that exhibited 78-95% identity to the closest published catechol 1,2-dioxygenase gene sequences. The two strains, Pseudomonas putida TX2 and Pseudomonas sp. TX1, contained catechol 1,2-dioxygenase genes also have catechol 2,3-dioxygenase genes. Our result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.     

Isolation and characterization of marine bacteria capable of utilizing phthalate

Eleven phthalate-degrading bacterial strains were isolated from seawater collected off the coast of Japan. The isolates were found to be most closely related to the marine bacterial genera Alteromonas, Citreicella, Marinomonas, Marinovum, Pelagibaca, Rhodovulum, Sulfitobacter, Thalassobius, Thalassococcus, Thalassospira, and Tropicibacter. For the first time, members of these genera were shown to be capable of growth on phthalate. The plate assay for visual detection of phthalate dioxygenase activity and PCR detection of a possible gene encoding 4,5-dihydroxyphthalate decarboxylase indicated that phthalate is degraded via 4,5-dihydroxyphthalate to protocatechuate in all the isolates.     

石油中长链烷烃微生物降解及分子机制研究进展

中长链烷烃是石油烃中的重要组成部分,由于其疏水性强、黏度大、化学活性低、难降解,是地下原油黏度大、石油采收率低、泄漏后长期污染生态环境的重要原因,因此成为提高石油采收率和石油污染环境治理中的重要降解目标。微生物降解中长链烷烃作为一种新型高效的绿色技术日益受到重视。本文总结了微生物降解中长链烷烃的间期适应与转运过程,与转运过程相关的膜蛋白,微生物好氧与厌氧降解的代谢途径,以及好氧降解过程中的基因调控机制,并对微生物降解中长链烷烃的研究方向提出了展望,以期为后续的相关研究工作提供参考。     

Diversity in surface colonization behavior in marine bacteria

Using laminar flow chambers and time-lapse video imaging, colonization of surfaces by four marine bacteria revealed a diverse range of morphological characteristics and cell-cell interactions. The strain SW5 formed a compact, multilayered single- and double-cell biofilm on hydrophobic surfaces but developed long multicellular chains on hydrophilic surfaces. The morphologically similar SW8 showed unusual proximal vertical packing of cells on both substrata.Vibrio sp strain S14 exhibited cyclical colonization-detachment events on both substrata.Pseudomonas sp strain S9 initially displayed reversible and then irreversible adhesion apparently triggered by a cell density phenomenon that led to the development of regular microcolonies on both substrata with individual cells translocating between the colonies. The length of time bacteria were exposed to and their density at a surface influenced behavioral traits, with diverse and distinctive species-specific behavioral events.     

Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme FMN: unveiling the long-chain alkane hydroxylase

LadA, a long-chain alkane monooxygenase, utilizes a terminal oxidation pathway for the conversion of long-chain alkanes (up to at least C₃₆) to corresponding primary alcohols in thermophilic bacillus Geobacillus thermodenitrificans NG80-2. Here, we report the first structure of the long-chain alkane hydroxylase, LadA, and its complex with the flavin mononucleotide (FMN) coenzyme. LadA is characterized as a new member of the SsuD subfamily of the bacterial luciferase family via a surprising structural relationship. The LadA:FMN binary complex structure and a LadA:FMN:alkane model reveal a hydrophobic cavity that has dual roles: to provide a hydrogen-bond donor (His138) for catalysis and to create a solvent-free environment in which to stabilize the C4a-hydroperoxyflavin intermediate. Consequently, LadA should catalyze the conversion of long-chain alkanes via the acknowledged flavoprotein monooxygenase mechanism. This finding suggests that the ability of LadA to catalyze the degradation of long-chain alkanes is determined by the binding mode of the long-chain alkane substrates. The LadA structure opens a rational perspective to explore and alter the substrate binding site of LadA, with potential biotechnological applications in areas such as petroleum exploration and treatment of environmental oil pollution.     

Diversity,abundance and expression of nitrite reductase (nirK)-like genes in marine thaumarchaea

Ammonia-oxidizing archaea (AOA) are widespread and abundant in aquatic and terrestrial habitats and appear to have a significant impact on the global nitrogen cycle. Like the ammonia-oxidizing bacteria, AOA encode a gene homologous to copper-containing nitrite reductases (nirK), which has been studied very little to date. In this study, the diversity, abundance and expression of thaumarchaeal nirK genes from coastal and marine environments were investigated using two mutually excluding primer pairs, which amplify the nirK variants designated as AnirKa and AnirKb. Only the AnirKa variant could be detected in sediment samples from San Francisco Bay and these sequences grouped with the nirK from Candidatus Nitrosopumilus maritimus and Candidatus Nitrosoarchaeum limnia. The two nirK variants had contrasting distributions in the water column in Monterey Bay and the California Current. AnirKa was more abundant in the epi- to mesopelagic Monterey Bay water column, whereas AnirKb was more abundant in the meso- to bathypelagic California Current water. The abundance and community composition of AnirKb, but not AnirKa, followed that of thaumarchaeal amoA, suggesting that either AnirKa is not exclusively associated with AOA or that commonly used amoA primers may be missing a significant fraction of AOA diversity in the epipelagic. Interestingly, thaumarchaeal nirK was expressed 10–100-fold more than amoA in Monterey Bay. Overall, this study provides valuable new insights into the distribution, diversity, abundance and expression of this alternative molecular marker for AOA in the ocean.     

Diversity and distribution of pigmented heterotrophic bacteria in marine environments

A systematic investigation of marine pigmented heterotrophic bacteria (PHB) based on the cultivation method and sequencing analysis of 16S rRNA genes was conducted in Chinese coastal and shelf waters and the Pacific Ocean. Both the abundance of PHB and the ratio of PHB to CFU decreased along trophic gradients from coastal to oceanic waters, with the highest values of 9.9 x 10(3) cell mL(-1) and 39.6%, respectively, in the Yangtze River Estuary. In contrast to the total heterotrophic bacteria (TB) and CFU, which were present in the whole water column, PHB were primarily confined to the euphotic zone, with the highest abundance of PHB and ratio of PHB to CFU occurring in surface water. In total, 247 pigmented isolates were obtained during this study, and the phylogenetic analysis showed a wide genetic diversity covering 25 genera of six phylogenetic classes: Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacilli, Flavobacteria and Sphingobacteria. PHB belonging to Alphaproteobacteria, Flavobacteria and Sphingobacteria were obtained mainly from the South China Sea and East China Sea; PHB from the Pacific Ocean water were predominantly affiliated with Gammaproteobacteria, and most isolates from the Yangtze River Estuary fell into the classes Actinobacteria and Bacilli. The isolates exhibited various colours (e.g. golden, yellow, red, pink and orange), with genus or species specificity. Furthermore, the pigment of PHB cells absorbed light mainly in the wavelength range between 450 and 550 nm. In conclusion, our work has revealed that PHB with broad genetic diversity are widely distributed in the marine environment, and may account for up to 39.6% of culturable bacteria, equivalent to 1.4% of the total microbial community. This value might even be underestimated because it is probable that not all pigmented bacteria were isolated. Their abundance and genetic distribution are heavily influenced by environmental properties, such as light and nutrition, suggesting that they have important roles in the marine ecosystem, especially in the absorption of visible light.     

Anaerobic degradation of polycyclic aromatic hydrocarbons and alkanes in petroleum-contaminated marine harbor sediments.

Although polycyclic aromatic hydrocarbons (PAHs) have usually been found to persist under strict anaerobic conditions, in a previous study an unusual site was found in San Diego Bay in which two PAHs, naphthalene and phenanthrene, were oxidized to carbon dioxide under sulfate-reducing conditions. Further investigations with these sediments revealed that methylnaphthalene, fluorene, and fluoranthene were also anaerobically oxidized to carbon dioxide in these sediments, while pyrene and benzo[a]pyrene were not. Studies with naphthalene indicated that PAH oxidation was sulfate dependent. Incubating the sediments with additional naphthalene for 1 month resulted in a significant increase in the oxidation of [14C]naphthalene. In sediments from a less heavily contaminated site in San diego Bay where PAHs were not readily degraded, naphthalene degradation could be stimulated through inoculation with active PAH-degrading sediments from the most heavily contaminated site. Sediments from the less heavily contaminated site that had been adapted for rapid anaerobic degradation of high concentrations of benzene did not oxidize naphthalene, suggesting that the benzene- and naphthalene-degrading populations were different. When fuels containing complex mixtures of alkanes were added to sediments from the two sites, there was significant degradation in the alkanes. [14C]hexadecane was also anaerobically oxidized to 14CO2 in these sediments. Molybdate, a specific inhibitor of sulfate reduction, inhibited hexadecane oxidation. These results demonstrate that a wide variety of hydrocarbon contaminants can be degraded under sulfate-reducing conditions in hydrocarbon-contaminated sediments, and they suggest that it may be possible to use sulfate reduction rather than aerobic respiration as a treatment strategy for hydrocarbon-contaminated dredged sediments.     

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.     

Isolation and identification of rumen bacteria capable of anaerobic phloroglucinol degradation.

Eight strains of rumen bacteria capable of degrading phloroglucinol (1,3,5-trihydroxybenzene) under anaerobic conditions were isolated from enrichment cultures of the bovine rumen microflora established in a prereduced medium containing 0.02 M phloroglucinol. Five of the strains were facultatively anaerobic Gram-positive streptococci which were identified as Streptococcus bovis. Three strains of obligately anaerobic Gram-positive cocci were assigned to the genus Coprococcus. Anaerobic cultures of the Streptococcus bovis strains in a 40% rumen fluid medium initially containing 0.02 M phloroglucinol degraded 50-80% of the substrate within 2 days, whereas cultures of the Coprococcus strains degraded more than 80% of the substrate under the same conditions. The Streptococcus bovis strains were incapable of degrading phloroglucinol in brain heart infusion or in the medium of de Man, Rogosa, and Sharpe (MRS broth) incubated aerobically.     

虾池中具有降解硝酸盐或亚硝酸盐能力的细菌多样性

水产养殖过程中,氮素积累日益严重,而其中亚硝酸盐由于转化速度低,毒性强,对养殖的危害更加突出。对从对虾养殖池中通过反硝化条件选择性富集培养得到的具有去除硝酸盐及亚硝酸盐能力的细菌进行筛选,结果分离到27株能够还原硝酸盐的异养细菌,其中24株在7d内能有效地降低硝酸盐和亚硝酸盐浓度;特别是LZX22、LZX27、LZX23、LZX21等4株使硝酸盐氮由起始的422．25mg／L降至4．00mg／L以下,亚硝酸盐浓度也降至0．40mg／L以下。对这些菌株进行16S rDNA系统发育分析,结果显示：27株菌分属于5个不同的类群,α-Proteobacteria（1）,γ-Proteobacteria（10）,Actinobacteria（12）,Firmicutes（3）,和Bacteroides（1）;它们在系统发育上分别与11个属相近,分别是Pseudomonas,Halomonas,Acinetobacter,Paracoccus,Arthrobacter,Microbacterium,Cellulosimicrobium,Bacillus,Stenotrophom,和Sphingobacterium。表明所分析的虾池中具有去除硝酸盐和亚硝酸盐能力的细菌具有较高的多样性,特别是多株细菌为首次报道具有去除硝酸盐和亚硝酸盐的能力,为下一步筛选亚硝酸盐高效去除细菌提供了丰富的菌种资源。     

Biodegradation of monohalogenated alkanes by soil NH(3)-oxidizing bacteria

Although cooxidative biodegradation of monohalogenated hydrocarbons has been well studied in the model NH₃-oxidizing bacterium, Nitrosomonas europaea, virtually no information exists about cooxidation of these compounds by native populations of NH₃-oxidizing bacteria. To address this subject, nitrifying activity was stimulated to 125–400 nmol NO₃ ^– produced g^–1 soil h^–1 by first incubating a Ca(OH)₂-amended, silt loam soil (pH 7.0±0.2) at field capacity (270 g H₂O kg^–1 soil) with 10 μmol NH₄ ⁺ g^–1 soil for 14 days, followed by another 10 days of incubation in a shaken slurry (2:1 water:soil, v/w) with periodic pH adjustment and maintenance of 10 mM NH₄ ⁺. These slurries actively degraded both methyl bromide (MeBr) and ethyl chloride (EtCl) at maximum rates of 20–30 nmol ml^–1 h^–1 that could be sustained for approximately 12 h. Although the MeBr degradation rates were linear for the first 10–12 h of incubation, they could not be sustained regardless of NH₄ ⁺ level and declined to zero over 20 h of incubation. The transformation capacity of the slurry enrichments (~1 μmol MeBr ml^–1 soil slurry) was similar to the value measured previously in cell suspensions of N. europaea with similar NH₃-oxidizing activity. Several MeBr-degrading characteristics of the nitrifying enrichments were found to be similar to those documented in the literature for MeBr-degrading methanotrophs and facultatively methylotrophic bacteria. Electronic Publication     

A novel synthesis of branched high-molecular-weight (C40+) long-chain alkanes

Many biological and geochemical questions remain concerning the structures, functions, and properties of naturally occurring high-molecular-weight (C40+) alkanes with various mid-chain alkylation patterns. Above C40, these alkanes are exceedingly difficult to separate and purify, and syntheses can be blocked by the low solubility of intermediates. To overcome these problems, a facile three-step synthesis employing the alkylation of 1,3-dithiane with a suitable alpha,omega-dibromoalkane was developed. Bisalkylation of the bis(dithianyl)alkane intermediate with the appropriate 1-bromoalkane and subsequent desulfurization with Raney nickel furnished the desired long-chain alkane. Long-chain alkanes modified at mid-chain and/or symmetrically near the chain termini (or unmodified, i.e., long-chain n-paraffins) are accessible by the selection of appropriate bromoalkanes. Nine mid-chain methylated (C38H78 to C53H108), one symmetrical terminal-chain dimethylated (C40H82), and four linear (C44H90 to C58H118) long-chain alkanes were synthesized by using this approach. High-temperature gas chromatography (HTGC) was found to have important advantages for evaluating the purity of the synthetic high-molecular-weight alkanes.