Phylogenomic analysis of the genus Alcanivorax: proposal for division of this genus into the emended genus Alcanivorax and two novel genera Alloalcanivorax gen. nov. and Isoalcanivorax gen. nov.

The members of the genus Alcanivorax are key players in the removal of petroleum hydrocarbons from polluted marine environments. More than half of the species were described in the last decade using 16S rRNA gene phylogeny and genomic-based metrics. However, the 16S rRNA gene identity (<94 %) between some members of the genus Alcanivorax suggested their imprecise taxonomic status. In this study, we examined the taxonomic positions of Alcanivorax species using 16S rRNA phylogeny and further validated them using phylogenomic-related indexes such as digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), average amino acid identity (AAI), percentage of conserved proteins (POCP) and comparative genomic studies. ANI and dDDH values confirmed that all the Alcanivorax species were well described at the species level. The phylotaxogenomic analysis showed that Alcanivorax species formed three clades. The inter-clade values of AAI and POCP were less than 70 %. The pan-genome evaluation depicted that the members shared 1223 core genes and its number increased drastically when analysed clade-wise. Therefore, these results necessitate the transfer of clade II and clade III members into Isoalcanivorax gen. nov. and Alloalcanivorax gen. nov., respectively, along with the emended description of the genus Alcanivorax sensu stricto.

High abundance of hydrocarbon-degrading Alcanivorax in plumes of hydrothermally active volcanoes in the South Pacific Ocean.

Species within the genus Alcanivorax are well known hydrocarbon-degraders that propagate quickly in oil spills and natural oil seepage. They are also inhabitants of the deep-sea and have been found in several hydrothermal plumes. However, an in-depth analysis of deep-sea Alcanivorax is currently lacking. In this study, we used multiple culture-independent techniques to analyze the microbial community composition of hydrothermal plumes in the Northern Tonga arc and Northeastern Lau Basin focusing on the autecology of Alcanivorax. The hydrothermal vents feeding the plumes are hosted in an arc volcano (Niua), a rear-arc caldera (Niuatahi) and the Northeast Lau Spreading Centre (Maka). Fluorescence in situ hybridization revealed that Alcanivorax dominated the community at two sites (1210-1565 mbsl), reaching up to 48% relative abundance (3.5 × 104 cells/ml). Through 16S rRNA gene and metagenome analyses, we identified that this pattern was driven by two Alcanivorax species in the plumes of Niuatahi and Maka. Despite no indication for hydrocarbon presence in the plumes of these areas, a high expression of genes involved in hydrocarbon-degradation was observed. We hypothesize that the high abundance and gene expression of Alcanivorax is likely due to yet undiscovered hydrocarbon seepage from the seafloor, potentially resulting from recent volcanic activity in the area. Chain-length and complexity of hydrocarbons, and water depth could be driving niche partitioning in Alcanivorax.

High-Resolution Small RNAs Landscape Provides Insights into Alkane Adaptation in the Marine Alkane-Degrader Alcanivorax dieselolei B-5

Alkanes are widespread in the ocean, and Alcanivorax is one of the most ubiquitous alkane-degrading bacteria in the marine ecosystem. Small RNAs (sRNAs) are usually at the heart of regulatory pathways, but sRNA-mediated alkane metabolic adaptability still remains largely unknown due to the difficulties of identification. Here, differential RNA sequencing (dRNA-seq) modified with a size selection (~50-nt to 500-nt) strategy was used to generate high-resolution sRNAs profiling in the model species Alcanivorax dieselolei B-5 under alkane (n-hexadecane) and non-alkane (acetate) conditions. As a result, we identified 549 sRNA candidates at single-nucleotide resolution of 5'-ends, 63.4% of which are with transcription start sites (TSSs), and 36.6% of which are with processing sites (PSSs) at the 5'-ends. These sRNAs originate from almost any location in the genome, regardless of intragenic (65.8%), antisense (20.6%) and intergenic (6.2%) regions, and RNase E may function in the maturation of sRNAs. Most sRNAs locally distribute across the 15 reference genomes of Alcanivorax, and only 7.5% of sRNAs are broadly conserved in this genus. Expression responses to the alkane of several core conserved sRNAs, including 6S RNA, M1 RNA and tmRNA, indicate that they may participate in alkane metabolisms and result in more actively global transcription, RNA processing and stresses mitigation. Two novel CsrA-related sRNAs are identified, which may be involved in the translational activation of alkane metabolism-related genes by sequestering the global repressor CsrA. The relationships of sRNAs with the characterized genes of alkane sensing (ompS), chemotaxis (mcp, cheR, cheW2), transporting (ompT1, ompT2, ompT3) and hydroxylation (alkB1, alkB2, almA) were created based on the genome-wide predicted sRNA-mRNA interactions. Overall, the sRNA landscape lays the ground for uncovering cryptic regulations in critical marine bacterium, among which both the core and species-specific sRNAs are implicated in the alkane adaptive metabolisms.

Responses of Alcanivorax species to marine alkanes and polyhydroxybutyrate plastic pollution: Importance of the ocean hydrocarbon cycles.

Understanding microbial responses to hydrocarbon and plastic pollution are crucial for limiting the detrimental impacts of environmental contaminants on marine ecosystems. Herein, we reported a new Alcanivorax species isolated from the North Atlantic Ocean capable of degrading alkanes and polyhydroxybutyrate (PHB) plastic (one of the emerging bioplastics that may capture the future plastic market). The whole-genome sequencing showed that the species harbors three types of alkane 1-monooxygenases (AlkB) and one PHB depolymerase (PhaZ) to initiate the degradation of alkanes and plastics. Growth profiling demonstrated that n-pentadecane (C15, the main alkane in the marine environment due to cyanobacterial production other than oil spills) and PHB could serve as preferential carbon sources. However, the cell membrane composition, PhaZ activity, and expression of three alkB genes were utterly different when grown on C15 and PHB. Further, Alcanivorax was a well-recognized alkane-degrader that participated in the ocean hydrocarbon cycles linking with hydrocarbon production and removal. Our discovery supported that the existing biogeochemical processes may add to the marine ecosystem's resilience to the impacts of plastics.

Mesopelagic microbial community dynamics in response to increasing oil and Corexit 9500 concentrations.

Marine microbial communities play an important role in biodegradation of subsurface plumes of oil that form after oil is accidentally released from a seafloor wellhead. The response of these mesopelagic microbial communities to the application of chemical dispersants following oil spills remains a debated topic. While there is evidence that contrasting results in some previous work may be due to differences in dosage between studies, the impacts of these differences on mesopelagic microbial community composition remains unconstrained. To answer this open question, we exposed a mesopelagic microbial community from the Gulf of Mexico to oil alone, three concentrations of oil dispersed with Corexit 9500, and three concentrations of Corexit 9500 alone over long periods of time. We analyzed changes in hydrocarbon chemistry, cell abundance, and microbial community composition at zero, three and six weeks. The lowest concentration of dispersed oil yielded hydrocarbon concentrations lower than oil alone and microbial community composition more similar to control seawater than any other treatments with oil or dispersant. Higher concentrations of dispersed oil resulted in higher concentrations of microbe-oil microaggregates and similar microbial composition to the oil alone treatment. The genus Colwellia was more abundant when exposed to multiple concentrations of dispersed oil, but not when exposed to dispersant alone. Conversely, the most abundant Marinobacter amplicon sequence variant (ASV) was not influenced by dispersant when oil was present and showed an inverse relationship to the summed abundance of Alcanivorax ASVs. As a whole, the data presented here show that the concentration of oil strongly impacts microbial community response, more so than the presence of dispersant, confirming the importance of the concentrations of both oil and dispersant in considering the design and interpretation of results for oil spill simulation experiments.

Genome sequence of an obligate hydrocarbonoclastic bacterium Alcanivorax marinus NMRL4 isolated from oil polluted seawater of the Arabian Sea.

Alcanivorax belongs to the hydrocarbonoclastic group of bacteria that are known for their preferential growth on alkanes and other related compounds. Here we report the genomic features of Alcanivorax marinus strain NMRL4 (=MCC 4632) isolated from oil polluted seawater of the Arabian Sea. Its 4,062,055 bp genome with 66.1% GC content encodes for 3935 coding sequences. The genome annotations of strain NMRL4 revealed the presence of multiple hydrocarbon degradation genes suggestive of its wider hydrocarbon substrate range. The strain encodes for three alkane monooxygenases, two cytochrome P450 and two flavin binding monooxygenases for degradation of short and long-chain alkanes. The genome shows capabilities for scavenging of nutrients, biofilm formation at oil-water interfaces, chemotaxis, motility and habitat specific adaptation. The genomic insights showed that the strain NMRL4 is an ideal candidate for bioremediation of pollutant petroleum hydrocarbons from the marine environment.

Alcanivorax profundimaris sp. nov., a Novel Marine Hydrocarbonoclastic Bacterium Isolated from Seawater and Deep-Sea Sediment.

Two novel Alcanivorax-related strains, designated ST75FaO-1T and 521-1, were isolated from the seawater of the South China Sea and the deep-sea sediment of the West Pacific Ocean, respectively. Both strains are Gram-stain-negative, rod-shaped, and non-motile, and grow at 10-40 °C, pH 5.0-10.0, in the presence of 1.0-15.0% (w/v) NaCl. Their 16S rRNA gene sequences showed 99.9% similarity. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that both strains belong to the genus Alcanivorax, and share 92.9-98.1% sequence similarity with all valid type strains of this genus, with the highest similarity being to type strain Alcanivorax venustensis DSM 13974T (98.0-98.1%). Digital DNA-DNA hybridization (dDDH) and average nucleotide identity values between strains ST75FaO-1T and 521-1 were 75.7% and 97.1%, respectively, while the corresponding values with A. venustensis DSM 13974T were only 25.4-25.6% and 82.4-82.7%, respectively. The two strains contained similar major cellular fatty acids including C16:0, C18:1 ω7c/ω6c, C19:0 cyclo ω8c, C16:1 ω7c/ω6c, C12:0 3-OH, and C12:0. The genomic G + C content of strains ST75FaO-1T and 521-1 were 66.3% and 66.1%, respectively. Phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids, and one unidentified polar lipid were present in both strains. On the basis of phenotypic and genotypic characteristics, the two strains represent a novel species within the genus Alcanivorax, for which the name Alcanivorax profundimaris sp. nov. is proposed. The type strain is ST75FaO-1T (= MCCC 1A17714T = KCTC 82142T).

Alcanivorax limicola sp. nov., isolated from a soda alkali-saline soil.

An alkaliphilic and aerobic bacterium, designated as strain JB21T, was isolated from a soda alkali-saline soil sample in Heilongjiang, Northeast China. Strain JB21T is a Gram-stain-negative, rod-shaped, non-motile and amylase-positive bacterium. Growth occurred at 15-45 °C (optimum, 35-37 °C), in the presence of 0-15.0% (w/v) NaCl (optimum, 1.0%) and at pH 6.5-10.5 (optimum, pH 8.5-9.5). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain JB21T was most closely related to type strains of the genus Alcanivorax, with the highest sequence similarity to Alcanivorax indicus SW127T (96.3%), and shared 95.4-93.1% sequence identity with other valid type strains of this genus. The major cellular fatty acids identified were C16:0 and summed feature 8 (C18:1ω6c and/or C18:1ω7c). The polar lipids comprised phosphatidylethanolamine, phosphatidylglycerol and one unidentified phospholipid. The genomic G + C content of strain JB21T was 61.3 mol%. The digital DNA-DNA hybridization (dDDH) estimation and average nucleotide identity (ANI) between strain JB21T and type strains of the genus Alcanivorax were 18.3-23.2% and 69.2-79.0%, respectively. On the basis of its phenotypic and phylogenetic characteristics, we suggest the creation of a new species within the Alcanivorax genus, named Alcanivorax limicola sp. nov., type strain JB21T (= CGMCC 1.16632T = JCM 33717T).

Complete genome sequence and comparative genome analysis of Alcanivorax sp. IO_7, a marine alkane-degrading bacterium isolated from hydrothermally-influenced deep seawater of southwest Indian ridge.

Genome of Alcanivorax sp. IO_7, an alkane degrading deep-sea bacteria isolated from hydrothermally-influenced Southwest Indian Ridge was sequenced and analysed. Genomic data mining revealed gene clusters for degrading n-alkane and cycloalkanes, including biosurfactant production. The strain was shown to grow on hexadecane as its sole carbon source, supporting the findings of genomic analysis. Presence of cyclohexanone monooxygenase among genomic islands suggest that this strain may have used gene transfer to enhance its hydrocarbon degradation ability. Genes encoding for heavy metal resistance, multidrug resistance and multiple natural product biosynthesis crucial for survival in the hydrothermally influenced deep sea environment were detected. In our comparative genome analysis, it was evident that marine Alcanivorax strains contain a suite of enzymes for n-alkane and haloalkanoate degradation. Comparative genome and genomic synteny analysis provided insights into the physiological features and adaptation strategies of Alcanivorax sp. IO_7 in the deep-sea hydrothermal environment.

Genomic features and copper biosorption potential of a new Alcanivorax sp. VBW004 isolated from the shallow hydrothermal vent (Azores, Portugal).

A new Alcanivorax sp. VBW004 was isolated from a shallow hydrothermal vent in Azores Island, Portugal. In this study, we determined VBW004 was resistant to copper. This strain showed maximum tolerance of copper concentrations up to 600 µg/mL. Based on 16S rRNA gene sequencing and phylogeny revealed that this strain was more closely related to Alcanivorax borkumensis SK2. We sequenced the genome of this strain that consist of 3.8 Mb size with a G + C content of 58.4 %. In addition, digital DNA-DNA hybridizations (dDDH) and the average nucleotide identities (ANI) analysis between Alcanivorax borkumensis SK2 and Alcanivorax jadensis T9 revealed that Alcanivorax sp. VBW004 belongs to new species. Functional annotation revealed that the genome acquired multiple copper resistance encoding genes that could assist VBW004 to respond to high Cu toxicity. Our results from biosorption analysis presumed that the VBW004 is an ecologically important bacterium that could be useful for copper bioremediation.

Point Mutations Lead to Increased Levels of c-di-GMP and Phenotypic Changes to the Colony Biofilm Morphology in Alcanivorax borkumensis SK2.

Alcanivorax borkumensis is a ubiquitous marine bacterium that utilizes alkanes as a sole carbon source. We observed two phenotypes in the A. borkumensis SK2 type strain: rough (R) and smooth (S) types. The S type exhibited lower motility and higher polysaccharide production than the R type. Full genome sequencing revealed a mutation in the S type involved in cyclic-di-GMP production. The present results suggest that higher c-di-GMP levels in the S type control the biofilm forming behavior of this bacterium in a manner commensurate with other Gram-negative bacteria.

Periodic and Spatial Spreading of Alkanes and Alcanivorax Bacteria in Deep Waters of the Mariana Trench.

In subduction zones, serpentinization and biological processes may release alkanes to the deep waters, which would probably result in the rapid spread of Alcanivorax However, the timing and area of the alkane distribution and associated enrichment of alkane-degrading microbes in the dark world of the deep ocean have not been explored. In this study, we report the richness (up to 17.8%) of alkane-degrading bacteria, represented by Alcanivorax jadensis, in deep water samples obtained at 3,000 to 6,000 m in the Mariana Trench in two cruises. The relative abundance of A. jadensis correlated with copy numbers of functional almA and alkB genes, which are involved in alkane degradation. In these water samples, we detected a high flux of alkanes, which probably resulted in the prevalence of A. jadensis in the deep waters. Contigs of A. jadensis were binned from the metagenomes for examination of alkane degradation pathways and deep sea-specific pathways, which revealed a lack of nitrate and nitrite dissimilatory reduction in our A. jadensis strains. Comparing the results for the two cruises conducted close to each other, we suggest periodic release of alkanes that may spread widely but periodically in the trench. Distribution of alkane-degrading bacteria in the world's oceans suggests the periodic and remarkable contributions of Alcanivorax to the deep sea organic carbon and nitrogen sources.IMPORTANCE In the oligotrophic environment of the Mariana Trench, alkanes as carbohydrates are important for the ecosystem, but their spatial and periodic spreading in deep waters has never been reported. Alkane-degrading bacteria such as Alcanivorax spp. are biological signals of the alkane distribution. In the present study, Alcanivorax was abundant in some waters, at depths of up to 6,000 m, in the Mariana Trench. Genomic, transcriptomic, and chemical analyses provide evidence for the presence and activities of Alcanivorax jadensis in deep sea zones. The periodic spreading of alkanes, probably from the subductive plates, might have fundamentally modified the local microbial communities, as well as perhaps the deep sea microenvironment.

Alcanivorax indicus sp. nov., isolated from seawater.

A Gram-stain-negative, motile, rod-shaped bacterium, designated SW127T, was isolated from a seawater sample collected from the Indian Ocean. Strain SW127T was aerobic, catalase- and oxidase-positive, and grew at 8-42 °C (optimum, 30 °C), at pH 5.5-9.0 (optimum, pH 7.5) and in the presence of 0.5-11.0 % (w/v) NaCl (optimum, 3.0-4.0 %). Comparative analysis of 16S rRNA gene sequences indicated that strain SW127T belonged to the genus Alcanivorax, and closely related to Alcanivorax pacificus MCCC 1A00474T (96.7 % sequence similarity). The predominant cellular fatty acids of strain SW127T were C16 : 0 and summed feature 8 (comprising C18 : 1ω7c and/or C18 : 1ω6c). Strain SW127T contained phosphatidylethanolamine and phosphatidylglycerol as the major polar lipids. The genomic DNA G+C content of strain SW127T was 62.8 mol%. On the basis of its phenotypic characteristics and phylogenetic data, strain SW127T represents a novel species of the genus Alcanivorax, for which the name Alcanivorax indicus sp. nov. is proposed. The type strain is SW127T (=CGMCC 1.16233T=KCTC 62652T).

Marinicella sediminis sp. nov., isolated from marine sediment.

A novel heterotrophic, Gram-stain-negative, aerobic, rod-shaped, pale yellow, non-motile and non-spore-forming bacterium, designated strain F2T, was isolated from marine sediment collected from the Weihai coast, Shandong Province, PR China. Optimal growth occurred at 33 °C (range, 10-37 °C), with 3.0-4.0 % (w/v) NaCl (1.0-8.0 %) and at pH 7.5-8.0 (pH 6.5-9.0). Q-8 was the sole respiratory quinone. The major polar lipids of strain F2T were phosphatidylmonomethylethanolamine, phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, two unidentified phospholipids and two unidentified polar lipids. The major cellular fatty acid in strain F2T was iso-C15 : 0. The genomic DNA G+C content of the strain was 48.1 mol%. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that strain F2T is most closely related to Marinicella litoralis JCM 16154T (97.5 %) and Marinicella pacifica sw153T (96.0 %). Based on the results of our polyphasic analysis, we conclude that strain F2T represents a novel species of the genus Marinicella, for which the name Marinicella sediminis sp. nov. is proposed. The type strain of the new species is F2T (=KCTC 42953T=MCCC 1H00149T).

Ketobacter alkanivorans gen. nov., sp. nov., an n-alkane-degrading bacterium isolated from seawater.

Strain GI5T was isolated from a surface seawater sample collected from Garorim Bay (West Sea, Republic of Korea). The isolated strain was aerobic, Gram-stain-negative, rod-shaped, motile by means of a polar flagellum, negative for catalase and weakly positive for oxidase. The optimum growth pH, salinity and temperature were determined to be pH 7.5-8.0, 3 % NaCl (w/v) and 25 °C, respectively; the growth ranges were pH 6.0-9.0, 1-7 % NaCl (w/v) and 18-40 °C. The results of phylogenetic analysis of 16S rRNA gene sequences indicated that GI5T clustered within the family Alcanivoracaceae, and most closely with Alcanivorax dieseloleiB-5T and Alcanivorax marinusR8-12T (91.9 % and 91.6 % similarity, respectively). The major cellular fatty acids in GI5T were C18 : 1ω7c/C18 : 1ω6c (44.45 %), C16 : 1ω6c/C16 : 1ω7c (14.17 %) and C16 : 0 (10.19 %); this profile was distinct from those of the closely related species. The major respiratory quinone of GI5T was Q-8. The main polar lipids were phosphatidylethanolamine and phosphatidylglycerol. Two putative alkane hydroxylase (alkB) genes were identified in GI5T. The G+C content of the genomic DNA of GI5T was determined to be 51.2 mol%. On the basis of the results of phenotypic, chemotaxonomic and phylogenetic studies, strain GI5T represents a novel species of a novel genus of the family Alcanivoracaceae, for which we propose the name Ketobacter alkanivorans gen. nov., sp. nov.; the type strain is GI5T (=KCTC 52659T=JCM 31835T).

Alcanivorax mobilis sp. nov., a new hydrocarbon-degrading bacterium isolated from deep-sea sediment.

A taxonomic study was carried out on strain MT13131T, which was isolated from deep-sea sediment of the Indian Ocean during the screening of oil-degrading bacteria. The chain length range of n-alkanes (C8 to C32) oxidized by strain MT13131T was determined in this study. The bacterium was Gram-negative, oxidase- and catalase-positive, single rod shaped, and motile by peritrichous flagella. Growth was observed at salinities of 1-12 % and at temperatures of 10-42 °C. The isolate was capable of Tween 20, 40 and 80 hydrolysis, but incapable of gelatin, cellulose or starch hydrolysis. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MT13131T belonged to the genus Alcanivorax, with highest sequence similarity to Alcanivorax marinus R8-12T (96.92 %), other species of genus Alcanivorax shared 92.96-96.69 % sequence similarity. The principal fatty acids were summed feature 3 (C16 : 1ω6c/ω7c), summed feature 8 (C18 : 1ω7c/ω6c), C16 : 0 and C12 : 0 3OH. The G+C content of the chromosomal DNA was 64.2 mol%. Phosphatidylglycerol, phosphatidylethanolamine, three aminolipids and three phospholipids were present. The combined genotypic and phenotypic data showed that strain MT13131T represents a novel species within the genus Alcanivorax, for which the name Alcanivorax mobilis sp. nov. is proposed, with the type strain MT13131T (=MCCC 1A11581T=KCTC 52985T).

Periodically spilled-oil input as a trigger to stimulate the development of hydrocarbon-degrading consortia in a beach ecosystem.

In this study, time-series samples were taken from a gravel beach to ascertain whether a periodic oil input induced by tidal action at the early stage of an oil spill can be a trigger to stimulate the development of hydrocarbon-degrading bacteria under natural in situ attenuation. High-throughput sequencing shows that the microbial community in beach sediments is characterized by the enrichment of hydrocarbon-degrading bacteria, including Alcanivorax, Dietzia, and Marinobacter. Accompanying the periodic floating-oil input, dynamic successions of microbial communities and corresponding fluctuations in functional genes (alkB and RDH) are clearly indicated in a time sequence, which keeps pace with the ongoing biodegradation of the spilled oil. The microbial succession that accompanies tidal action could benefit from the enhanced exchange of oxygen and nutrients; however, regular inputs of floating oil can be a trigger to stimulate an in situ "seed bank" of hydrocarbon-degrading bacteria. This leads to the continued blooming of hydrocarbon-degrading consortia in beach ecosystems. The results provide new insights into the beach microbial community structure and function in response to oil spills.

Differential expression of the three Alcanivorax borkumensis SK2 genes coding for the P450 cytochromes involved in the assimilation of hydrocarbons.

Alcanivorax borkumensis, a marine bacterium highly specialized in degrading linear and branched alkanes, plays a key ecological role in the removal of marine oil spills. It contains several alternative enzyme systems for terminal hydroxylation of alkanes, including three P450 cytochromes (P450-1, P450-2 and P450-3). The present work shows cytochrome P450-1 to be expressed from the promoter of the upstream gene fdx. Promoter Pfdx was more active when C8 -C18 n-alkanes or pristane were assimilated than when pyruvate was available. The product of ABO_0199 (named CypR) was identified as a transcriptional activator of Pfdx . The inactivation of cypR impaired growth on tetradecane, showing the importance of the fdx-P450-1 and/or cypR genes. P450-2 expression was low-level and constitutive under all conditions tested, while that of P450-3 from promoter P450-3 was much higher when cells assimilated pristane than when n-alkanes or pyruvate were available. However, the inactivation of P450-3 had no visible impact on pristane assimilation. Cyo terminal oxidase, a component of the electron transport chain, was found to stimulate promoter PP450-3 activity, but it did not affect promoters Pfdx or PP450-2 . A. borkumensis, therefore, appears to carefully coordinate the expression of its multiple hydrocarbon degradation genes using both specific and global regulatory systems.

Diversity and antimicrobial activity of bacteria isolated from different Brazilian coral species.

Corals harbor a wide diversity of bacteria associated with their mucus. These bacteria can play an important role in nutrient cycling, degradation of xenobiotics and defense against pathogens by producing antimicrobial compounds. However, the diversity of the cultivable heterotrophic bacteria, especially in the Brazilian coral species, remains poorly understood. The present work compares the diversity of cultivable bacteria isolated from the mucus and surrounding environments of four coral species present along the Brazilian coast, and explores the antibacterial activity of these bacteria. Bacteria belonging to the phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were isolated. The mucus environment presented a significantly different bacteria composition, compared to the water and sediment environments, with high abundance of Alcanivorax, Acinetobacter, Aurantimonas and Erythrobacter. No difference in the inhibition activity was found between the isolates from mucus and from the surrounding environment. Eighty-three per cent of the bacteria isolated from the mucus presented antimicrobial activity against Serratia marcescens, an opportunistic coral pathogen, suggesting that they might play a role in maintaining the health of the host. Most of the bacteria isolates that presented positive antimicrobial activity belonged to the genus Bacillus.

Alcanivorax nanhaiticus sp. nov., isolated from deep sea sediment.

A taxonomic study was carried out on strain 19-m-6T, which was isolated from deep sea sediment of the South China Sea during the screening of alkane-degrading bacteria. The isolate was Gram-reaction-negative, and oxidase- and catalase- positive. On the basis of 16S rRNA gene sequence similarity, strain 19-m-6T was shown to belong to the genus Alcanivorax, related to Alcanivorax jadensis T9T (97.5 %), Alcanivorax hongdengensis A-11-3T (97.3 %), A. lcanivorax borkumensis SK2T (96.6 %) and seven other species of the genus Alcanivorax(93.9-95.4 %). Average nucleotide identity values between strain 19-m-6T and A. jadensis T9T, A. hongdengensis A-11-3T and A. borkumensis SK2T were 85.12, 85.87 and 84.35 %, respectively. The estimated DNA-DNA hybridization values between strain 19-m-6T and these three type strains were 22.0, 22.6 and 21.2 %, respectively. Four alkane hydroxylase (alkB) genes were obtained from the draft genome sequence. The G+C content of the chromosomal DNA was 56.44 mol%. The major fatty acids were C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, one aminolipid, three phospholipids, two glycolipids and two aminophospholipids. According to its phenotypic features, fatty acid composition and 16S rRNA gene sequence, the novel strain fitted well into the genus Alcanivorax, but could be clearly distinguished from all other known Alcanivorax species described to date. The nameAlcanivorax nanhaiticus sp. nov. is thus proposed, with 19-m-6T (=MCCC 1A05629T=KCTC 52137T) as the type strain.