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Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD) genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest kcat/Km value toward 1,3-dibromopropane (Km = 0.82 mM, kcat/Km = 16.43 mM-1.s-1). DadB aggregates fast in the buffers with pH less than or equal to 7.0, while keeps stable in monomer form when pH greater than or equal to 7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications.

Five psychrotolerant Alcanivorax spp. strains were isolated from Antarctic coastal waters. Strains were screened for molecular and physiological properties and analyzed regarding their growth capacity. Partial 16S rDNA, alk-B1, and P450 gene sequencing was performed. Biolog EcoPlates and the API 20E test were used to evaluate metabolic and biochemical profiles. Bacterial growth in sodium acetate was determined at 4, 15, 20, and 25 °C to evaluate the optimal temperature. Furthermore, the ability of each strain to grow in a hydrocarbon mixture at 4 and 25 °C was assayed. Biosurfactant production tests (drop-collapse and oil spreading) and emulsification activity tests (E24) were also performed. Concerning results of partial gene sequencing (16S rDNA, alk-B1, and P450), a high similarity of the isolates with the same genes isolated from other Alcanivorax spp. strains was observed. The metabolic profiles obtained by Biolog assays showed no significant differences in the isolates compared to the Alcanivorax borkumensis wild type. The results of biodegradative tests showed their capability to grow at different temperatures. All strains showed biosurfactant production and emulsification activity. Our findings underline the importance to proceed in the isolation and characterization of Antarctic hydrocarbon-degrading bacterial strains since their biotechnological and environmental applications could be useful even for pollution remediation in polar areas.

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. Although barely detectable in unpolluted environments, A. borkumensis becomes the dominant microbe in oil-polluted waters. A. borkumensis SK2 has a streamlined genome with a paucity of mobile genetic elements and energy generation–related genes, but with a plethora of genes accounting for its wide hydrocarbon substrate range and efficient oil-degradation capabilities. The genome further specifies systems for scavenging of nutrients, particularly organic and inorganic nitrogen and oligo-elements, biofilm formation at the oil-water interface, biosurfactant production and niche-specific stress responses. The unique combination of these features provides A. borkumensis SK2 with a competitive edge in oil-polluted environments. This genome sequence provides the basis for the future design of strategies to mitigate the ecological damage caused by oil spills.

The environmental accumulation of plastics worldwide is a consequence of the durability of the material. Alternative polymers, marketed as biodegradable, present a potential solution to mitigate their ecological damage. However, understanding of biodegradability has been hindered by a lack of reproducible testing methods. We developed a novel method to evaluate the biodegradability of plastic samples based on the monitoring of bacterial respiration in aqueous media via the quantification of CO2 produced, where the only carbon source available is from the polymer. Rhodococcus rhodochrous and Alcanivorax borkumensis were used as model organisms for soil and marine systems, respectively. Our results demonstrate that this approach is reproducible and can be used with a variety of plastics, allowing comparison of the relative biodegradability of the different materials. In the case of low-density polyethylene, the study demonstrated a clear correlation between the molecular weight of the sample and CO2 released, taken as a measure of biodegradability.

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).

Bacterial cytochrome P450 enzymes in cytochrome P450 (CYP)153 family were recently reported as fatty acid ω-hydroxylase. Among them, CYP153As from Marinobacter aquaeolei VT8 (CYP153A33), Alcanivorax borkumensis SK2 (CYP153A13), and Gordonia alkanivorans (CYP153A35) were selected, and their specific activities and product yields of ω-hydroxy palmitic acid based on whole cell reactions toward palmitic acid were compared. Using CamAB as redox partner, CYP153A35 and CYP153A13 showed the highest product yields of ω-hydroxy palmitic acid in whole cell and in vitro reactions, respectively. Artificial self-sufficient CYP153A35-BMR was constructed by fusing it to the reductase domain of CYP102A1 (i.e., BM3) from Bacillus megaterium , and its catalytic activity was compared with CYP153A35 and CamAB systems. Unexpectedly, the system with CamAB resulted in a 1.5-fold higher yield of ω-hydroxy palmitic acid than that using A35-BMR in whole cell reactions, whereas the electron coupling efficiency of CYP153A35-BM3 reductase was 4-fold higher than that of CYP153A35 and CamAB system. Furthermore, various CamAB expression systems according to gene arrangements of the three proteins and promoter strength in their gene expression were compared in terms of product yields and productivities. Tricistronic expression of the three proteins in the order of putidaredoxin (CamB), CYP153A35, and putidaredoxin reductase (CamA), i.e., A35-AB2, showed the highest product yield from 5 mM palmitic acid for 9 h in batch reaction owing to the concentration of CamB, which is the rate-limiting factor for the activity of CYP153A35. However, in fed-batch reaction, A35-AB1, which expressed the three proteins individually using three T7 promoters, resulted with the highest product yield of 17.0 mM (4.6 g/L) ω-hydroxy palmitic acid from 20 mM (5.1 g/L) palmitic acid for 30 h.

To design bioprocesses utilising hydrocarbon-metabolising organisms (HMO) as biocatalysts, the effect of the organism on the hydrodynamics of bubble column reactor (BCR), such as gas holdup, needs to be investigated. Therefore, this study investigates the first use of an HMO, Alcanivorax borkumensis SK2, as a solid phase in the operation and hydrodynamics of a BCR. The study investigated the gas holdup in 3-phase and 4-phase systems in a BCR under ranges of superficial gas velocities (UG) from 1 to 3 cm/s, hydrocarbon (chain length C13-21) concentrations (HC) of 0, 5, and 10% v/v and microbial concentrations (MC) of 0, 0.35, 0.6 g/l. The results indicated that UG was the most significant parameter, as gas holdup increases linearly with increasing UG from 1 to 3 cm/s. Furthermore, the addition of hydrocarbons into the air-deionized water -SK2 system showed the highest increase in the gas holdup, particularly at high UG (above 2 cm/s). The solids (yeast, cornflour, and SK2) phases had differing effects on gas holdup, potentially due to the difference in surface activity. In this work, SK2 addition caused a reduction in the fluid surface tension in the bioprocess which therefore resulted in an increase in the gas holdup in BCR. This work builds upon previous investigations in optimising the hydrodynamics for bubble column hydrocarbon bioprocesses for the application of alkane bioactivation.

Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (NaVSp1) PD forms a stand-alone, ion selective pore (NaVSp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the NaVSp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel NaVBh1 (NaChBac), creates a calcium-selective pore-only channel, CaVSp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile NaVs: one from the hydrocarbon degrader Alcanivorax borkumensis (NaVAb1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (NaVAe1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.

The overuse of antibiotics as veterinary feed additives is potentially contributing to a significant reservoir of antibiotic resistance in agricultural farmlands via the application of antibiotic-contaminated manure. Vermicomposting of swine manure using housefly larvae is a promising biotechnology for waste reduction and control of antibiotic pollution. To determine how vermicomposting influences antibiotic resistance traits in swine manure, we explored the resistome and associated bacterial community dynamics during larvae gut transit over 6 days of treatment. In total, 94 out of 158 antibiotic resistance genes (ARGs) were significantly attenuated (by 85%), while 23 were significantly enriched (3.9-fold) following vermicomposting. The manure-borne bacterial community showed a decrease in the relative abundance of Bacteroidetes , and an increase in Proteobacteria , specifically Ignatzschineria , following gut transit. ARG attenuation was significantly correlated with changes in microbial community succession, especially reduction in Clostridiales and Bacteroidales . Six genomes were assembled from the manure, vermicompost (final product) and gut samples, including Pseudomonas, Providencia , Enterococcus , Bacteroides and Alcanivorax . Transposon-linked ARGs were more abundant in gut-associated bacteria compared with those from manure and vermicompost. Further, ARG-transposon gene cassettes had a high degree of synteny between metagenomic assemblies from gut and vermicompost samples, highlighting the significant contribution of gut microbiota through horizontal gene transfer to the resistome of vermicompost. In conclusion, the larvae gut microbiome significantly influences manure-borne community succession and the antibiotic resistome during animal manure processing.

The massive influx of crude oil into the Gulf of Mexico during the Deepwater Horizon (DWH) disaster triggered dramatic microbial community shifts in surface oil slick and deep plume waters. Previous work had shown several taxa, notably DWH Oceanospirillales , Cycloclasticus and Colwellia , were found to be enriched in these waters based on their dominance in conventional clone and pyrosequencing libraries and were thought to have had a significant role in the degradation of the oil. However, this type of community analysis data failed to provide direct evidence on the functional properties, such as hydrocarbon degradation of organisms. Using DNA-based stable-isotope probing with uniformly 13 C-labelled hydrocarbons, we identified several aliphatic ( Alcanivorax , Marinobacter )- and polycyclic aromatic hydrocarbon ( Alteromonas , Cycloclasticus , Colwellia )-degrading bacteria. We also isolated several strains ( Alcanivorax , Alteromonas , Cycloclasticus , Halomonas , Marinobacter and Pseudoalteromonas ) with demonstrable hydrocarbon-degrading qualities from surface slick and plume water samples collected during the active phase of the spill. Some of these organisms accounted for the majority of sequence reads representing their respective taxa in a pyrosequencing data set constructed from the same and additional water column samples. Hitherto, Alcanivorax was not identified in any of the previous water column studies analysing the microbial response to the spill and we discuss its failure to respond to the oil. Collectively, our data provide unequivocal evidence on the hydrocarbon-degrading qualities for some of the dominant taxa enriched in surface and plume waters during the DWH oil spill, and a more complete understanding of their role in the fate of the oil.