Dynamics of a microbial community during an effective boost MEOR trial using high-throughput sequencing.

Using 454 pyrosequencing of 16S rRNA gene amplicons, microbial communities in samples of injection water and production water during a serial microbial enhanced oil recovery (MEOR) field trial in a water flooded high pour point oil reservoir were determined. There was a close microbial community compositional relationship between the injection water and the successful first round MEOR processed oil reservoir which was indicated by the result of 43 shared dominant operational taxonomic units detected in both the injection water and the production water. Alterations of microbial community after the injection of boost nutrients showed that microbes giving positive responses were mainly those belonging to the genera of Comamonas, Brevundimonas, Azospirillum, Achromobacter, Pseudomonas, and Hyphomonas, which were detected both in the injection water and in the production water and usually detected in oil reservoir environments or associated with hydrocarbon degradation. Additionally, microbes only dominant in the production waters were significantly inhibited with a sharp decline in their relative abundance. Based on these findings, a suggestion of re-optimization of the boost nutrients, targetting the microbes co-existing in the injection water and the oil reservoir and having survival ability in both surface and subsurface environments, rather than simple repeats for the subsequent in situ MEOR applications was proposed.

Draft genome sequence of Paenibacillus sp. strain A2.

Paenibacillus sp. strain A2 is a Gram-negative rod-shaped bacterium isolated from a mixture of formation water and petroleum in Daqing oilfield, China. This facultative aerobic bacterium was found to have a broad capacity for metabolizing hydrocarbon and organosulfur compounds, which are the main reasons for the interest in sequencing its genome. Here we describe the features of Paenibacillus sp. strain A2, together with the genome sequence and its annotation. The 7,650,246 bp long genome (1 chromosome but no plasmid) exhibits a G+C content of 54.2 % and contains 7575 protein-coding and 49 RNA genes, including 3 rRNA genes. One putative alkane monooxygenase, one putative alkanesulfonate monooxygenase, one putative alkanesulfonate transporter and four putative sulfate transporters were found in the draft genome.

Isolation and characterization of a crude oil degrading bacteria from formation water: comparative genomic analysis of environmental Ochrobactrum intermedium isolate versus clinical strains.

In this study, we isolated an environmental clone of Ochrobactrum intermedium, strain 2745-2, from the formation water of Changqing oilfield in Shanxi, China, which can degrade crude oil. Strain 2745-2 is aerobic and rod-shaped with optimum growth at 42 °C and pH 5.5. We sequenced the genome and found a single chromosome of 4 800 175 bp, with a G+C content of 57.63%. Sixty RNAs and 4737 protein-coding genes were identified: many of the genes are responsible for the degradation, emulsification, and metabolizing of crude oil. A comparative genomic analysis with related clinical strains (M86, 229E, and LMG3301(T)) showed that genes involved in virulence, disease, defense, phages, prophages, transposable elements, plasmids, and antibiotic resistance are also present in strain 2745-2.

Permanent draft genome sequence of Bacillus flexus strain T6186-2, a multidrug-resistant bacterium isolated from a deep-subsurface oil reservoir.

Previous studies suggest that antibiotic resistance genes have an ancient origin, which is not always linked to the use of antibiotics but can be enhanced by human activities. Bacillus flexus strain T6186-2 was isolated from the formation water sample of a deep-subsurface oil reservoir. Interestingly, antimicrobial susceptibility testing showed that this strain is susceptible to kanamycin, however, resistant to ampicillin, erythromycin, gentamicin, vancomycin, fosfomycin, fosmidomycin, tetracycline and teicoplanin. To explore our knowledge about the origins of antibiotic resistance genes (ARGs) in the relatively pristine environment, we sequenced the genome of B. flexus strain T6186-2 as a permanent draft. It represents the evidence for the existence of a reservoir of ARGs in nature among microbial populations from deep-subsurface oil reservoirs.

Permanent draft genome sequence of Geobacillus thermocatenulatus strain GS-1.

Geobacillus thermocatenulatus strain GS-1 is a thermophilic bacillus having a growth optimum at 60°C, capable of degrading alkanes. It was isolated from the formation water of a high-temperature deep oil reservoir in Qinghai oilfield, China. Here, we report the draft genome sequence with an estimated assembly size of 3.5Mb. A total of 3371 protein-coding sequences, including monooxygenase, alcohol dehydrogenase, aldehyde dehydrogenase, fatty acid-CoA ligase, acyl-CoA dehydrogenase, enoyl-CoA hydrogenase, hydroxyacyl-CoA dehydrogenase and thiolase, were detected in the genome, which are involved in the alkane degradation pathway. Our results may provide insights into the genetic basis of the adaptation of this strain to high-temperature oilfield ecosystems.

Genome sequence of Brevibacillus agri strain 5-2, isolated from the formation water of petroleum reservoir.

Brevibacillus agri strain 5-2 was isolated from the formation water of a deep oil reservoir in Changqing Oilfield, China. This bacterium was found to have a capacity for degrading tetradecane, hexadecane and alkanesulfonate. To gain insights into its efficient metabolic pathway for degrading hydrocarbon and organosulfur compounds, here, we report the high quality draft genome of this strain. Two putative alkane 1-monooxygenases, one putative alkanesulfonate monooxygenase, one putative alkanesulfonate transporter, one putative sulfate permease and five putative sulfate transporters were identified in the draft genome. The genomic data of strain 5-2 may provide insights into the mechanism of microorganisms adapt to the petroleum reservoir after chemical flooding.

Genomovar assignment of Pseudomonas stutzeri populations inhabiting produced oil reservoirs.

Oil reservoirs are specific habitats for the survival and growth of microorganisms in general. Pseudomonas stutzeri which is believed to be an exogenous organism inoculated into oil reservoirs during the process of oil production was detected frequently in samples from oil reservoirs. Very little is known, however, about the distribution and genetic structure of P. stutzeri in the special environment of oil reservoirs. In this study, we collected 59 P. stutzeri 16S rRNA gene sequences that were identified in 42 samples from 25 different oil reservoirs and we isolated 11 cultured strains from two representative oil reservoirs aiming to analyze the diversity and genomovar assignment of the species in oil reservoirs. High diversity of P. stutzeri was observed, which was exemplified in the detection of sequences assigned to four known genomovars 1, 2, 3, 20 and eight unknown genomic groups of P. stutzeri. The frequent detection and predominance of strains belonging to genomovar 1 in most of the oil reservoirs under study indicated an association of genomovars of P. stutzeri with the oil field environments.

Microbial diversity in long-term water-flooded oil reservoirs with different in situ temperatures in China.

Water-flooded oil reservoirs have specific ecological environments due to continual water injection and oil production and water recycling. Using 16S rRNA gene clone library analysis, the microbial communities present in injected waters and produced waters from four typical water-flooded oil reservoirs with different in situ temperatures of 25 °C, 40 °C, 55 °C and 70 °C were examined. The results obtained showed that the higher the in situ temperatures of the oil reservoirs is, the less the effects of microorganisms in the injected waters on microbial community compositions in the produced waters is. In addition, microbes inhabiting in the produced waters of the four water-flooded oil reservoirs were varied but all dominated by Proteobacteria. Moreover, most of the detected microbes were not identified as indigenous. The objective of this study was to expand the pictures of the microbial ecosystem of water-flooded oil reservoirs.

Improvement of crystal solubility and increasing toxicity against Caenorhabditis elegans by asparagine substitution in block 3 of Bacillus thuringiensis crystal protein Cry5Ba.

The crystal proteins from Bacillus thuringiensis are widely used for their specific toxicity against insects and nematodes. The highly conserved sequence blocks play an important role in Cry protein stability and flexibility, the basis of toxicity. The block 3 in Cry5Ba subfamily has a shorter sequence (only 12 residues) and more asparagine residues than that of others which harbor about 48 residues but only one asparagine. Based on the theoretical structure model of Cry5Ba, all three asparagines in block 3 are closely located in the interface of putative three domains, implying their probable importance in structure and function. In this study, all three asparagines in Cry5Ba2 block 3 were individually substituted with alanine by site-directed mutagenesis. The wild-type and mutant proteins were overexpressed and crystallized in acrystalliferous B. thuringiensis strain BMB171. However, the crystals formed in one of the mutants, designated N586A, abnormally disappeared and dissolved into the culture supernatant once the sporulation cells lysed, whereas the Cry5Ba crystal and the other mutant crystals were stable. The mutant N586A crystal, isolated from sporulation cells by the ultrasonic process, was found to be easily dissolved at wide range of pH value (5.0 to 10.0). Moreover, the toxicity assays showed that the mutant N586A exhibited nearly 9-fold-higher activity against nematodes and damaged the host's intestine more efficiently than the native Cry5Ba2. These data support the presumption that the amide residue Asn586 at the interface of domains might adversely affect the protein flexibility, solubility and resultant toxicity of Cry5Ba.

Synergistic activity between Bacillus thuringiensis Cry6Aa and Cry55Aa toxins against Meloidogyne incognita.

Plant-parasitic nematodes are the most destructive group of plant pathogens worldwide and are extremely challenging to control. Some Bacillus thuringiensis crystal proteins are highly toxic to the plant-parasitic nematode Meloidogyne incognita. In this study, the nematicidal crystal proteins Cry6Aa, Cry5Ba and Cry55Aa were tested against M. incognita to select the best toxin combination for its management. The results showed that a combination of Cry6Aa and Cry55Aa showed significant synergistic toxicity against M. incognita, and the highest synergistic effect (five times the expected toxicity of the two toxins calculated from their separate toxicities) was observed when they were combined in a 1:1 ratio. Furthermore, ligand blot analyses of the interaction between total proteins of M. incognita and the three toxins showed many different signal bands, indicating that there is a range of host proteins with which the toxins can interact. One explanation of the observed synergism is that the toxins damage the host in diverse ways, and they may thus act cooperatively and thereby show greater toxicity in combination. Our discovery provides an effective strategy for controlling M. incognita by using a combination of Cry6Aa and Cry55Aa.

Determination of plasmid copy number reveals the total plasmid DNA amount is greater than the chromosomal DNA amount in Bacillus thuringiensis YBT-1520.

Bacillus thuringiensis is the most widely used bacterial bio-insecticide, and most insecticidal crystal protein-coding genes are located on plasmids. Most strains of B. thuringiensis harbor numerous diverse plasmids, although the plasmid copy numbers (PCNs) of all native plasmids in this host and the corresponding total plasmid DNA amount remains unknown. In this study, we determined the PCNs of 11 plasmids (ranging from 2 kb to 416 kb) in a sequenced B. thuringiensis subsp. kurstaki strain YBT-1520 using real-time qPCR. PCNs were found to range from 1.38 to 172, and were negatively correlated to plasmid size. The amount of total plasmid DNA (∼8.7 Mbp) was 1.62-fold greater than the amount of chromosomal DNA (∼5.4 Mbp) at the mid-exponential growth stage (OD(600) = 2.0) of the organism. Furthermore, we selected three plasmids with different sizes and replication mechanisms to determine the PCNs over the entire life cycle. We found that the PCNs dynamically shifted at different stages, reaching their maximum during the mid-exponential growth or stationary phases and remaining stable and close to their minimum after the prespore formation stage. The PCN of pBMB2062, which is the smallest plasmid (2062 bp) and has the highest PCN of those tested, varied in strain YBT-1520, HD-1, and HD-136 (172, 115, and 94, respectively). These findings provide insight into both the total plasmid DNA amount of B. thuringiensis and the strong ability of the species to harbor plasmids.