Philip Hendry

Microbial biodiversity in a Malaysian oil field and a systematic comparison with oil reservoirs worldwide

Microbial diversity within formation water and oil from two compartments in Bokor oil reservoir from a Malaysian petroleum oil field was examined. A total of 1,056 16S rRNA gene clones were screened from each location by amplified ribosomal DNA restriction analysis. All samples were dominated by clones affiliated with Marinobacter, some novel Deferribacteraceae genera and various clones allied to the Methanococci. In addition, either Marinobacterium- or Pseudomonas-like operational taxonomic units were detected from either compartment. A systematic comparison with the existing pertinent studies was undertaken by analysing the microbial amplicons detected and the PCR primers used. The analyses demonstrated that bacterial communities were site specific, while Archaea co-occurred more frequently. Amplicons related to Marinobacter, Marinobacterium and Pseudomonas were detected in a number of the studies examined, suggesting they may be ubiquitous members in oil reservoirs. Further analysis of primers used in those studies suggested that most primer pairs had fairly broad but low matches across the bacterial and archaeal domains, while a minority had selective matches to certain taxa or low matches to all the microbial taxa tested. Thus, it indicated that primers may play an important role in determining which taxa would be detected.

Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability

Background Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs specifically. As such they have potential as laboratory reagents, diagnostics and therapeutics. Despite having been extensively studied for 15 years or so, their wide application is hampered by their instability in biological media, and by the poor translation of cleavage studies on short substrates to long RNA molecules. This work describes a systematic study aimed at addressing these two issues. Results A series of hammerhead ribozyme derivatives, varying in their hybridising arm length and size of helix II, were tested in vitro for cleavage of RNA derived from the carbamoyl phosphate synthetase II gene of Plasmodium falciparum. Against a 550-nt transcript the most efficient (t1/2 = 26 seconds) was a miniribozyme with helix II reduced to a single G-C base pair and with twelve nucleotides in each hybridising arm. Miniribozymes of this general design were targeted to three further sites, and they demonstrated exceptional cleavage activity. A series of chemically modified derivatives was prepared and examined for cleavage activity and stability in human serum. One derivative showed a 103-fold increase in serum stability and a doubling in cleavage efficiency compared to the unmodified miniribozyme. A second was almost 104-fold more stable and only 7-fold less active than the unmodified parent. Conclusion Hammerhead ribozyme derivatives in which helix II is reduced to a single G-C base pair cleave long RNA substrates very efficiently in vitro. Using commonly available phosphoramidites and reagents, two patterns of nucleotide substitution in this derivative were identified which conferred both good cleavage activity against long RNA targets and good stability in human serum.

Reanalysis and Simulation Suggest a Phylogenetic Microarray Does Not Accurately Profile Microbial Communities

The second generation (G2) PhyloChip is designed to detect over 8700 bacteria and archaeal and has been used over 50 publications and conference presentations. Many of those publications reveal that the PhyloChip measures of species richness greatly exceed statistical estimates of richness based on other methods. An examination of probes downloaded from Greengenes suggested that the system may have the potential to distort the observed community structure. This may be due to the sharing of probes by taxa; more than 21% of the taxa in that downloaded data have no unique probes. In-silico simulations using these data showed that a population of 64 taxa representing a typical anaerobic subterranean community returned 96 different taxa, including 15 families incorrectly called present and 19 families incorrectly called absent. A study of nasal and oropharyngeal microbial communities by Lemon et al (2010) found some 1325 taxa using the G2 PhyloChip, however, about 950 of these taxa have, in the downloaded data, no unique probes and cannot be definitively called present. Finally, data from Brodie et al (2007), when re-examined, indicate that the abundance of the majority of detected taxa, are highly correlated with one another, suggesting that many probe sets do not act independently. Based on our analyses of downloaded data, we conclude that outputs from the G2 PhyloChip should be treated with some caution, and that the presence of taxa represented solely by non-unique probes be independently verified.

Small Efficient Hammerhead Ribozymes

The hammerhead ribozyme is able to cleave RNA in a sequence-specific manner. These ribozymes are usually designed with four basepairs in helix II, and with equal numbers of nucleotides in the 5′ and 3′ hybridizing arms that bind the RNA substrate on either side of the cleavage site. Here guidelines are given for redesigning the ribozyme so that it is small, but retains efficient cleavage activity. First, the ribozyme may be reduced in size by shortening the 5′ arm of the ribozyme to five or six nucleotides; for these ribozymes, cleavage of short substrates is maximal. Second, the internal double-helix of the ribozyme (helix II) may be shortened to one or no basepairs, forming a miniribozyme or minizyme, respectively. The sequence of the shortened helix+loop II greatly affects cleavage rates. With eight or more nucleotides in both the 5′ and the 3′ arms of a miniribozyme containing an optimized sequence for helix+loop II, cleavage rates of short substrates are greater than for analogous ribozymes possessing a longer helix II. Cleavage of genelength RNA substrates may be best achieved by miniribozymes.

Complete Genome Sequence of a Nonculturable Methanococcus maripaludis Strain Extracted in a Metagenomic Survey of Petroleum Reservoir Fluids

Extraction of genome sequences from metagenomic data is crucial for reconstructing the metabolism of microbial communities that cannot be mimicked in the laboratory. A complete Methanococcus maripaludis genome was generated from metagenomic data derived from a thermophilic subsurface oil reservoir. M. maripaludis is a hydrogenotrophic methanogenic species that is common in mesophilic saline environments. Comparison of the genome from the thermophilic, subsurface environment with the genome of the type species will provide insight into the adaptation of a methanogenic genome to an oil reservoir environment.

Design of Hybridizing Arms in Hammerhead Ribozymes

Hammerhead ribozymes as typically used in their trans-cleaving form are defined by their hybridizing arms, the remainder of the molecule being essentially constant. A major determinant of the nature of the hybridizing arms is their sequence, which is in turn determined by the sequence of the RNA at the cleavage site that is targeted. Therefore, the first step in designing hybridizing arms is to choose a target site. There are a number of methods that have been used. We will outline the techniques used without going into detail, because at least some of the techniques are the subject of other chapters in this book.

Influence of Helix Length on Cleavage Efficiency of Hammerhead Ribozymes

The cleavage rates of RNA substrates by trans-acting, hammerhead ribozymes are controlled by interactions between helices I and II. The interactions are affected by the relative lengths of these two double helices and by unpaired nucleotides protruding beyond helix I, either in the substrate or the ribozyme strand. Maximum cleavage rates are observed for ribozyme–substrate complexes with three or more base pairs in helix II and six or less base pairs in helix I. However, for these helix combinations, rates fall sharply with unpaired nucleotides at the end of helix I. Cleavage rates by ribozymes with one or two base pairs in helix II increase as helix I is lengthened, and are unaffected by unpaired nucleotides on the end. Since miniribozymes, with one base pair in helix II, efficiently cleave long RNA transcripts under physiological conditions, they represent the optimal design for the simple hammerheads for application in vivo.

Functional and genetic characterization of hydrocarbon biodegrader and exopolymer-producing clones from a petroleum reservoir metagenomic library

ABSTRACT Microbial degradation of petroleum is a worldwide issue, which causes physico-chemical changes in its compounds, diminishing its commercial value. Biosurfactants are chemically diverse molecules that can be produced by several microorganisms and can enable microbial access to hydrocarbons. In order to investigate both microbial activities, function-driven screening assays for biosurfactant production and hydrocarbon biodegradation were carried out from a metagenomic fosmid library. It was constructed from the total DNA extracted from aerobic and anaerobic enrichments from a Brazilian biodegraded petroleum sample. A sum of 10 clones were selected in order to evaluate their ability to produce exopolymers (EPS) with emulsifying activity, as well as to characterize the gene sequences, harbored by the fosmid clones, through 454 pyrosequencing. Functional analyses confirmed the ability of some clones to produce surfactant compounds. Regarding hydrocarbon as microbial carbon sources, n-alkane (in mixture or not) and naphthalene were preferentially consumed as substrates. Analysis of sequence data set revealed the presence of genes related to xenobiotics biodegradation and carbohydrate metabolism. These data were corroborated by the results of hydrocarbon biodegradation and biosurfactant production detected in the evaluated clones.