David Mead

Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli

Despite recent advances in sequencing, complete finishing of large genomes and analysis of novel proteins they encode typically require cloning of specific regions. However, many of these fragments are extremely difficult to clone in current vectors. Superhelical stress in circular plasmids can generate secondary structures that are substrates for deletion, particularly in regions that contain numerous tandem or inverted repeats. Common vectors also induce transcription and translation of inserted fragments, which can select against recombinant clones containing open reading frames or repetitive DNA. Conversely, transcription from cloned promoters can interfere with plasmid stability. We have therefore developed a novel Escherichia coli cloning vector (termed ‘pJAZZ’ vector) that is maintained as a linear plasmid. Further, it contains transcriptional terminators on both sides of the cloning site to minimize transcriptional interference between vector and insert. We show that this vector stably maintains a variety of inserts that were unclonable in conventional plasmids. These targets include short nucleotide repeats, such as those of the expanded Fragile X locus, and large AT—rich inserts, such as 20-kb segments of genomic DNA from Pneumocystis, Plasmodium, Oxytricha or Tetrahymena. The pJAZZ vector shows decreased size bias in cloning, allowing more uniform representation of larger fragments in libraries.

One-plasmid tunable coexpression for mycobacterial protein-protein interaction studies.

A single plasmid that allows controlled coexpression has been developed for use in mycobacteria. The tetracycline inducible promoter, PtetO, was used to provide tetracycline‐dependent induction of one gene, while the Psmyc, Pimyc, or Phsp promoters were used to provide three different levels of constitutive expression of a second gene. The functions of these four individual promoters were established using green fluorescent protein (GFP) and a newly identified red fluorescence inducible protein from Geobacillus sterothermophilus strain G1.13 (RFIP) as reporters. The tandem use of GFP and RFIP as reporter genes allowed optimization of the tunable coexpression in Mycobacterium smegmatis; either time at a fixed inducer concentration or changes in inducer concentration could be used to control the protein:protein ratio. This single vector system was used to coexpress the two‐protein Mycobacterium tuberculosis stearoyl‐CoA Δ9 desaturase complex (integral membrane desaturase Rv3229c and NADPH oxidoreductase Rv3230c) in M. smegmatis. The catalytic activity was found to increase in a manner corresponding to increasing the level of Rv3230c relative to a fixed level of Rv3229c. This system, which can yield finely tuned coexpression of the fatty acid desaturase complex in mycobacteria, may be useful for study of other multicomponent complexes. Furthermore, the tunable coexpression strategy used herein should also be applicable in other species with minor modifications.

Functional Metagenomics of a Replicase from a Novel Hyperthermophilic Aquificales Virus

Bacteriophage and viral replisomes typically require fewer proteins to replicate their genome compared to their cellular counterparts and are therefore model systems for studying this fundamental and ubiquitous process. Replication elements also tend to be arranged in a cluster or operon, as opposed to the distributed replication genes found in Bacteria and Archaea. A gene encoding a DNA polymerase with innate reverse transcriptase activity was previously isolated from an uncultivated Octopus hot spring viral metagenome sample collected in Yellowstone National Park. This report describes the complete metagenomic sequence of Octopus Spring OS3173 virus, novel structural variants, and new functionally active polymerase derivatives. The 37,256 bp dsDNA circular viral genome contains 48 open reading frames, with numerous genes associated with replication, including a full-length, polyprotein-like variant of the polymerase. OS3173 is predicted to infect an Aquificales host, as multiple clustered regularly interspaced short palindromic repeat (CRISPR) sequences matching seven locations in the virus genome are found within a pink filament streamer microbial community metagenome from Octopus Spring. Bioinformatic analysis of the DNA surrounding the CRISPR spacer region matches portions of a cultivated Thermocrinis ruber genome from the same location with high sequence identity. Enzymatic screening of large-insert clones yielded numerous polyprotein-containing genes encoding active thermostable variants from this virus, confirming the functional diversity of the polymerase in its native habitat. One variant demonstrated robust PCR capabilities compared to the original “wild-type” enzyme.