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The integron-gene cassette system contributes to multiple antibiotic resistance in bacteria and is likely to be of broader evolutionary significance. However, the majority of integron diversity consists of chromosomal integrons (CIs), with mostly unknown phenotypes, which are poorly characterized. A pUC-based reporter plasmid (pUS23) was developed containing a recombination site [aadB 59 base element (59-be)] upstream of promoterless aadB [gentamicin (Gm) resistance] and gfp (green fluorescence) genes, and this construct was used to investigate the recombination and expression activities of the CI in Pseudomonas stutzeri strain Q. Electroporation of pUS23 into P. stutzeri Q gave ampicillin-resistant transformants, which yielded [Gm.sup.R] green fluorescent recombinants after plating on Gm medium. Site-specific integration of pUS23 at attl was detected by [P.sub.c]R in 8% of [Gm.sup.R] colonies and the frequency of attl integration was estimated as 2.0 x [10.sup.-8] per P. stutzeri Q(pUS23) cell. RT-[P.sub.c]R confirmed integron-mediated expression of aadB in one recombinant strain (Q23-17) and a promoter ([P.sub.c]) was localized to the 5' end of the intl gene. The integrated pUS23 and flanking integron DNA were cloned from genomic DNA of strain Q23-17 and sequenced, confirming that site-specific integration of the entire reporter plasmid had occurred at the attl site. An insertion sequence (ISPst5; IS5 family) was discovered in the vector backbone of the reporter plasmid integrated at attl and also in a pUS23 derivative recovered as a plasmid in Escherichia coli JM 109. This is the first demonstration that wild-type CIs can capture gene cassettes and express cassette-associated genes.

Author(s): Jessica L. Green (corresponding author) [1, 4]; Andrew J. Holmes [2]; Mark Westoby [1]; Ian Oliver [3]; David Briscoe [1]; Mark Dangerfield [1]; Michael Gillings [1]; Andrew J. Beattie [...]

Researchers have developed a technique for detecting bacterial genes in environmental samples even when a genetic sequence is lacking. It is a PCR assay based on the gene cassettes associated with integrons. Most of the gene cassettes contained open reading frames that were not homologous to any known sequence.

Researchers have discovered an unknown group of methanotrophic bacteria that participates in the soil sink for atmospheric methane. Monooxygenase gene analysis indicates that the bacteria belong to the alpha subclass of Proteobacteria and are only distantly related to other methane-oxidizing strains.

The methanotrophs have a lot of biotechnological potential and are important in the global methane cycle. In an experiment, methane-oxidizing bacteria were utilized to assess the specificity and efficiency of rRNA-targeted group-specific probes. Methanotroph group specific probes did not give a signal with samples from these enrichments, though a methanol dehydrogenase structural gene was detected. Enrichment on methane combined with the screening with the broad specific methanol dehydrogenase probe facilitated detection of novel methanotrophs that were not detected with the original suit of methanotroph group-specific probes.

Research has been conducted on Pseudomonas stutzeri. The isolation of these integron-containing bacteria from soil samples has been carried out, and the results demonstrate that integrons are present in some but not all strains.

Trauma- and stress-related disorders are clinically heterogeneous and associated with substantial genetic risk. Understanding the biological origins of heterogeneity of key intermediate phenotypes such as cognition and emotion can provide novel mechanistic insights into disorder pathogenesis. Performing quantitative genetics in animal models is a tractable strategy for examining both the genetic basis of intermediate phenotypes and functional testing of candidate quantitative traits genes (QTGs). Here, existing and newly collected data were used for collaborative genome-wide mapping of cued fear acquisition and expression in 65 mouse strains from the BXD genetic reference panel. For fear acquisition, we identified a significant locus on chromosome (Chr) 10 and eight suggestive loci on Chr 2, 4, 5, 11, 13, and 15. For fear expression, we identified one significant and another highly suggestive locus on Chr 13, as well as four suggestive loci on Chr 10, 11, and X. Across these loci, 60 putative QTGs were identified. The quantitative trait locus on distal Chr 13 contained a single, highly promising gene at the location of the peak likelihood ratio statistic score. The gene, hyperpolarization-activated cyclic nucleotidegated channel 1 (Hcn1), regulates neuronal excitability. Validation experiments using behavioral pharmacology revealed that functional Hcn channels in the basolateral amygdala are necessary for conditioned fear acquisition and expression. Hcn1, together with the other candidate QTGs, thus provide new targets for neurobiological and treatment studies of fear learning and trauma- and stress-related disorders. [ABSTRACT FROM AUTHOR]

The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH4) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH4 and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure-function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C2-C4 alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis-this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3 nmol min-1 per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C2-C4 alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible. [ABSTRACT FROM AUTHOR]

Integrons facilitate the evolution of complex phenotypes by physical and transcriptional linkage of genes. They can be categorized as chromosomal integrons (CIs) or mobile resistance integrons (MRIs). The significance of MRIs for the problem of multiple antibiotic resistance is well established. CIs are more widespread, but their only demonstrated significance is as a reservoir of gene cassettes for MRIs. In characterizing CIs associated with Pseudomonas, we discovered a subfamily of insertion sequences, termed the IS1111-attC group, that insert into the recombination sites of gene cassettes (attC site) by site-specific recombination. IS1111-attC elements appear to have recently spread from Pseudomonas species to clinical class 1 integrons. Such elements are expected to significantly impact integrons. To explore this further, we examined CIs in 24 strains representing multiple levels of evolutionary divergence within the genus Pseudomonas. Cassette arrays frequently had a degenerated "footprint" of an IS1111-attC group element at their terminus and in three cases were occupied by multiple functional IS1111-attC elements. Within Pseudomonas spp. the IS-integron interaction appears to follow an evolutionarily rapid cycle of infection, expansion, and extinction. The final outcome is extinction of the IS element and modification of the right-hand boundary of the integron. This system represents an unusual example of convergent evolution whereby heterologous families of site-specific recombinases of distinct genetic elements have adopted the same target site. The interactions described here represent a model for evolutionary processes that offer insights to a number of aspects of the biology of integrons and other mosaic genetic elements. [ABSTRACT FROM AUTHOR]