Harold W. Stokes

Structure and function of 59‐base element recombination sites associated with mobile gene cassettes

The integration of gene cassettes into integrons is effected by site‐specific recombination catalysed by an integrase, IntI, encoded by the integron. The cassette‐associated recombination sites, 59‐base elements, are not highly conserved and vary in length from 57 to 141u2003bp. They can be identified by their location and the relationship of over 20u2003bp at their outer ends to consensus sequences that are imperfect inverted repeats of one another. The recombination cross‐over occurs close to one end of the 59‐base element, within a conserved core site with the consensus sequence GTTAGGC or GTTRRRY. By introducing single‐base changes at each of these positions in the aadB 59‐base element, bases that are critical for site activity were identified. The recombination cross‐over was also localized to a unique position between the adjacent G and T residues. Changes introduced in the conserved AAC of the inverse core site (GCCTAAC or RYYYAAC) located at the opposite end of the 59‐base element also reduced site activity but to a lesser extent. Sequences of rare recombinants revealed an alternative position for strand exchange and led to the conclusion that 59‐base elements comprise two simple sites, analogous to those recognized by other integrases, with each simple site made up of a pair of inversely oriented IntI binding domains separated by a spacer of 7 or 8u2003bp. Re‐examination of the sequences of all known 59‐base elements revealed that this simple site configuration was present at both the left and right ends in all 59‐base elements. The identity of bases in the spacer is not required for efficient recombination and the cross‐over is located at one end of the spacer, suggesting that during IntI1‐mediated recombination only one strand exchange occurs.

Integrons: novel DNA elements which capture genes by site-specific recombination.

Integrons are unusual DNA elements which include a gene encoding a site-specific DNA recombinase, a DNA integrase, and an adjacent site at which a wide variety of antibiotic resistance and other genes are found as inserts. One or more genes can be found in the insert region, but each gene is part of an independent gene cassette. The inserted genes are expressed from a promoter in the conserved sequences located 5′ to the genes, and integrons are thus natural expression vectors. A model for gene insertion in which circular gene cassettes are inserted individually via a single site-specific recombination event has been proposed and verified experimentally. The gene cassettes include a gene coding region and, at the 3′ end of the gene an imperfect inverted repeat, a 59-base element. The 59-base elements are a diverse family of elements which function as sites recognized by the DNA integrase. Site-specific insertion of individual genes thus represents a further mechanism which contributes to the evolution of the genomes of Gram-negative bacteria and their plasmids and transposons.Members of the most studied class of integrons, which include thesulI gene in the conserved sequences, are believed to be mobile DNA elements on the basis that they are found in many independent locations, and a discrete boundary is found at the outer end of the 5′-conserved segment. However, the length of the 3′-conserved segment is variable in the integrons examined to date, and it is likely that this variability has arisen as the result of insertion and deletion events. Though the true extent of the 3′-conserved segment remains to be determined, it seems likely that these integrons are mobile DNA elements. The second known class of integrons comprises members of the Tn7 transposon family.

Gene Cassette PCR: Sequence-Independent Recovery of Entire Genes from Environmental DNA

ABSTRACT The vast majority of bacteria in the environment have yet to be cultured. Consequently, a major proportion of both genetic diversity within known gene families and an unknown number of novel gene families reside in these uncultured organisms. Isolation of these genes is limited by lack of sequence information. Where such sequence data exist, PCR directed at conserved sequence motifs recovers only partial genes. Here we outline a strategy for recovering complete open reading frames from environmental DNA samples. PCR assays were designed to target the 59-base element family of recombination sites that flank gene cassettes associated with integrons. Using such assays, diverse gene cassettes could be amplified from the vast majority of environmental DNA samples tested. These gene cassettes contained complete open reading frames, the majority of which were associated with ribosome binding sites. Novel genes with clear homologies to phosphotransferase, DNA glycosylase, methyl transferase, and thiotransferase genes were identified. However, the majority of amplified gene cassettes contained open reading frames with no identifiable homologues in databases. Accumulation analysis of the gene cassettes amplified from soil samples showed no signs of saturation, and soil samples taken at 1-m intervals along transects demonstrated different amplification profiles. Taken together, the genetic novelty, steep accumulation curves, and spatial heterogeneity of genes recovered show that this method taps into a vast pool of unexploited genetic diversity. The success of this approach indicates that mobile gene cassettes and, by inference, integrons are widespread in natural environments and are likely to contribute significantly to bacterial diversity.

Gene cassettes encoding resistance to quaternary ammonium compounds: a role in the origin of clinical class 1 integrons?

DNA sequencing, phylogenetic and mapping studies suggest that the class 1 integron found in pathogens arose when one member of the diverse family of environmental class 1 integrons became embedded into a Tn402 transposon. However, the timing of this event and the selective forces that first fixed the newly formed element in a bacterial lineage are still unknown. Biocides have a longer use in clinical practice than antibiotics, and a qac (quaternary ammonium compound) resistance gene, or remnant thereof, is a normal feature of class 1 integrons recovered from clinical isolates. Consequently, it is possible that the initial selective advantage was conferred by resistance to biocides, mediated by qac. Here, we show that diverse qac gene cassettes are a dominant feature of cassette arrays from environmental class 1 integrons, and that they occur in the absence of any antibiotic resistance gene cassettes. They are present in arrays that are dynamic, acquiring and rearranging gene cassettes within the arrays. The abundance of qac gene cassettes makes them a likely candidate for participation in the original insertion into Tn402, and as a source of a readily selectable phenotype. More broadly, the increasing use of qac and other biocides at the present time seems likely to promote the fixation of further novel genetic elements, with unpredictable and potentially adverse consequences for human health and agriculture.

Integron-encoded IntI integrases preferentially recognize the adjacent cognate attI site in recombination with a 59-be site.

Integrons have the capacity to capture small mobile elements known as gene cassettes, and this reaction is catalysed by integron‐encoded IntI integrases. IntI integrases form a distinct family within the tyrosine recombinase superfamily and include a characteristic additional domain that is well conserved. Two different IntI enzymes were used to examine their ability to recognize heterologous attI sites in both integration and excision assays. IntI1 and IntI3 are 59% identical and catalyse both integrative and excisive recombination between a cassette‐associated 59‐be site and the cognate attI1 or attI3 site. Integrative recombination events involving a 59‐be and a non‐cognate attI site, attI2 and attI3 for IntI1 or attI1 and attI2 for IntI3, were detected extremely rarely. In cassette excision assays, the non‐cognate attI3 site was recognized by IntI1, but attI1 was not well recognized by IntI3. The purified IntI1 and IntI3 proteins bound strongly only to their cognate attI site.

Identification of a chloroplast-encoded secA gene homologue in a chromophytic alga: possible role in chloroplast protein translocation.

SummarySecA is one of seven Sec proteins that comprise the prokaryotic protein translocation apparatus. A chloroplast-encoded secA gene has been identified from the unicellular chromophytic alga Pavlova lutherii. The gene predicts a protein that is related to the SecA proteins of Escherichia coli and Bacillus subtilis. The presence of secA, as well as the previously described secY and hsp70 genes, on the chloroplast genome of P. lutherii suggests that this eukaryotic organism utilises protein translocation mechanisms similar to those of bacterial cells.

The integron In1 in plasmid R46 includes two copies of the oxa2 gene cassette

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.

Genetic Exchange in Natural Microbial Communities

Genetic exchange between bacteria was first observed over 60 years ago (Griffith, 1928). In recent years, considerable advances have been made in the understanding of the molecular mechanisms involved in bacterial gene transfer. We now have a clear understanding of the three basic mechanisms of genetic exchange in bacteria: conjugation, transformation, and transduction. Most of these studies were, however, performed using pure cultures of bacteria and genetic transfer was regarded largely as a laboratory phenomenon (De Flaun et al., 1990). More recently, genetic exchange by each of these mechanisms has been demonstrated in a variety of natural environments (Table I).

Heat shock Hsp70 protein is chloroplast-encoded in the chromophytic alga Pavlova lutherii

Heat shock proteins are ubiquitous and highly conserved. Recently they have become implicated in the import of proteins into organelles. All the heat shock genes characterized to date, however, are known or assumed to be encoded in the nuclear genome even if the corresponding protein can be localised in the mitochondrion or chloroplast. In contrast, we identify here an hsp70 gene in the unicellular chromophytic alga Pavlova lutherii which is located on the chloroplast genome. Localisation of this gene to the chloroplast chromosome is confirmed by Southern blot analysis and pulse-field gel electrophoresis which also reveals that the length of the P. lutherii chloroplast chromosome is 115 kb. We compare the predicted protein of this hsp70 gene with that of maize and of the analogous proteins in the prokaryotic organisms Escherichia coli and Synechocystis PCC6803. The greatest identity is found with the cyanobacterium Synechocystis PCC6803.

Characterisation of a chloroplast‐encoded sec Y homologue and atpH from a chromophytic alga Evidence for a novel chloroplast genome organisation

sec Y is a prokaryotic gene that encodes the SecY protein, an integral membrane component of the prokaryotic protein translocation apparatus. A chloroplast‐encoded sec Y homologue has been identified in the unicellular, chromophytic alga, Pavlova lutherii. The gene predicts a protein composed of ten membrane‐spanning regions, that is approximately 25% homologous and 50% similar to bacterial and plastid SecY proteins. The sec Y gene from P. lutherii is independent of the ribosomal protein (rp) gene cluster to which it is closely linked in other organisms. In P. lutherii sec Y is located 5′ to atpI and atpH. Since, in higher plants the atpIHFA gene cluster and the rp gene cluster are separated by approximately 50 kb, we conclude, this indicates a novel chloroplast gene arrangement in P. lutherii.