Integrase-directed recovery of functional genes from genomic libraries

Large population sizes, rapid growth and 3.8 billion years of evolution firmly establish microorganisms as a major source of the planet''s biological and genetic diversity. However, up to 99% of the microorganisms in a given environment cannot be cultured. Culture-independent methods that directly access the genetic potential of an environmental sample can unveil new proteins with diverse functions, but the sequencing of random DNA can generate enormous amounts of extraneous data. Integrons are recombination systems that accumulate open reading frames (gene cassettes), many of which code for functional proteins with enormous adaptive potential. Some integrons harbor hundreds of gene cassettes and evidence suggests that the gene cassette pool may be limitless in size. Accessing this genetic pool has been hampered since sequence-based techniques, such as hybridization or PCR, often recover only partial genes or a small subset of those present in the sample. Here, a three-plasmid genetic strategy for the sequence-independent recovery of gene cassettes from genomic libraries is described and its use by retrieving functional gene cassettes from the chromosomal integron of Vibrio vulnificus ATCC 27562 is demonstrated. By manipulating the natural activity of integrons, we can gain access to the caches of functional genes amassed by these structures.     

Marker Gene Controversy in Transgenic Plants

Biosafety implications of selectable marker genes that are integrated into the transgenic plants are discussed. In the laboratory, selectable marker genes are used at two stages to distinguish transformed cells out of a large population of nontransformed cells: 1) initial assembly of gene cassettes is generally done in E. coli on easily manipulatable plasmid vectors that contain the selectable marker genes which often code for antibiotic inactivating enzymes, and 2) Then the gene cassettes are inserted into the plant genome by various transformation methods. For selection of transformed plant cells, antibiotic and herbicide resistance genes are widely used. Consequently, transgenic plants can end up with DNA sequences of selectable markers that are functional in E. coli and plants. The potential for horizontal gene transfer of selectable markers from transgenic plants to other organisms both in the environment and in the intestine of humans and animals is evaluated. Mechanisms and consequences of the transfer of marker genes from plants to other organisms is examined. Strategies to avoid marker genes in plants are discussed. It is possible to avoid the use of controversial selectable markers in the construction of transgenic plants.     

Characterization and chromosomal mapping of antimicrobial resistance genes in Salmonella enterica serotype typhimurium

Two hundred and twenty-six Salmonella enterica serotype Typhimurium isolates were examined for the presence of integron-associated gene cassettes. All but two of the non-DT104 isolates, together with DT104 isolates, contained gene cassettes. Amplicons of 1.5 kbp each were found in two non-DT104 isolates, encoding a dhfrI gene (trimethoprim resistance) linked to an aadA gene (streptomycin and spectinomycin resistance), by site-specific recombination. DT104 isolates of resistance (R) type ACSSuT possessed the recently described 1.0- and 1.2-kbp gene cassettes. Macrorestriction analysis with XbaI and DNA probing mapped ant(3")-1a, bla(PSE-1), and dhfrI genes to large multiresistant gene clusters in a DT170a isolate and a DT193 isolate. In contrast, all DT104 isolates (R-type ACSSuT) possessed a conserved 10-kbp Xba1 DNA fragment. Our study highlights the occurrence of integrons (and antimicrobial resistance determinants) among serotype Typhimurium isolates other than DT104. Larger and previously unrecognized multiresistance gene clusters were identified in these isolates by DNA probing.     

Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by ϕC31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. ϕC31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition.     

Mobile gene cassettes: a fundamental resource for bacterial evolution

Horizontal gene transfer increases genetic diversity in prokaryotes to a degree not allowed by the limitations of reproduction by binary fission. The integron/gene cassette system is one of the most recently characterized examples of a system that facilitates horizontal gene transfer. This system, discovered in the context of multidrug resistance, is recognized in a clinical context for its role in allowing pathogens to adapt to the widespread use of antibiotics. Recent studies suggest that gene cassettes are common and encode functions relevant to many adaptive traits. To estimate the diversity of mobile cassettes in a natural environment, a molecular technique was developed to provide representative distributions of cassette populations at points within a sampling area. Subsequently, statistical methods analogous to those used for calculating species diversity were employed to assess the diversity of gene cassettes within the sample area in addition to gaining an estimate of cassette pool size. Results indicated that the number of cassettes within a 5x10-m sample area was large and that the overall mobile cassette metagenome was likely to be orders of magnitude larger again. Accordingly, gene cassettes appear to be capable of mobilizing a significant genetic resource and consequently have a substantial impact on bacterial adaptability.     

Gene cassettes from the insert region of integrons are excised as covalently closed circles

Integrons are DNA elements which generally include one or more discrete gene cassettes inserted at a specific site. We have recently proposed a model for the acquisition and dissemination of genes found in the insert region of integrons, which requires the existence of circularized gene cassettes. Evidence for the existence of covalently closed circular molecules consisting of one or more gene cassettes has now been obtained. Low levels of small molecules which hybridize to probes specific for individual gene cassettes were detected in plasmid DNA isolated from cells containing a plasmid which includes an integron fragment with three gene cassettes aacC1, orfE and aadA2. These molecules were only detected when the gene encoding the integron DNA integrase was also present and are thus products of site-specific cassette excision. The excised cassettes have been shown to be in the form of covalently closed supercoiled circles, by digestion with restriction enzymes exonuclease III and DNase I. The circular excision products detected included either one cassette, aadA2 or orfE, two cassettes, aacC1 and orfE or all three cassettes. The predicted sequence of the recombinant junction in the excised aadA2 cassette confirmed that excision was precise. The predicted unique sequences of the 59-base elements associated with individual genes in the circular cassette form were compiled, and the sequences of the seven-base core sites which flank 59-base elements are now, with few exceptions, exact inverted repeats.     

Targeted transgene insertion into human chromosomes by adeno-associated virus vectors

Efficient methods are needed for the precise genetic manipulation of diploid human cells, in which cellular senescence and low conventional gene targeting rates limit experimental and therapeutic options. We have shown previously that linear, single-stranded DNA vectors based on adeno-associated virus (AAV) could accurately introduce small (<20 bp) genetic modifications into homologous human chromosomal sequences. Here we have used AAV vectors to introduce large (>1 kb) functional transgene cassettes into the hypoxanthine phosphoribosyl transferase (HPRT) and Type I collagen (COL1A1) loci in normal human fibroblasts. The transgene cassettes are inserted at high frequencies (1% of the total cell population under optimal conditions) and without secondary mutations. Selection for the inserted transgene cassette can be used to enrich for targeting events, such that >70% of surviving cells have undergone gene targeting with an appropriately designed vector. This approach should prove useful both for functional genomic analysis in diploid human cells and for therapeutic gene targeting.     

Site-specific insertion of gene cassettes into integrons

Site-specific insertion of gene cassettes into the insert region of integrons has been demonstrated. Insertion was only observed if the integron DNA integrase was expressed in the recipient cell and if the cassette DNA was ligated prior to transformation. The essential ligation products were resistant to treatment with exonuclease III, indicating that they were closed circular molecules. Insertion of cassettes into integron fragments containing either no insert (one recombination site), or one gene cassette (two recombination sites), was demonstrated. In the latter case, insertion occurred predominantly at the core site located 5′ to the resident cassette, which corresponds to the only site available when no insert is present in the recipient. When DNA molecules including two gene cassettes were used, insertion of only one of the gene cassettes was generally observed, suggesting that resolution of the circular molecule to generate two independent circular cassettes occurred more rapidly than insertion into the recipient integron.     

Conservation of Gene Cassettes among Diverse Viruses of the Human Gut

Viruses are a crucial component of the human microbiome, but large population sizes, high sequence diversity, and high frequencies of novel genes have hindered genomic analysis by high-throughput sequencing. Here we investigate approaches to metagenomic assembly to probe genome structure in a sample of 5.6 Gb of gut viral DNA sequence from six individuals. Tests showed that a new pipeline based on DeBruijn graph assembly yielded longer contigs that were able to recruit more reads than the equivalent non-optimized, single-pass approach. To characterize gene content, the database of viral RefSeq proteins was compared to the assembled viral contigs, generating a bipartite graph with functional cassettes linking together viral contigs, which revealed a high degree of connectivity between diverse genomes involving multiple genes of the same functional class. In a second step, open reading frames were grouped by their co-occurrence on contigs in a database-independent manner, revealing conserved cassettes of co-oriented ORFs. These methods reveal that free-living bacteriophages, while usually dissimilar at the nucleotide level, often have significant similarity at the level of encoded amino acid motifs, gene order, and gene orientation. These findings thus connect contemporary metagenomic analysis with classical studies of bacteriophage genomic cassettes. Software is available at https://sourceforge.net/projects/optitdba/.     

Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA.

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

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.     

Biolistic transformation of wheat: increased production of plants with simple insertions and heritable transgene expression

The feasibility of map-based cloning in wheat has been demonstrated recently, opening new perspectives for a better understanding of wheat plant biology and for accelerating wheat improvement in the coming decades. To validate the function of candidate genes, an efficient transformation system is needed. Here, we have performed two methods for wheat transformation using particle bombardment that ensures the production of transgenic plants with simple integration patterns for research purposes and stable transgene expression for accurate and rapid validation of gene function. To establish this method, we used the bar and pmi selectable genes either as part of whole plasmids, gene cassettes (obtained by PCR or purified on agarose gels), or as dephosphorylated cassettes. The analysis of about 300 transgenic plants showed that the use of gene cassettes or dephosphorylated gene cassettes leads to a majority (50–60 %) of simple integration events. This is significantly higher than the number of simple events obtained with whole plasmids (9–25 %). Moreover, the decrease of the quantity of DNA from 500 to 5 ng/µl for PCR-amplified cassettes used for transformation increased the number of single integration events. The transformation efficiency remained stable at 2.5 %, and a higher number of plants expressing the transgenes were obtained with the dephosphorylated cassette. No correlation was observed between the complexity of the events and stability of expression of the transgene, suggesting that plasmid sequences could be involved on transgene silencing. The inheritability of the transgene was demonstrated in T1 and T2 generations. These results show that biolistic transformation of dephosphorylated gene cassettes provides an easy and efficient route to produce backbone vector-free transgenic wheat carrying and expressing intact and single transgenes.     

Characterization and Chromosomal Mapping of Antimicrobial Resistance Genes in Salmonella enterica Serotype Typhimurium

Two hundred and twenty-six Salmonella enterica serotype Typhimurium isolates were examined for the presence of integron-associated gene cassettes. All but two of the non-DT104 isolates, together with DT104 isolates, contained gene cassettes. Amplicons of 1.5 kbp each were found in two non-DT104 isolates, encoding a dhfrI gene (trimethoprim resistance) linked to an aadA gene (streptomycin and spectinomycin resistance), by site-specific recombination. DT104 isolates of resistance (R) type ACSSuT possessed the recently described 1.0- and 1.2-kbp gene cassettes. Macrorestriction analysis with XbaI and DNA probing mapped ant(3")-1a, bla_PSE-1, and dhfrI genes to large multiresistant gene clusters in a DT170a isolate and a DT193 isolate. In contrast, all DT104 isolates (R-type ACSSuT) possessed a conserved 10-kbp Xba1 DNA fragment. Our study highlights the occurrence of integrons (and antimicrobial resistance determinants) among serotype Typhimurium isolates other than DT104. Larger and previously unrecognized multiresistance gene clusters were identified in these isolates by DNA probing.     

Efficient microprojectile bombardment-mediated transformation of rice using gene cassettes

This study was aimed at determining whether gene cassettes (promoter-coding sequence-terminator) can be efficiently used in microprojectile acceleration-mediated co-transformation of rice in the place of whole plasmids, and to what extent their use influences the integration and expression of the co-transferred gene of interest. Two non-linked marker genes (yfp and hph) were co-introduced by microprojectile bombardment into cells of embryogenic calli in three separate experiments. Three different DNA structures were compared for their ability to transiently and stably transform rice cells: supercoiled or linearized whole-plasmid DNA, gene cassette DNA and single-stranded gene cassette DNA coated with Escherichia coli single-stranded binding (SSB) proteins. Our results demonstrate that microprojectile bombardment-mediated transformation of rice using gene cassettes is possible without significantly reducing transformation efficiency in comparison to the use of whole-plasmid DNA. Furthermore, no obvious difference in transgene integration pattern and inheritance was observed among plants transformed with gene cassettes compared to those transformed with the whole plasmid, except that concatemerization of molecules prior to integration was rarely observed in gene cassette transformants. Received: 4 April 2001 / Accepted: 13 August 2001     

Inhibition of hepatitis B virus replication with linear DNA sequences expressing antiviral micro-RNA shuttles

RNA interference (RNAi) may be harnessed to inhibit viral gene expression and this approach is being developed to counter chronic infection with hepatitis B virus (HBV). Compared to synthetic RNAi activators, DNA expression cassettes that generate silencing sequences have advantages of sustained efficacy and ease of propagation in plasmid DNA (pDNA). However, the large size of pDNAs and inclusion of sequences conferring antibiotic resistance and immunostimulation limit delivery efficiency and safety. To develop use of alternative DNA templates that may be applied for therapeutic gene silencing, we assessed the usefulness of PCR-generated linear expression cassettes that produce anti-HBV micro-RNA (miR) shuttles. We found that silencing of HBV markers of replication was efficient (>75%) in cell culture and in vivo. miR shuttles were processed to form anti-HBV guide strands and there was no evidence of induction of the interferon response. Modification of terminal sequences to include flanking human adenoviral type-5 inverted terminal repeats was easily achieved and did not compromise silencing efficacy. These linear DNA sequences should have utility in the development of gene silencing applications where modifications of terminal elements with elimination of potentially harmful and non-essential sequences are required.     

Generation of Campylobacter fetus S-layer protein diversity utilizes a single promoter on an invertible DNA segment

Wild-type strains of Campylobacter fetus contain a monomolecular array of surface layer proteins (SLPs) and vary the antigenicity of the predominant SLP expressed. Reciprocal recombination events among the eight genomic SLP gene cassettes, which encode 97- to 149 kDa SLPs, permit this variation. To explore whether SLP expression utilizes a single promoter, we created mutant bacterial strains using insertional mutagenesis by rescue of a marker from plasmids. Experimental analysis of the mutants created clearly indicates that SLP expression solely utilizes the single sapA promoter, and that for variation C. fetus uses a mechanism of DNA rearrangement involving inversion of a 6.2 kb segment of DNA containing this promoter. This DNA inversion positions the sapA promoter immediately upstream of one of two oppositely oriented SLP gene cassettes, leading to its expression. Additionally, a second mechanism of DNA rearrangement occurs to replace at least one of the two SLP gene cassettes bracketing the invertible element. As previously reported promoter inversions in prokaryotes, yeasts and viruses involve alternate expression of at most two structural genes, the ability of C. fetus to use this phenomenon to express one of multiple cassettes is novel.     

High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes

We have developed a simple binary vector construction system for the simultaneous expression of multiple genes in plants. Up to three independent gene cassettes can be easily integrated into one binary vector using the MultiSite Gateway System. Using this system, we produced transgenic rice plants that accumulated high levels of the hypocholesterolaemic peptide lactostatin (IIAEK) in endosperm. Binary vectors were constructed that could accommodate up to three independent modified glutelin gene cassettes encoding multimer lactostatin in the variable regions. Eight construct permutations were used for rice transformation. We measured the accumulation of lactostatin expressed as a glutelin fusion protein in the mature seeds of 105 independent transgenic rice lines. A general correlation was observed between accumulation level and gene number in the vector constructs, indicating that a higher accumulation of lactostatin was obtained from transgenic rice plants containing the maximum number of gene inserts. These results indicate that this strategy is applicable for the selection of transgenic lines containing large amounts of bioactive peptides in rice seeds.     

Nested DNA inversion of Campylobacter fetus S-layer genes is recA dependent.

Wild-type strains of Campylobacter fetus are covered by a monomolecular array of surface layer proteins (SLPs) critical for virulence. Each cell possesses eight SLP gene cassettes, tightly clustered in the genome, that encode SLPs of 97 to 149 kDa. Variation of SLP expression occurs by a mechanism of nested DNA rearrangement that involves the inversion of a 6.2-kb sapA promoter-containing element alone or together with one or more flanking SLP gene cassettes. The presence of extensive regions of identity flanking the 5' and 3' ends of each SLP gene cassette and of a Chi-like recognition sequence within the 5' region of identity suggests that rearrangement of SLP gene cassettes may occur by a generalized (RecA-dependent) homologous recombination pathway. To explore this possibility, we cloned C. fetus recA and created mutant strains by marker rescue, in which recA is disrupted in either S+ or S- strains. These mutants then were assessed for their abilities to alter SLP expression either in the presence or absence of a complementary shuttle plasmid harboring native recA. In contrast to all previously reported programmed DNA inversion systems, inversion in C. fetus is recA dependent.     

Structural organization and mechanisms of mobility of the gene cassette coding for resistance to antibiotics and bacterial virulence factors

The review is focussed on two types of gene cassettes which are significant in bacterial variability. The first type are cassettes with antibiotic resistance genes; these are the smallest mobile genetic elements including a gene (most commonly an antibiotic resistance gene) and a short sequence acting as a recombination site. Sometimes these cassettes contain genes not responsible for antibiotic resistance but their functions are not yet known. The second type contains large clusters of genes coding for bacterial virulence factors. They were termed "pathogenicity islands" due to their difference in the percentage of G-C pairs in comparison with bacterial chromosomes, in which they are contained. The structural organization and mechanisms of mobility of various types of gene cassettes are discussed.