Integration of a laterally acquired gene into a cell network important for growth in a strain of Vibrio rotiferianus
© Labbate et al; licensee BioMed Central Ltd. 2011
Received: 5 July 2011
Accepted: 18 November 2011
Published: 18 November 2011
Lateral Gene Transfer (LGT) is a major contributor to bacterial evolution and up to 25% of a bacterium's genome may have been acquired by this process over evolutionary periods of time. Successful LGT requires both the physical transfer of DNA and its successful incorporation into the host cell. One system that contributes to this latter step by site-specific recombination is the integron. Integrons are found in many diverse bacterial Genera and is a genetic system ubiquitous in vibrios that captures mobile DNA at a dedicated site. The presence of integron-associated genes, contained within units of mobile DNA called gene cassettes makes up a substantial component of the vibrio genome (1-3%). Little is known about the role of this system since the vast majority of genes in vibrio arrays are highly novel and functions cannot be ascribed. It is generally regarded that strain-specific mobile genes cannot be readily integrated into the cellular machinery since any perturbation of core metabolism is likely to result in a loss of fitness.
In this study, at least one mobile gene contained within the Vibrio rotiferianus strain DAT722, but lacking close relatives elsewhere, is shown to greatly reduce host fitness when deleted and tested in growth assays. The precise role of the mobile gene product is unknown but impacts on the regulation of outermembrane porins. This demonstrates that strain specific laterally acquired mobile DNA can be integrated rapidly into bacterial networks such that it becomes advantageous for survival and adaptation in changing environments.
Mobile genes that are highly strain specific are generally believed to act in isolation. This is because perturbation of existing cell machinery by the acquisition of a new gene by LGT is highly likely to lower fitness. In contrast, we show here that at least one mobile gene, apparently unique to a strain, encodes a product that has integrated into central cellular metabolic processes such that it greatly lowers fitness when lost under those conditions likely to be commonly encountered for the free living cell. This has ramifications for our understanding of the role mobile gene encoded products play in the cell from a systems biology perspective.
The integron includes a site-specific recombination system that integrates and expresses genes present on mobile elements called gene cassettes . The integron platform is defined by three characteristics: an integrase gene (intI) whose product encodes a site-specific integrase, IntI, an attachment site (attI) at which point DNA sequences are inserted and a promoter (Pc) which expresses genes within the gene cassettes inserted at attI . Gene cassettes can be inserted into the integron as individual units but multiple events can lead to large tandem arrays. Integrons are best known for their role in the spread of antibiotic resistance genes in clinical environments . These clinical integrons harbour 1-6 gene cassettes and are often associated with mobile elements such as resistance plasmids and transposons . However, integrons are diverse genetic elements found in approximately 10% of environmental bacteria . In these bacteria, integrons are found in chromosomal locations and rarely carry antibiotic resistance gene cassettes indicating a general role in evolution.
Vibrio is a genus of highly adaptable bacteria found in diverse marine-associated niches . This adaptability is partly driven by lateral gene transfer (LGT), a process believed to be particularly important in this genus since the recent finding that Vibrio cholerae and other vibrios naturally take up DNA from the environment [5, 6]. In the vibrio, integron cassette arrays can comprise well in excess of 100 cassettes . Thus, the integron is a significant source of laterally acquired DNA in vibrio consisting of 1-3% of the total genome and generates genetic diversity even among closely related strains . For example, it is the only identified genomic region that differs between some strains responsible for the current V. cholerae pandemic . It has also been recently suggested that integron associated gene pools in the vibrios are important in adaptation to local environmental and ecological conditions .
Recent additional studies have provided new insight into the biology of vibrio integrons. The SOS stress response induces transcription of the integron-integrase increasing the rate of insertion, excision and shuffling of gene cassettes . Furthermore, the majority of gene cassettes in a 116-cassette array  located in the Vibrio rotiferianus strain DAT722  were found to be transcribed due to the presence of promoters distributed throughout the array . Thus, cassette transcription is not absolutely dependent on being near Pc. Collectively these findings suggest the integron provides a more prominent role in vibrio adaptation than previously thought.
Approximately 75% of integron associated gene cassette products in Vibrio species are novel with the remainder being designated with a putative function based on the presence of known domains through in silico analysis  or, to a very limited extent, by protein structural analysis . The novelty of gene cassette products has made them difficult targets to study. However, like most mobile DNA, gene cassettes are believed to provide their host with accessory phenotypes imparting a niche-specific advantage. The exemplar of this phenomenon is antibiotic resistance, where most of the genes driving resistance adaptation are highly mobile . This has also been supported by the handful of novel gene cassettes that have been characterised proving them to be functional and include genes potentially involved in pathogenesis in V. cholerae [14, 16–18]. It is easy to understand how a protein carrying out a single biochemical reaction, for example the chemical inactivation of an antibiotic, can act in isolation and confer a strong selective advantage when the containing cell is in an environment where a toxic compound is present. This argument can also be extended to self contained sets of genes (operons) that encode pathways conferring resistance to such things as mercury and chromate which have also been captured and spread by mobilizing elements. It is largely believed that highly mobile genes would be confined to such function-types since laterally acquired genes that influence core metabolic functions are likely to lower fitness when first captured . However we show here that at least one of eight novel cassettes associated with a vibrio integron encodes a product that is integrated into cell membrane porin regulation such that its loss would impact on cell fitness under physiological conditions that would normally be encountered by the free living host.
Deletion of cassettes reduces growth on some carbon sources
To confirm that the dramatic reduction in fitness of d8-60a was a result of the loss of a mobile cassette and not the consequence of a spontaneous mutation elsewhere in the genome of the isolate selected for analysis, two other independent mutants, d8-60b and d8-60c, comprising loss of the same cassettes were constructed and examined for their growth characteristics. The results for these two mutants showed significant growth impairment in minimal medium although not in a manner identical to d8-60a. In glucose, both d8-60b and d8-60c had significant lag phases of up to 14 hours compared to wild type DAT722 and d8-60a but thereafter grew to achieve wild type cell densities at 24 hours (Figure 2B). In pyruvate, d8-60b and d8-60c showed reduced growth rates compared to DAT722 although they were significantly better than d8-60a (Figure 2C).
Cassette deletions change the outermembrane protein profiles of cells
The mutants d8-60b and d8-60c had very similar porin profiles, a result consistent with the similar growth phenotypes displayed by these mutants. In 2M + pyruvate conditions, a significant down-regulation of the maltoporin (band 2) and the OmpU-like porin (band 5) but an up-regulation of OmpU (band 3) was observed when compared to the wild-type (Figure 5B). In 2M + LB nutrient medium, these mutants had reduced levels of the maltoporin (band 2) and the presence of the putative porin (band 4) protein in replacement of the OmpU-like porin (band 5) compared to the wild-type (Figure 5C).
Expression of a single gene cassette in transmaintains normal growth after generation of strains with deleted cassettes
Since mutant d16-60 (cassettes 16 to 60 deleted) had normal growth phenotypes compared to the wild-type, at least one cassette gene located between cassettes 7 and 16 has a strong pleiotropic affect. All eight cassettes within this region, except cassette 11, encode small hypothetical proteins with homology only to other cassette proteins. Therefore, nothing could be inferred regarding their putative function. However, cassette 11 includes a gene, encoding a 257 amino acid protein with pfam http://pfam.sanger.ac.uk/ identifying two domains; 1) an uncharacterized NERD domain at residues 31-150 that has weak homology to nucleases and is commonly associated with other protein domains involved in DNA processing , 2) a DNA-binding C4-zinc finger domain at residues 216-257 found in topoisomerase I proteins and involved in removing excessive negative supercoils from DNA . Based on this bioinformatics analysis one possible biochemical function of the cassette 11 gene product is as a DNA topoisomerase. In addition, experiments with a mutated topoisomerase I (topA) gene have described phenotypes that are similar to those observed in the d8-60 mutants. Most notably, in characterized topA mutants, this includes the requirement for a compensatory mutation, emergence of spontaneous mutants and alterations in the composition of outermembrane porin proteins [23–28].
The integron/gene cassette system is broadly dispersed amongst the Proteobacteria and is found in about 10% of sequenced genomes . In the vibrios it is ubiquitous with arrays generally being especially large. Despite the fact that the integron gene cassette "metagenome" pool is very large [29, 30], little is known about what the encoded proteins do beyond the enormous contribution some cassette proteins make to the antibiotic resistance problem . A conventional understanding of cell metabolism would suggest they encode accessory phenotypes providing their host with a niche-specific advantage. Antibiotic resistance is a classic example of this since cassettes containing antibiotic resistance genes quite clearly provide a selective advantage in clinical environments where antibiotics are frequently used . These highly mobilized genes frequently cross phylogenetic boundaries and a single gene can protect a cell from toxic compounds irrespective of the metabolic context in which it finds itself. The same phenomenon can extend to some adaptive genes that are part of a "self contained" unit as is the case, for example, in operons on transposons that confer mercury resistance .
The vibrios represent a diverse group of marine organisms and members of this group have very large cassette arrays. A typical vibrio cassette array comprises more than 100 novel genes . Moreover, they represent the most dynamic component of the genome. In V. cholerae, pandemic strains that are otherwise indistinguishable by most phylogenetic typing techniques can still have very disparate cassette arrays . Similarly, this is true for enclosed symbiotic communities of vibrios . This highly mobile pool of genes, in a metagenomic sense, therefore number in at least the thousands and probably orders of magnitude more . What do all these genes do? Many probably comprise functions that are metabolically independent of the rest of the cell in a manner analogous to antibiotic and heavy metal resistance genes. However, we show for the first time, that at least one mobile gene product can influence other aspects of core cell metabolism. In DAT722 this influence is such that at least one gene within the deleted region is highly adapted to this cell line to the extent that its loss reduces fitness to the point where the host cell is barely viable. The target gene or genes was contained to within a contiguous set of eight cassettes within the DAT722 array. Each of these cassettes contained a single predicted protein (Figure 1 and ). All of the predicted proteins are novel in that identical proteins are not present in any other known bacterium. Further, seven of the eight predicted proteins are highly novel to the point where they can only be described as hypothetical proteins. The remaining predicted protein, derived from cassette 11, is also novel although it contains a domain related to the DNA topoisomerase I family of proteins.
Although the precise function of this cassette protein needs to be established experimentally, the data generated was consistent with the hypothesis that the cassette 11 gene product was integrated into an essential cell network in the wild type DAT722. In particular, the fact that supplying this product alone in trans via pMAQ1082 preserved the wild type phenotype after subsequent deletion of cassettes 8 - 16 unambiguously points to an essential role in the cell porin regulatory network.
Overall, this study emphasizes the importance of LGT in bacterial evolution and that this process can bring rapid adaptation not only through acquisition of novel functional genes, but more importantly through gain of genes that alter a cell's regulatory network. Thus, mobile genes can be adaptive over very short time scales such that their loss can threaten the viability of the cell through the disruption of a core metabolic process. This is in contrast to the generally held view that mobile DNA contributes to cell fitness by providing additional protein/s that act largely independently of core cell networks. Also, this data reinforces the point that large integron arrays are not solely dependent on Pc for transcription since this cluster of genes if relatively distal to this promoter. It is clear therefore that despite the enormous increase in genomics and proteomic data in recent years, much is still to be learnt about the full of gamut of proteins necessary for important cell metabolic processes.
Strains, growth conditions and DNA purification
List of strains and plasmids
Strain or plasmid
Reference or source
V. rotiferianus DAT722
DAT722; Spontaneous SmR mutant.
DAT722-Sm; Single recombination cross-over of pVSD2 into cassette 61, KmR
DAT722-Sm; Δcassettes 8-60, SmR, KmR
DAT722-Sm; Δcassettes 8-60, SmR, KmR
DAT722-Sm; Δcassettes 8-60, SmR, KmR. Spontaneous mutant of d8-60b.
DAT722-Sm; Δcassettes 8-60, SmR, KmR
DAT722-Sm; Δcassettes 8-60, SmR, KmR. Spontaneous mutant of d8-60c.
DAT722-Sm; Δcassettes 16-60, SmR, KmR
F' proAB lacI q ZΔM15 Tn10/recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relAi, TcR
SY327 λ pir
Δ(lac pro) argE (Am) rif nalA recA56
SM10 λ pir
thi thr leu tonA lacY supE recA::RP4-2-Tc::Mu, Tcr KmR
Cloning vector, KmR
Cloning vector, ApR
pGEM-T Easy carrying a 1834-bp fragment. The fragment was created using fusion PCR and consists of, in order, a 448-bp of paralog group 1 sequence, a 964-bp fragment containing aphA1 and a 410-bp paralog group 2 sequence abutted by salI restriction sites.
Mobilisable sacB counter-selectable suicide vector, ApR
ColE1 oriV; RP4tra + RP4 oriT; CmR; helper plasmid in triparental matings
Low copy IPTG-inducible expression vector, CmR
salI fragment from pMAQ1080 cloned into the unique salI site of pCVD442.
pJAK16 containing cassette 11
Primers used in this study
GTC GAC CAA AAT TTG GCT GCT TGT TG
Paralog 1 gene cassettes in Vibrio rotiferianus DAT722
CAT CAG AGA TTT TGA GAC ACA ACC CGA GCG ACA ATT TTA AGC
Paralog 1 gene cassettes in Vibrio rotiferianus DAT722
GGC AGA GCA TTA CGC TGA TCA AAG GTC ATA AGT TTT GGT G
Paralog 2 gene cassettes in Vibrio rotiferianus DAT722
GTC GAC CAT GCG CTA CTT CTA TTT ATG C
Paralog 2 gene cassettes in Vibrio rotiferianus DAT722
GTT GTG TCT CAA AAT CTC TGA TG
aphA1 in pLOW2 (F)
TCA GCG TAA TGC TCT GCC
aphA1 in pLOW2 (R)
TGA GCT ACC ACA AGC AAG G
Cassette 5 in Vibrio rotiferianus DAT722 (F)
AAA GCG GTT ACA TTC GGG
Cassette 5 in Vibrio rotiferianus DAT722 (R)
ACA TAT GTA GAC CCT GTG CG
Cassette 25 in Vibrio rotiferianus DAT722 (F)
CAT TTT AAG TCG GCT CTT CC
Cassette 25 in Vibrio rotiferianus DAT722 (R)
GTA GGT AAT TTC GGC TTC TCG
Cassette 62 in Vibrio rotiferianus DAT722 (R)
TGC GCA ATA TAT CGC AAG AG
Cassette 25 in Vibrio rotiferianus DAT722 (F)
GCC GTC CAT AGT CGT CAT TT
Cassette 25 in Vibrio rotiferianus sp. DAT722 (R)
TTT TGG ATC CGA ATA GGG AAA ATC CGT G
Gene from cassette 11 in V. rotiferianus DAT722 (F)
TTT TCT GCA GTT AGT TGA ATT GTT TCA CAG C
Gene from cassette 11 in V. rotiferianus DAT722 (R)
DAT722 cassette analysis and strain construction
The cassette array of DAT722 is fully sequenced  and consists of 116 gene cassettes although there are 94 different cassette types due to the presence of paralogous cassettes . For the deletion of cassettes by homologous recombination, the presence of paralogous cassettes in different positions of the array was exploited. Two of the paralogous cassette types were selected based on their position in the array. The first paralogous cassette type (group 1) is in positions 6, 7, 15, 27, 49, 66, 71, 76, 77 and 111. The second paralogous group (group 2) is in positions 34, 61, 83, 87, 90, 93 and 105. Using fusion PCR, a 1834 bp DNA fragment consisting of, in order, a portion of group 1 sequence (448 bp), the aphA1 gene from pLOW2 (964 bp) and a portion of group 2 sequence (410 bp) was amplified and cloned into pGEM-T Easy producing pMAQ1080. The fragment was excised from pMAQ1080 using salI and cloned into the salI site of the sacB-counter selectable suicide vector pCVD442 to create pMAQ1081. Homologous recombination (allele replacement) was used to replace cassettes between group 1 and group 2 cassettes with the 1834 bp fragment created by fusion PCR. Plasmid pMAQ1081 was conjugated into DAT722-Sm using E. coli SM10 as a donor with recombinants selected on LB20 medium supplemented with 100 μg/ml and 25 μg/ml of kanamycin and streptomycin respectively. A merodiploid (designated MD7) was isolated with pMAQ1081 recombining into cassette 61 of the integron cassette array (see Figure 1). An overnight culture of MD7 was inoculated into fresh LB20 at a dilution of 10-6 and grown until turbidity was evident (~ 6 hours). For selection of double cross-over recombinants, a dilution series of the MD7 culture was plated onto LB medium containing 0.4% NaCl, 10% sucrose and 100 μg/ml kanamycin. Using primers targeting unique cassettes outside the expected deletions (Table 2), colonies were screened for the presence of deletions between 6/7 and 61, 15 and 61, 27 and 61 and 49 and 61. In the case of the mutants d8-60a, d8-60b, d8-60c, all three generated identical length PCR products by this method indicating identical deletion end points.
Membrane protein analysis
The outer membrane proteins (OMPs) were extracted as previously described  using equal number of cells (equivalent to 5 ml of cells diluted to an OD600 of 5.0). The membrane pellet was resuspended in 200 μl of SDS sample buffer containing 5 mM tributylphosphine and 20 mM acrylamide for reduction and alkylation of proteins . The solubilized proteins were diluted 1:5 in SDS sample buffer and 5 μl subject to polyacrylamide gel electrophoresis using a Criterion XT precast gel (4-12% Bis-Tris; Bio-Rad). Protein gels were stained with Flamingo protein stain (Bio-Rad) and imaged using a Pharos FX Plus Molecular Imager (Bio-Rad). Flamingo stained protein gels were post-stained with colloidal Coomassie G-250 stain and proteins of interest excised for identification by LC-MS/MS as previously described . PEAKS software (Bioinformatics Solutions Inc.) was used to directly search peptides against a protein sequence FASTA output derived from the V. rotiferianus DAT722 genome . The highest PEAKS score (percentage based on a p-value < 0.05) was taken as the closest peptide match. The full sequence of identified proteins is given in the additional file 1.
This work was supported by a grant from the National Health and Medical Research Council of Australia. ML is supported by an ithree Institute Postdoctoral Fellowship.
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