Naturally transformable bacteria acquire chromosomal DNA from related species at lower frequencies than from cognate DNA sources. To determine how genome location affects heterogamic transformation in bacteria, we inserted an nptI marker into random chromosome locations in 19 different strains of the Acinetobacter genus (>24% divergent at the mutS/trpE loci). DNA from a total of 95 nptI-tagged isolates was used to transform the recipient Acinetobacter baylyi strain ADP1. A total of >1300 transformation assays revealed that at least one nptI-tagged isolate for each of the strains/species tested resulted in detectable integration of the nptI marker into the ADP1 genome. Transformation frequencies varied up to 10,000-fold among independent nptI insertions within a strain. The location and local sequence divergence of the nptI flanking regions were determined in the transformants. Heterogamic transformation depended on RecA and was hampered by DNA mismatch repair. Our studies suggest that single-locus-based studies, and inference of transfer frequencies from general estimates of genomic sequence divergence, is insufficient to predict the recombination potential of chromosomal DNA fragments between more divergent genomes. Interspecies differences in overall gene content, and conflicts in local gene organization and synteny are likely important determinants of the genomewide variation in recombination rates between bacterial species.

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HORIZONTAL gene transfer (HGT) contributes to bacterial evolution by providing access to DNA evolved and retained in separate species or strains (COHAN 1994a,b; BERGSTROM et al. 2000; OCHMAN et al. 2000; FEIL et al. 2001; KOONIN 2003; LAWRENCE and HENDRICKSON 2003; FRASER et al. 2007). Multilocus sequence typing (MLST) has provided strong evidence for frequent transfer and recombination of chromosomal DNA between related bacterial strains within the same species (MAIDEN et al. 1998; ENRIGHT et al. 2002). HGT occurring by natural transformation allows bacteria to exploit the presence of nucleic acids in their environment for the purposes of nutrition, DNA repair, reacquisition of lost genes, and/or acquisition of novel genetic diversity (REDFIELD 1993; MEHR and SEIFERT 1998; DUBNAU 1999; CLAVERYS et al. 2000; SZÖLLÖSI et al. 2006; JOHNSEN et al. 2009). It can be inferred from observations of the presence of extracellular DNA in most environments that bacteria are constantly exposed to DNA from a variety of sources, without such exposure necessarily producing observable changes in the genetic compositions of bacterial populations over evolutionary time (THOMAS and NIELSEN 2005; NIELSEN et al. 2007a,b).

The absence of sequence similarity between the donor DNA and the DNA of the recipient bacterium is the strongest barrier to the horizontal acquisition of chromosomal genes in bacteria (MATIC et al. 1996; VULIC et al. 1997; MAJEWSKI 2001; TOWNSEND et al. 2003) as illegitimate recombination occurs only at extremely low frequencies in bacteria (HÜLTER and WACKERNAGEL 2008a). Single-locus transfer models have been extensively applied and have demonstrated a log-linear decrease in recombination frequencies with increasing sequence divergence for Bacillus subtilis (ROBERTS and COHAN, 1993; ZAWADZKI et al. 1995), Acinetobacter baylyi (YOUNG and ORNSTON 2001), Escherichia coli (SHEN and HUANG 1986; VULIC et al. 1997), and Streptococcus pneumoniae (MAJEWSKI et al. 2000). For instance, heterogamic transformation between nonmutator isolates at the rpoB locus of B. mojavensis is undetectable at sequence divergences >16.7% (ZAWADZKI et al. 1995) and between S. pneumoniae isolates with sequence divergences >18% (MAJEWSKI et al. 2000). In A. baylyi, the nonmutator sequence divergence limit for detectable transformation at the pcaH locus of strain ADP1 was found to be 20% (YOUNG and ORNSTON 2001), and up to 24% overall divergence yielded transformants at 16S rRNA loci in strain DSM587 (STRÄTZ et al. 1996).

Several recent studies also show that short stretches (<200 bp) of DNA sequence identity can facilitate additive or substitutive integration of longer stretches (>1000 bp) of heterologous DNA in bacteria (PRUDHOMME et al. 1991, 2002; DE VRIES and WACKERNAGEL 2002; HÜLTER and WACKERNAGEL 2008a). Thus, the uptake of DNA in bacteria can facilitate larger substitutions within gene sequences and the integration of additional DNA material on the basis of recombination initiated in flanking DNA stretches (either at one or both ends) with high sequence similarity (NIELSEN et al. 2000). On the other hand, segments of heterologous DNA interrupting the synteny of homologous DNA have also been shown to be a barrier in intraspecies transformation in S. pneumoniae (PASTA and SICARD 1996, 1999).

The various studies of the interspecies transfer potential of single genes demonstrate that the immediate local sequence divergence of the transferred locus is of high importance in determining recombination frequencies in hosts up to 20% divergent (at the housekeeping gene level). However, it can be hypothesized that the broader structural, organizational, and biochemical properties of the genome region surrounding a particular locus will determine its transfer potential to more divergent host species (COHAN 2001; LAWRENCE 2002). The interspecies transfer potential of various genome regions/loci between more diverged species (>20% at the housekeeping gene level) may therefore differ substantially from a log-linear model (determined experimentally for more closely related species) as local gene organization becomes less conserved with evolutionary time. The barriers to gene exchange between divergent bacterial species is likely a combination of inefficient recombination due to both mismatched base pairs (the main determinator in the log-linear model) and conflicting gene order and organization across the local recombining DNA regions. In addition, selective barriers due to negative effects on host fitness of the transferred DNA regions may become increasingly important for the removal of recombination events from the bacterial population. Recent bioinformatics-based genome analysis of E. coli and Salmonella genomes suggests various parts of the bacterial genome may have different suceptibilities to undergo evolutionarily successful recombination leading to temporal fragmentation of speciation (LAWRENCE 2002; RETCHLESS and LAWRENCE 2007). Nevertheless, few studies have experimentally tested the effect of variable species and chromosome locations of genes on their transfer potential between bacteria (RAVIN and CHEN 1967; RAVIN and CHAKRABARTI 1975; SIDDIQUI and GOLDBERG 1975; COHAN et al. 1991; HUANG et al. 1991; FALL et al. 2007).

Here, we determine to what extent genome location contributes to sexual isolation between the recipient A. baylyi strain ADP1 and 19 sequence divergent (24–27% divergent at the mutS/trpE loci) donor Acinetobacter strains and species (carrying a selectable nptI gene in a total of 95 random genome locations).

Bacterial strains and cultures:

The Acinetobacter wild-type strains used in this study are listed in Table 1. Other strains employed were derivatives of A. baylyi ADP1 described previously [Rp^R (rifampicin resistant, rif^R; NIELSEN et al. 1997); recA::tetA (tetracycline resistant, DE VRIES and WACKERNAGEL 2002); mutS:: (streptomycin resistant; YOUNG and ORNSTON 2001)] or carried mutations described previously (recBCD, recJ, sbcCD, ruvC, KICKSTEIN et al. 2007; HARMS and WACKERNAGEL 2008; HÜLTER and WACKERNAGEL 2008b) crossed into the wild-type background (KICKSTEIN et al. 2007). A. baylyi control strains that carried chromosomally located kanamycin-resistance (km^R) were KTG (Rp^R chr::nptII) (NIELSEN et al. 1997) and ADP1200-2, which is ADP1200 (KOK et al. 1999) with a km^R cassette [(ACIAD3309)::nptIII; km^R]. Except for the mutS:: mutant, none of the strains used are known mutators. All bacteria were grown in Luria-Bertani medium (LB) at 32° with aeration. A. baylyi Rp^R was grown in LB with 50 µg/ml rifampicin (LBR) except when noted.

View this table: In this window In a new window   TABLE 1 Sequence divergence among Acinetobacter spp. strains based on pairwise comparisons of 3331 bp of concatenated sequence of the the trpE and mutS housekeeping genes (lower left triangle) and 1294 bp of the 16S rRNA gene (upper right triangle)

 

Sequence divergence estimates:

Each strain was subjected to DNA sequence determination (BigDye chemistry) at the 16S rRNA, trpE, and mutS loci. The trpE and mutS genes were amplified from DNA using degenerate primers as described previously (YOUNG and ORNSTON 2001). The PCR products obtained were cloned into pCR2.1 TOPO plasmid (Invitrogen), end sequenced using M13 primers, and further primer walked until completion. The 16S rRNA gene was amplified with universal 16S primers and sequenced from ExoSAP-IT (USB Chemicals) treated PCR product. The accession numbers of all 16S rRNA, mutS and trpE sequences are given in Table 1.

The representativeness of the mutS and trpE genes of overall housekeeping gene divergence between species was assessed by comparing an additional 20 other protein coding genes (from the sequenced A. baylyi ADP1 genome to the A. baumannii AYE genome, accession no. CU459141). Ten genes were chosen for comparison on the basis of their previous use in MLST analysis (JOLLEY et al. 2004; BARTUAL et al. 2005; ECKER et al. 2006); gyrB, rpoD, gltA, recA, pgi, groEL, adk, efp, mutY, and ppa. An additional 10 genes were identified at random using the true random number generator located at www.random.org. (ACIAD nos. 600, 674, 819, 927, 1213, 1970, 2330, 2513, 2535, and 3140). The identified genes were included if they had annotated homolog genes in the genome of A. baumannii AYE as identified by the Artemis Comparison Tool (CARVER et al. 2005). Pairwise comparisons were performed at the nucleotide level with the software MegAlign (Lasergene package v. 8, DNASTAR, Madison, WI) by the clustalW matrix and the percentage of identity calculated.

Generation of an Acinetobacter library with random nptI insertions:

Each of the 20 Acinetobacter wild-type strains used in this study was subjected to random transposon insertion using the EZ::TN KAN-2 Tnp Transposome kit (Epicentre Technologies) according to the manufacturer's protocol. After selection for growth on LBK up to 13 random km^R colonies from each parent strain were isolated, cultured for analysis and DNA isolation, and stored in 20% (v/v) glycerol at –70°.

DNA isolation and characterization of the Acinetobacter library:

All strains used in this study, including wild type and those containing nptI-insertions, were screened for verification of kanamycin sensitivity or acquired resistance using kanamycin E-test strips (AB Biodisk). Genomic DNA was isolated from bacterial cultures using Genomic-tip 500-g DNA tips (QIAGEN). DNA was resuspended in sterile TE buffer, pH 8.0, and the DNA concentration measured in duplicate using a spectrophotometer SpectraMax 190 (Molecular Devices) or Nanodrop ND-1000 (Nanodrop Technologies). The concentration of each sample was accordingly adjusted to 0.5 µg/µl and samples were stored at –70°. Uniform quality of DNA was confirmed by agarose gel electrophoresis.

Each wild-type Acinetobacter strain and all transposon-carrying isolates were tested by PCR for the absence or presence of the nptI gene using the primers EZTN-KAN-2LP480 5'-ATT CAG GTG AAA ATA TTG TTG ATG C-3' and EZTN-KAN-2RP1154 5'-CAC GGT TGA TGA GAG CTT TG-3' and HotStar Taq Master Mix (QIAGEN). The 675-bp internal nptI segment (bases 480–1154) was amplified with the following parameters: 95° for 15 min, 30 cycles of 95° for 1 min, 65.5° for 1 min, and 72° for 1 min, 72° for 10 min, and 4° until sample analysis. Southern blot hybridization of EcoRI digested genomic DNA was performed to determine the copy number, and uniqueness of nptI insertions in each km^R isolate. EcoRI does not cut within the nptI gene. A 398-bp internal segment from the nptI sequence found in the EZ::TNKAN-2 Transposome construct was amplified by PCR using the primers EZTNKAN-2LP147 5'-ATT CAA CGG GAA ACG TCT TG-3' and EZTNKAN-2RP545 5'-AAC AGG AAT CGA ATG CAA CC-3' with the following parameters: 95° for 15 min, 30 cycles of 95° for 30 sec, 62° for 20 sec, 72° for 30 sec, 72° for 10 min, and 4° until analysis. The 398-bp PCR product was purified using the QIAQuick PCR purification kit (QIAGEN) and labeled using the random-primed DIG-labeling kit (Boehringer Mannheim). Five micrograms of genomic DNA was digested with EcoRI (New England Biolabs) in fivefold excess. Purified EcoRI-digested DNA was separated by agarose gel electrophoresis for 18 hr at 35 V in a 0.8% agarose gel. DIG-labeled DNA molecular weight marker II (Roche Diagnostics) was included as a size marker. Separated DNA fragments were transferred to a positively charged nylon membrane (Roche Diagnostics). Hybridizations were carried out at 68°, and detection performed using the DIG luminescence detection kit (Boehringer Mannheim).

Individual isolates producing hybridization signals indistinguishable from other isolates of the same parent strain were retained as unique clones only if their transformation frequency and (ultimately) flanking sequences (see below) were unique; those isolates with indistinguishable hybridization signals and identical flanking sequences were concluded to be clonal and all but one were excluded from use in transformation experiments.

Preparation of naturally competent cells:

Competent cells were cultivated by diluting an overnight culture of the appropriate recipient strain 1:100 into 100 ml LB or LBR. The culture was incubated at 32° with 200 rpm shaking until the OD_600nm 0.6 (filter transformation) or until the titer reached 1 x 10⁹ ml^–1 determined using a counting chamber (liquid transformation). Cells were pelleted by centrifugation in a SS-34 rotor (Sorvall) for 15 min at 4000 rpm and resuspended in LB amended with 15% (v/v) glycerol to yield a cell density of 1.0 x 10⁹ cells ml^–1 (filter transformation) or 1.0 x 10¹⁰ cells ml^–1 (liquid transformation). One-milliliter aliquots of the suspensions were stored at –70° until use.

Transformation experiments on filter discs and statistical analyses:

Filter transformations were performed as described previously (RAY and NIELSEN 2005). All transformation frequencies are reported as the number of transformants per viable recipient cell (transformants per recipient), with a minimum of three replicates each (Table 2). Average transformation frequency and 95% confidence intervals for each isolate were calculated from all filter transformations for that isolate. Single replicates that were below the limit of detection were excluded from the calculations of the frequency, resulting in the most conservative estimation of transformation frequency for these isolates. For isolates from which no transformants were obtained for any replicates, the average limit of detection was instead calculated. Where the number of replicates was greater than two and the error did not overlap the limit of detection, the 95% confidence interval was calculated for each nptI-tagged isolate to determine statistical significance of variance between individual isolates of the same parent Acinetobacter strain. When the transformation frequency was below detection limits, the specific limit of detection was calculated to be less than the resulting transformation frequency where one transformant is found among the total recipient bacteria enumerated for one filter.

View this table: In this window In a new window   TABLE 2 Transformation frequencies of Acinetobacter baylyi ADP1 RpR obtained with individually nptI-tagged Acinetobacter spp. isolates and their sequenced flanking regions

 
A positive control using A. baylyi strain KTG (km^R) chromosomal DNA was included to verify recipient cell competence and consistency of incubation conditions. Negative controls consisted of untreated sterile filters, or recipient cells treated with sterile saline instead of transforming DNA. Finally, DNA from the kanamycin-sensitive wild-type strain was included as a negative control in each experiment. Random recipient and transformant colonies were in some experiments checked for the absence or presence of the nptI gene by PCR analysis of heat-treated cell lysates (675-bp internal nptI target segment, see above).

Transformation experiments in liquid medium:

Experiments involving A. baylyi ADP1 DNA metabolism mutant strains as recipients were performed as liquid transformation assays in 2–500 ml LB as described previously (DE VRIES and WACKERNAGEL 1998). When the recipient was the recA mutant, a minimum of 5 km^R transformants obtained with each donor DNA were verified as recA by tetracycline resistance and sensitivity to UV irradiation. Km^R transformants obtained with the mutS recipient strain were confirmed by their streptomycin resistance (minimum five per experiment).

Direct sequencing of genomic DNA to determine the location of nptI insertions:

Genomic sequencing was performed as described (WANG et al. 2000) with the following modifications: 20-µl sequencing reactions consisted of 8 µl BigDye v3.1 sequencing mix (Applied Biosystems), 5 pmol of primer (left end, EZTN-KAN2RP292 5'-AAC TCT GGC GCA TCG GGC TTC C-3' or right end, EZTN-KAN2LP1046 5'-TTG AAG GAT CAG ATC ACG CAT CTT CCC G-3'), and 3.5 µg DNA. Both left and right end flanking sequencing were determined for a total of 95 transformants, and 13 transformants obtained with isogenic A. baylyi nptI-tagged DNA. Seven of the initial transformants were excluded from further study as the right- and left-end flanking sequences did not concur. Sequencing reactions were run with the following parameters: 95° for 5 min followed by 100 cycles of 99° for 30 sec, 55° for 10 sec, and 60° for 4 min, followed by 4° until ethanol precipitation and subsequent analysis on an ABI 3100 Sequencer. Sequencing files were edited using Sequencher v.4.2.2 (Gene Codes). Comparison of these sequences to the A. baylyi was performed using the BLAST (blastn) algorithm (ALTSCHUL et al. 1997).

Statistical analyses:

To test for significance of heterogeneity of transformation frequency among nptI-tagged isolates per donor Acinetobacter strain, one-way analysis of variance (ANOVA) was performed on log₁₀-transformed transformation frequencies using SYSTAT v.11 (SYSTAT Software). The ANOVA was followed by post hoc multiple comparisons (Tukey HSD adjustment) to locate the differences. Transformation frequencies below the limit of detection were excluded from statistical analyses. Multiple regression analysis was performed using SPSS v.13 (SPSS). Average transformation frequencies were log₁₀ transformed prior to analysis and included as the dependent variable. The three independent variables in the multiple regression (estimated global sequence divergence, based on trpE and mutS sequences, local sequence divergence, and chromosomal position (pos.) relative to the origin of replication) were centered and standardized before model estimation. Only those isolates having real values for all four variables were included in the analysis, thus excluding isolates having transformation frequencies below the limit of detection, as well as those with no BLAST-identifiable homology to the ADP1 genome.

Relative fitness measurements and calculations:

Pairwise competition experiments were used to estimate the relative fitness of transformants. Each competition was performed in triplicate with independent starting cultures. Colonies were inoculated into 3-ml S2 minimal medium (JUNI 1974) with 2% lactic acid and incubated overnight (37°, 225 rpm). These cultures were then diluted 1:10 in 0.9% NaCl, and equal quantity of each competitor (150 µl) was transferred into 2.7-ml prewarmed S2 media supplied with 0.1 mg/ml DNase. A 100-µl sample of the mixture was immediately taken to determine the initial cell density of the two competitors (N₀). The mixed culture was then incubated for 24 hr at 37°, 225 rpm. The culture was diluted and plated onto LB agar (with or without 25 µg/ml kanamycin) to estimate the final cell density of the two competitors (N₁). From the initial and final densities, the population growth of each competitor, known as its Malthusian parameter (LENSKI et al. 1991; ROZEN et al. 2007) were determined using the equation M = ln (N₁/N₀). The relative fitness of each isolate is ratio of the Malthusian parameter of the transformant to that of the A. baylyi isogenic km^s strain. Statistical tests were performed with SPSS (v.15) software. For the theoretical predictions of the potential effects of reduced fitness on transformation frequencies, the following equation was used: t 1/s ln [(1 – q_t)q₀/(1 – q₀)q_t] (LEVIN et al. 1997).

Sequence divergence within the Acinetobacter spp. donor collection:

The 16S rRNA data gave 3.2–5.4% divergence levels of the donor strains from the recipient strain, indicating that all donor strains belonged to different species than the recipient A. baylyi (Table 1). The Acinetobacter spp. donor isolates were 23.9–26.5% sequence divergence at the housekeeping gene level from the recipient A. baylyi strain on the basis of a comparison of 3331 bp DNA sequence obtained from the mutS and trpE genes. Between all strains, the divergence was between 0.1 and 27.2% (Table 1). The representativeness of the mutS and trpE genes of overall housekeeping gene divergence was assessed by a comparison of the sequence divergence estimates obtained for these two alleles with divergence estimates obtained for 20 other genes, all compared to the sequenced A. baumannii AYE strain. For the 10 genes selected due to their use in MLST analyses (providing a conservative estimate), divergences ranged from 14.2 to 29.1% (mean divergence 19.1%). For the 10 randomly identified genes, sequence divergence ranged from 21.5 to 34.4% (mean divergence 27.4%).

Characterization of the Acinetobacter spp. nptI insertion events:

AB Biodisk E-tests confirmed kanamycin sensitivity for all 20 wild-type Acinetobacter strains and recipient strain ADP1 Rp^R with minimal inhibitory concentrations <32 µg/ml kanamycin (data not shown). In contrast, all nptI-tagged isolates generated from these wild-type strains were resistant to >64 µg/ml kanamycin. To confirm the presence of the nptI gene in each isolate, a 675-bp internal fragment of the nptI gene was amplified by PCR (no PCR product was obtained with untagged strains; data not shown). Southern hybridization analysis of EcoRI-digested genomic DNA, using a DIG-labeled nptI probe, revealed positive hybridization signals for all km^R isolates. None of the wild-type DNA samples produced a hybridization signal (data not shown).

Determination of the nptI flanking sequences in donor isolates:

A summary of the donor isolates used, the identified nptI insertion loci and transformation frequencies is given in Table 2. For seven additional transformants obtained, discrepancies between right- and left-border sequences when compared to ADP1 sequence precluded their inclusion in the final analysis. Two of these seven excluded isolates transformed ADP1 Rp^R at high relative frequencies (>1 x 10^–7 transformants per recipient). BLAST analysis (ALTSCHUL et al. 1997) of the nptI-flanking sequences in these isolates identified either high similarity to nonneighboring regions in the ADP1 genome or to the ADP1 genome only on one side of the nptI gene insertion. Insertion–deletion events (DE VRIES and WACKERNAGEL 2002; PRUDHOMME et al. 2002) or differences in gene content and synteny between donor and recipient may explain these observations.

Effect of DNA sequence divergence on transformation frequency in A. baylyi ADP1:

The different individual nptI insertion events created in the divergent species and one isogenic Acinetobacter strain were used to transform the naturally competent A. baylyi ADP1 Rp^R (Figures 1 and 2). The transformation frequencies are shown in Table 2. The highest, statistically robust transformation frequency observed for a single isolate was 6.8 (±6.0) x 10^–6 with donor DNA from isolate 8 of A. calcoaceticus AZR54, which has an estimated divergence of 24.1% from ADP1. Although this was the highest heterogamic transformation frequency measured, it was still 150- to 300-fold lower than homogamic transformation frequency (Table 2). The range of transformation frequencies between the randomly tagged km^R isolates for each parent strain ranged from a 1.5-fold difference between the highest and the lowest frequencies (A. sp. AC511B, 2 isolates) to a >9100-fold difference (A. calcoaceticus AZR583, 8 isolates) (Table 2; Figure 3).

View larger version (12K): In this window In a new window Download PPT slide   FIGURE 1.— Average transformation frequencies as a function of estimated sequence divergence (%) from recipient A. baylyi ADP1 of the 95 unique nptI-tagged insertion events (T-bars: standard deviation). The sequence divergence estimate is based on comparison of the mutS and trpE loci between donor and recipient strains. The data points below the dashed line (approximate detection limit) indicate transformation assays for loci from which no transformants were obtained.

 

View larger version (13K): In this window In a new window Download PPT slide   FIGURE 2.— (A) Linear map of the recipient strain ADP1 (solid horizontal bar) showing the transformation frequencies obtained with the heterologous donor DNA (nptI tagged). The transformation frequency is indicated relative to the position of the recipient origin [0 indicates oriV of the 3599 kbp ADP1 genome (x-axis)] (T-bars: standard deviation). Bars below the dashed line (approximate detection limit) indicate strains/loci for which no transformants were obtained. Solid arrow, the position of the rpoB gene (rifR locus); open arrows, seven rRNA loci. (B) Transformation frequencies of the strains with nptI insertion in rRNA operons with unresolved integration sites.

 

View larger version (10K): In this window In a new window Download PPT slide   FIGURE 3.— Average transformation frequency among individual nptI-tagged isolates/loci of strain Acinetobacter sp. AZR54 (A) or AZR583 (B) (numbers on the x-axis refer to the individual isolates; see Table 2). Error bars represent 95% confidence interval of all replicates per isolate. One-way ANOVA of all 8 isolates demonstrated significant heterogeneity at the 95% confidence level (P < 0.001).

 

Locus-specific effects on transformation in strain ADP1:

The 95 transformants for which the nptI flanking DNA sequences were obtained in this study can be roughly categorized into four groups. The first group consisted of isolates that had high local flanking gene similarity to A. sp. ADP1 (as determined using the BLAST algorithm) (ALTSCHUL et al. 1997) and transformed ADP1 Rp^R at relative high frequency (>1 x 10^–6). Of these, the highest transformation frequencies were observed for isolates with nptI insertions in ribosomal RNA (rrn) operons: A. sp. 01B0, isolate 2; A. sp. 63A1, isolate 7; A. sp 81A1, isolate 2; A. sp. AZR54, isolate 8; and A. calcoaceticus AZR583, isolate 9. Exceptions were A. calcoaceticus AZR583, isolate 4, which transformed ADP1 Rp^R at a frequency of 2.8 (±0.4) x 10^–6 at tufA (encoding the protein chain elongation factor EF-Tu; ACIAD0885) locus; and A. sp. 26B2, isolate 3, which contained an nptI insertion in an rrn operon (ACIADrRNA23S), but transformed strain ADP1 Rp^R at an average frequency of 1.5 (± 0.6) x 10^–8.

The second category consisted of 8 isolates with no BLAST-identifiable similarity to ADP1 that transformed ADP1 Rp^R at low or undetectable frequencies: A. sp. 48A1, isolate 4; A. sp. 62A1, isolate 1; A. sp. 66A1, isolate 3; A. sp. 71A1, isolate 3; A. sp. A06, isolate 4; A. calcoaceticus AZR583, isolate 1; and A. sp. P1-6, isolates 5 and 8. This result is not unexpected and corroborates previous studies demonstrating the necessity of sequence similarity between donor and recipient for transformation (SHEN and HUANG 1986; DE VRIES and WACKERNAGEL 1998; NIELSEN et al. 2000; DE VRIES et al. 2001; MAJEWSKI 2001; KAY et al. 2002; TEPFER et al. 2003).

The third category consisted of 34 isolates with BLAST-identifiable similarity to ADP1 that transformed ADP1 Rp^R poorly. For these isolates, the presence of regions with DNA sequence similarity (>75%) to ADP1 was insufficient to facilitate recombination at frequencies substantially higher than the limit of detection (<10^–9). Fourteen of these 34 isolates did not transform ADP1 Rp^R at detectable frequencies.

The fourth category consists of 5 isolates that transformed ADP1 Rp^R at detectable frequencies but without any BLAST-identifiable sequence similarity to ADP1: A. sp. 62A1, isolate 4; A. sp. 81A1, isolate 1; A. sp. A06, isolates 1 and 8; and A. sp. P1-6, isolate 10. With the exception of A. sp. 62A1, isolate 4 (transformation frequency of 1.2 ± 0.1 x 10^–7), these isolates transformed ADP1 Rp^R at frequencies <3.0 x 10^–8.

In our study, the recombination joints lie in most cases outside of the sequenced flanking DNA region. The acquired donor DNA present in the transformant populations may span several thousand base pairs (beyond the nptI locus) since genomic DNA fragments were used as the donor DNA source. Previous studies of the size of DNA fragments integrated during transformation in Acinetobacter sp. (PALMEN and HELLINGWERF 1997) and B. subtilis (ZAWADZKI and COHAN 1995) demonstrated that larger fragments (up to several thousand base pairs) integrate at high frequencies. Multiple regression analysis also showed that global sequence divergence does not contribute significantly to the observed variation in transformation frequency (Table 3). According to the same multiple regression analysis, both local sequence divergence and chromosomal position (relative to the origin of replication) are significantly negatively correlated to transformation frequency and account for 7.6 and 9.2% of observed variation, respectively (Table 3).

View this table: In this window In a new window   TABLE 3 Multiple regression analysis determining the contributions of estimated global sequence divergence (global SD), local sequence divergence (local SD), and chromosomal location relative to the origin of replication (location) to observed variation in transformation frequency (dependent variable)

 

Influence of the DNA metabolism on heterogamic transformation frequency:

To determine how selected cellular components affect homologous and heterologous transformation, we used several mutant A. baylyi strains as recipients in transformation experiments as listed in Table 4. A recA mutation decreased the homologous recombination frequency 1000-fold when using donor DNA from an isogenic strain. With a heterogamic donor DNA source, the decrease relative to wild type was also 1000-fold. Exonuclease RecBCD deficiency led to an 10-fold decrease in transformation frequency during transformation with homologous DNA (in accordance with previous reports; KICKSTEIN et al. 2007; HARMS and WACKERNAGEL 2008). The decrease relative to wild type was approximately the same with different sequence-divergent donor DNA preparations (Table 4). Mutations in the recJ, sbcCD, and ruvC genes (encoding the RecJ exonuclease, SbcCD DNase, and Holliday junction resolvase, respectively) had little effect on homologous or heterologous transformation. In a strain with MutS deficiency (mutator phenotype), the heterologous transformation frequencies with different donor DNA preparations were increased 50- to 100-fold relative to wild type, while the homologous transformation frequency was indistinguishable from that of the wild-type strain. An increase of recombination in mutator strains has also been observed previously in interspecies HGT (RAYSSIGUIER et al. 1989; ZAHRT et al. 1994; HUMBERT et al. 1995; YOUNG and ORNSTON 2001). The observations suggest that >98% of the heterogamic recombination events are prevented by the DNA mismatch repair system.

View this table: In this window In a new window   TABLE 4 Homogamic and heterogamic transformation frequencies of recombination mutants of A. baylyi strain ADP1 relative to wild type

 

Absence of effect of minor variation in DNA concentration on transformation frequency:

The ADP1 Rp^R strain was transformed with different concentrations of donor DNA from four individual strains and insertion events (63A1, isolate 6; 66A1, isolate 1; AZR54, isolate 5; and ADP1, isolate 5). The concentrations of DNA ranged from 10 ng to 50 µg per 1.0 x 10⁸ competent recipient cells. Saturation of the transformation frequency for all donor strains occurred at DNA concentrations 10 µg (data not shown) indicating that minor variations in DNA concentrations would not affect the number of transformants recovered.

Absence of mutagenic effect of DNA in the transformation process:

The uptake of heterologous donor DNA may hypothetically have a mutagenic effect that could impact the transformation frequency (BULL et al. 2000). Over several years of study, we have never observed spontaneous mutation to km^R (50 µg/ml) in the recipient strain BD413 (isogenic to ADP1) (NIELSEN et al. 1997; 2000). Moreover, the absence of mutagenic effects of added DNA sources were also demonstrated by the fact that the frequency of mutation to rifampicin resistance was not significantly different (P > 0.05) among ADP1 cells after treatment with saline, ADP1 wild-type DNA, herring sperm DNA, or after treatment with strain KTG (km^R) DNA as positive control (data not shown).

Noninterference of rif^R marker in detecting km^R transformants:

The donor DNA isolates were km^R, but rifampicin sensitive, a simultaneous recombination at the mutant rpoB (rif^R) host locus during transformation employing ADP1 Rp^R as recipient strain would therefore result in transformants that were km^R (nptI positive) but rifampicin sensitive. Such transformants would be excluded from the transformant counts as all transformants were selected on media containing both kanamycin and rifampicin, which would result in an underestimation of recombination frequencies. We identified strains with km^R insertions in genes that have homologous counterparts that are most closely located upstream and downstream of the rpoB locus of A. baylyi ADP1 (ACIAD0307, pos. 302,457–306,545; Figure 2) which were A. sp. P1-6, isolate 4 (argH, pos. 281,279–282,712; 20 kbp upstream of rpoB), and A. sp. 85A1, isolate 3 (ACIAD0526, pos. 511,425–512,096; >200 kbp downstream of rpoB), respectively (Table 2, Figure 2). Transformation of A. baylyi ADP1 Rp^R by DNA of either of these strains did not result in significantly (n = 3, P > 0.4 for both experiments) different transformation frequencies for selection for km^R and rif^R, or km^R alone (data not shown). Moreover, transformation assays using ADP1 Rp^R competent cells as recipient and five additional different donor DNA sources (A. sp. AC423D, isolate 5; A. sp. 62A1, isolate 5; A. sp. 81A1, isolate 2; A. calcoaceticus AZR583, isolate 9; and A. baylyi KTG), demonstrated no significant difference (n = 3, P > 0.20 for all samples) between selection on media with and without rifampicin (data not shown). The distribution of transformation frequencies across the entire length of the A. baylyi genome does not suggest a specific rpoB interference on frequencies (Figure 2).

Fitness effects of nptI acquisitions in transformants:

The recombination with nptI sequences inserted at various loci in the donor genomes could potentially lead to reduced absolute fitness by causing lethal or severe growth rate reducing effects on the transformant ADP1 cells and introduce bias in our observations. However, the total number of recipient cells remained constant over all experiments and slow-growing colonies were rarely seen on the nutrient-rich media used for plating transformants. The bacterial cells grow only 6–8 generations (10⁸ to 10¹⁰ cells) in competition during the filter transformations. The limited number of cell divisions taking place in growth competition before plating of single colonies will thus not cause reductions in the relative fitness of transformants (vs. the recipients) to dictate the recombination frequencies measured. For instance, a 30% reduction in relative fitness (of transformants compared to recipient strain) would produce a <10-fold difference in the transformation frequencies observed (see MATERIALS AND METHODS). This estimate is based on a model of constant selection in a mixed population of transformants and recipients (LEVIN et al. 1997), with the following parameters: transformants have a 30% (s = 0.30) fitness cost as compared to the recipient, an initial frequency of 1 x 10^–7 (q₀), and undergo 6.7 generations (t) of binary fission in transformation assays before growth of individual cells on selective media. The bacteria undergo between 3.3 and 6.7 generations on the filter (as calculated from a 10- to 100-fold observed increase in total CFU). However, transformants arising in the later part of the transformation period will be even less affected by relative fitness differences. Experimental measurements showed that nine independent transformants had a mean relative fitness of 0.87 (CI 0.80–0.95). The mean relative fitness ranged from 0.61 (CI 0.56–0.67) for isolate 3, strain AZR54; 0.92 (0.87–0.97) for isolate 4; and 0.94 (CI 0.87–1.0) for isolate 8, strain AZR583. Previous studies have reported a mean 3% reduction in fitness for Tn10 insertions in the E. coli genome (ELENA et al. 1998).

We have demonstrated that A. baylyi ADP1 can, through natural transformation, access DNA from a range of related bacterial species, with estimated chromosomal DNA sequence divergence up to 27% (on the basis of the mutS and trpE loci), and that the gene transfer potential and, hence, sexual isolation varies up to almost 10,000-fold, depending on the genome location of the selected locus. The frequency range reported here (Table 2) is unlikely to represent the full variation among loci and species due to the limited number of insertion events examined. Some loci from divergent species transferred into the ADP1 strain with frequencies only 150- to 300-fold lower than the frequencies observed for transformation between isogenic ADP1 strains (Table 2). In contrast, the presence of the nptI gene in other loci did not facilitate its transfer into A. baylyi ADP1 at detectable frequencies (detection limit approximately one transformant per 10¹⁰ recipients). The broadest range observed was for A. calcoaceticus AZR583, (9 isolates compared), for which transformation frequencies ranged from below detection limit (<3.4 [±0.5] x 10^–10, isolate 3) to 3.1 (±0.2) x 10^–6 transformants per recipient (isolate 4), a difference of almost 10,000-fold (Figures 1 and 2). Our results show that local sequence divergence and chromosomal position (relative to the origin of replication) together account for only 17% of variation in transformation frequency (R = 0.415, R² = 0.172, Adjusted R² = 0.131).

Previous studies have applied single-locus transfer models to establish a log-linear relationship between increasing DNA divergence (between species usually <20% divergent at the transferred locus) and reduced gene transfer frequencies (VULIC et al. 1997; MAJEWSKI et al. 2000). Such studies may not fully predict the potential of horizontal transfer of genes situated in different genome regions of more divergent bacterial species (>20%). Our study suggests that the recombination potential between particular loci of divergent but related species depends upon the genetic properties of the <5000 to >20,000 bp of DNA sequence spanning the loci in consideration.

The 1000-fold decrease of heterogamic transformation in a recA strain suggests that the homologous recombination machinery facilitates the recombination events observed in our study. RecA initiates recombination by facilitating strand exchange at identical or highly similar DNA segments (20–27 bp; minimal efficient processing segments, MEPS) (SHEN and HUANG 1986; MAJEWSKI et al. 2000).

The highest heterogamic transformation frequencies were, in our study, obtained for markers integrated in highly conserved genes (e.g., genes of the protein biosynthesis apparatus like rrn operons, tuf, and rimM; Table 2), in all of which the number of MEPS is probably higher than at less conserved loci. Six of the 95 isolates tested contained nptI insertions in an rrn operon. The A. sp. ADP1 genome contains seven rrn operons, two of which are within immediate flanking proximity of the strain ADP1 origin of replication [at 22,000 bp and 3,560,000 bp (Figure 2)]. High transformation frequencies {>10⁶, except for strain 26B2, isolate 3 [1.5 (±0.6) x 10^–8]} were obtained when recombination occurred in an rrn operon. Sequence conservation of rrn genes among diverse bacterial lineages suggests high recombination potential between them when present in multiple copies and if individual copies are expendable (WILSON and YOUNG 1972; COLE and SAINT-GIRONS 1994; STRÄTZ et al. 1996). Multiple regression analysis of 65 of the nptI-tagged isolates tested in this study corroborate earlier hypotheses that genes located closer to the origin of replication may have higher transfer potential. This result is unsurprising as sequence-conserved housekeeping genes are often clustered within close proximity to the origin of replication (SUVOROV and FERRETTI 2000; BRASSINGA et al. 2001). A biased distribution of DNA uptake sequences toward particular housekeeping genes in bacteria with sequence specific uptake may cause similar effects (DAVIDSEN et al. 2004). The observed slightly skewed distribution of recombination efficiency in our study may also be due to the oriV-distal location of large catabolic islands in the ADP1 strain, which may be specific to the recipent strain used (BARBÉ et al. 2004) and therefore do not provide opportunities for homologous recombination between other strains.

The local sequence divergence of the regions immediately flanking the nptI insert in the donor isolates was determined after sequencing and alignment with corresponding regions in the published ADP1 genome (BARBÉ et al. 2004). In general, we observed that high similarity between donor and recipient most commonly occurred as shorter subgenic homologous regions of variable length and similarity (Table 2). Recombination joints in three isolates (obtained with DNA from A. sp. A06, isolate 10; A. sp. AZR54, isolate 8; A. calcoaceticus AZR583, isolate 9) were identified within the 500 bp sequenced (data not shown). Surprisingly, further examination of our data indicates that the recombination joint lies far from the selected nptI insert in the majority of the transformants. Extensive primer walking has shown that the lengths of the acquired heterologous DNA fragment often extended >3000 bp and some integration events extends above 15,000 bp (O. G. Wikmark, K. Harms, J. Ray, K. M. Nielsen, unpublished results). Thus the predictive role of sequence similarity (in immediate flanking DNA regions) for the interspecies transfer potential of a particular gene may be less obvious, and is likely to vary substantially among transformable species and donor loci (ZAWADZKI and COHAN, 1995; LINZ et al. 2000; FALUSH et al. 2001).

The extent of conservation of DNA sequence composition and gene order may be uneven across the bacterial donor genomes used in our study, reflecting variation in evolutionary history, directional selection, and intra- and intergenomic recombination (LAWRENCE 2002; FRASER et al. 2009). The genetic complexity of such recombining regions (>15 kb) caused by a combination of (i) variation in insertion lengths where also the initial (multiple) DNA synapses formation may differ from the final recombination junctions, (ii) variable DNA sequence similarity and GC% over the recombination area, (iii) variable extent of maintained gene order patterns and conservation, (iv) frequent occurrence of local inversions and frame shifts, and (v) potential lethal effects of host fitness of some of the recombination products (that may be determined by the size of the recombined regions that can vary randomly according to the size of the incoming DNA molecule) precludes an in-depth mechanistic analysis and unambiguous identification of a single factor governing sexual isolation in bacteria.

In conclusion, the presented data provide experimental evidence that only defined regions of a bacterial chromosome are susceptible to recombination between a particular recipient and donor bacterial species of >20% divergence.

The authors thank David Young, Nicholas Ornston, Lenie Dijkshoorn, Johann de Vries, and Wilfried Wackernagel for the generous donation of the Acinetobacter spp. strains used in this study. Special thanks to David Young for providing primer sequences and DNA sequence of many of the housekeeping alleles, to Thomas Bøhn and Raul Primicerio for assistance with statistical analyses, and to Anne-Hilde Conradi for technical assistance. This work was initiated in the laboratory of Daniel Hartl. J.L.R., P.J.J., and K.M.N. acknowledge financial support from the Research Council of Norway.

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. GQ178006–GQ178024 (trpE), GQ178025–GQ178043 (mutS), and GQ178044–GQ178062 (16S rRNA).

ALTSCHUL, S. F., T. L. MADDEN, A. A. SCHAFFER, J. ZHANG, Z. ZHANG et al., 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402.[Abstract/Free Full Text]

BARTUAL, S. G., H. SEIFERT, C. HIPPLER, M. A. LUZON, H. WISPLINGHOFF et al., 2005 Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. J. Clin. Microbiol. 43: 4382–4390.[Abstract/Free Full Text]

BARBÉ, V., D. VALLENET, N. FONKNECHTEN, A. KREIMEYER, S. OZTAS et al., 2004 Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res. 32: 5766–5779.[Abstract/Free Full Text]

BERGSTROM, C. T., M. LIPSITCH and B. R. LEVIN, 2000 Natural selection, infectious transfer and the existence conditions for bacterial plasmids. Genetics 155: 1505–1519.[Abstract/Free Full Text]

BRASSINGA, A. K., R. SIAM and G. T. MARCZYNASKI, 2001 Conserved gene cluster at replication origins of the alpha-proteobacteria Caulobacter crescentus and Rickettsia prowazekii. J. Bacteriol. 183: 1824–1829.[Abstract/Free Full Text]

BULL, H. J., G. J. MCKENZIE, P. J. HASTINGS and S. ROSENBERG, 2000 Evidence that stationary-phase hypermutation in the Escherichia coli chromosome is promoted by recombination. Genetics 154: 1427–1437.[Abstract/Free Full Text]

CARVER, T. J., K. M. RUTHERFORD, M. BERRIMAN, M. A. RAJANDREAM, B. G. BARRELL et al., 2005 ACT: the Artemis Comparison Tool. Bioinformatics 21: 3422–3423.[Abstract/Free Full Text]

CLAVERYS, J.-P., M. PRUDHOMME, I. MORTIER-BARRIERE and B. MARTIN, 2000 Adaptation to the environment: Streptococcus pneumoniae, a paradigm for recombination-mediated genetic plasticity? Mol. Microbiol. 35: 251–259.[CrossRef][Medline]

COHAN, F. M., M. S. ROBERTS and E. C. KING, 1991 The potential for genetic exchange by transformation within a natural population of Bacillus subtilis. Evolution 45: 1393–1421.[CrossRef]

COHAN, F. M., 1994a Genetic exchange and evolutionary divergence in prokaryotes. Trends Ecol. Evol. 9: 175–180.[CrossRef]

COHAN, F. M., 1994b The effects of rare but promiscuous genetic exchange on evolutionary divergence in prokaryotes. Am. Nat. 143: 965–986.[CrossRef]

COHAN, F. M., 2001 Bacterial species and speciation. Syst. Biol. 50: 513–524.[Abstract/Free Full Text]

COLE, S., and I. SAINT-GIRONS, 1994 Bacterial genomics. FEMS Microbiol. Lett. 14: 139–160.[CrossRef]

DAVIDSEN, T., E. A. RØDLAND, K. LAGESEN, E. SEEBERG, T. ROGNES et al., 2004 Biased distribution of DNA uptake sequences towards genome maintenance genes. Nucleic Acids Res. 32: 1050–1058.[Abstract/Free Full Text]

DE VRIES, J., and W. WACKERNAGEL, 1998 Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation. Mol. Gen. Genet. 257: 606–613.[CrossRef][Medline]

DE VRIES, J., P. MEIER and W. WACKERNAGEL, 2001 The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly depends on homologous sequences in the recipient cells. FEMS Microbiol. Lett. 195: 211–215.[Medline]

DE VRIES, J., and W. WACKERNAGEL, 2002 Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination. Proc. Natl. Acad. Sci. USA 99: 2094–2099.[Abstract/Free Full Text]

DUBNAU, D., 1999 DNA uptake in bacteria. Annu. Rev. Microbiol. 53: 217–244.[CrossRef][Medline]

ECKER, J. A., C. MASSIRE, T. A. HALL, R. RANKEN, T. T. PENNELLA et al., 2006 Identification of Acinetobacter species and genotyping of Acinetobacter baumannii by multilocus PCR and mass spectrometry. J. Clin. Microbiol. 44: 2921–2932.[Abstract/Free Full Text]

ELENA, S. F., L. EKUNWE, E. HAJELA, S. A. ODEN and R. E. LENSKI, 1998 Distribution of fitness effects caused by random insertion mutations in Escherichia coli. Genetica 102/103: 349–358.

ENRIGHT, M. C., D. A. ROBINSON, G. RANDLE, E. J. FEIL, H. GRUNDMANN et al., 2002 The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc. Natl. Acad. Sci. USA 99: 7687–7692.[Abstract/Free Full Text]

FALL, S., A. MERCIER, F. BERTOLLA, A. CALTEAU, L. GUEGUEN et al., 2007 Horizontal gene transfer regulation in bacteria as a "spandrel" of DNA repair mechanisms. PLOS One 2: e1055.[CrossRef][Medline]

FALUSH, D., C. KRAFT, N. S. TAYLOR, P. CORREA, J. G. FOX et al., 2001 Recombination and mutation during long-term gastric colonization by Helicobacter pylori: estimates of clock rates, recombination size and monimal age. Proc. Natl. Acad. Sci. USA 98: 15056–15061.[Abstract/Free Full Text]

FEIL, E. J., E. C. HOLMES, D. E. BESSEN, M.-S. CHAN, N. P. J. DAY et al., 2001 Recombination within natural populations of pathogenic bacteria: Short-term empirical estimates and long-term phylogenetic consequences. Proc. Natl. Acad. Sci. USA 98: 182–187.[Abstract/Free Full Text]

FRASER, C., W. P. HANAGE and B. G. SPRATT, 2007 Recombination and the nature of bacterial speciation. Science 315: 476–480.[Abstract/Free Full Text]

FRASER, C., E. J. ALM, M. F. POLZ, B. G. SPRATT and W. P. HANAGE, 2009 The bacterial species challenge: making sense of genetic and ecological diversity. Science 323: 741–746.[Abstract/Free Full Text]

HARMS, K., and W. WACKERNAGEL, 2008 The RecBCD and SbcCD DNases suppress homology-facilitated illegitimate recombination during natural transformation in Acinetobacter baylyi. Microbiology 154: 2437–2445.[Abstract/Free Full Text]

HUANG, L. C., E. A. WOOD and M. M. COX, 1991 A bacterial model system for chromosomal targeting. Nucleic Acids Res. 19: 443–448.[Abstract/Free Full Text]

HÜLTER, N., and W. WACKERNAGEL, 2008a Foreign DNA integration occurs by double illegitimate recombination events through two different mechanisms during natural transformation of Acinetobacter baylyi. Mol. Microbiol. 67: 984–995.[CrossRef][Medline]

HÜLTER, N., and W. WACKERNAGEL, 2008b Frequent integration of short homologous DNA tracks during Acinetobacter baylyi transformation and influence of transcription and DNases. Microbiology 154: 3676–3685.[Abstract/Free Full Text]

HUMBERT, O., M. PRUDHOMME, R. HAKENBECK, C. G. DOWSON and J. P. CLAVERYS, 1995 Homeologous recombination and mismatch repair during transformation in Streptococcus pneumoniae: saturation of the Hex mismatch repair system. Proc. Natl. Acad. Sci. USA 92: 9052–9056.[Abstract/Free Full Text]

JOHNSEN, P. J., D. DUBNAU and B. LEVIN, 2009 Episodic selection and the maintenance of competence and natural transformation in Bacillus subtilis. Genetics 181: 1521–1533.[Abstract/Free Full Text]

JOLLEY, K. A., M. S. CHAN and M. C. J. MAIDEN, 2004 mlstdbNet: distributed multi-locus sequence typing (MLST) databases. BMC Bioinformatics 5: 86.[CrossRef][Medline]

JUNI, E., 1974 Simple genetic transformation assay for rapid diagnosis of Moraxella osloensis. Appl. Microbiol. 27: 16–24.[Medline]

KAY, E., T. M. VOGEL, F. BERTOLLA, R. NALIN and P. SIMONET, 2002 In situ transfer of antibiotic resistance genes from transgenic (transplastomic) tobacco plants to bacteria. Appl. Environ. Microbiol. 68: 3345–3351.[Abstract/Free Full Text]

KICKSTEIN, E., K. HARMS and W. WACKERNAGEL, 2007 Deletions of recBCD or recD influence genetic transformation differently and are lethal together with a recJ deletion in Acinetobacter baylyi. Microbiology 153: 2259–2270.[Abstract/Free Full Text]

KOK, R. G., D. M. YOUNG and L. N. ORNSTON, 1999 Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation. Appl. Environ. Microbiol. 65: 1675–1680.[Abstract/Free Full Text]

KOONIN, E., 2003 Horizontal gene transfer: the path to maturity. Mol. Microbiol. 50: 725–727.[CrossRef][Medline]

LAWRENCE, J. G., 2002 Gene transfer in bacteria: speciation without species? Theor. Pop. Biol. 61: 449–460.[CrossRef][Medline]

LAWRENCE, J. G., and H. HENDRICKSON, 2003 Lateral gene transfer: when will adolescence end? Mol. Microbiol. 50: 739–749.[CrossRef][Medline]

LENSKI, R. E., M. R. ROSE, S. C. SIMPSON and S. C. TADLER, 1991 Long-term experimental evolution in Escherichia coli. I. Adaptation and divergence during 2000 generations. Am. Nat. 138: 1315–1341.[CrossRef]

LEVIN, B. R., M. LIPSITCH, V. PERROT, S. SCHRAG, R. ANTIA et al., 1997 The population genetics of antibiotic resistance. Clin. Infect. Dis. 24: S9–S16.[Medline]

LINZ, B., M. SCHENKER, P. ZHU and M. ACHTMAN, 2000 Frequent interspecies genetic exchange between commensal neisseriae and Neisseria meningitidis. Mol. Microbiol. 36: 1049–1058.[CrossRef][Medline]

MAIDEN, M. C. J., J. A. BYGRAVES, E. FEIL, G. MORELLI, J. E. RUSSELL et al., 1998 Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA 95: 3140–3145.[Abstract/Free Full Text]

MAJEWSKI, J., P. ZAWADZKI, P. PICKERILL, F. M. COHAN and C. G. DOWSON, 2000 Barriers to genetic exchange between bacterial species: Streptococcus pneumoniae transformation. J. Bacteriol. 182: 1016–1023.[Abstract/Free Full Text]

MAJEWSKI, J., 2001 Sexual isolation in bacteria. FEMS Microbiol. Lett. 199: 161–169.[CrossRef][Medline]

MATIC, I., F. TADDEI and M. RADMAN, 1996 Genetic barriers among bacteria. Trends Microbiol. 4: 69–73.[CrossRef][Medline]

MEHR, I. J., and H. S. SEIFERT, 1998 Differential roles of homologous recombination pathways in Neisseria gonorrhoeae pilin antigenic variation, DNA transformation and DNA repair. Mol. Microbiol. 30: 697–710.[CrossRef][Medline]

NIELSEN, K. M., M. D. M. VAN WEERELT, T. N. BERG, A. M. BONES, A. HAGLER et al., 1997 Natural transformation and availability of transforming DNA to Acinetobacter calcoaceticus in soil microcosms. Appl. Environ. Microbiol. 63: 1945–1952.[Abstract/Free Full Text]

NIELSEN, K. M., J. D. VAN ELSAS and K. SMALLA, 2000 Transformation of Acinetobacter sp. strain BD413 (pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants. Appl. Environ. Microbiol. 66: 1237–1242.[Abstract/Free Full Text]

NIELSEN, K. M, P. J. JOHNSEN, D. BENSASSON and D. DAFFONCHIO, 2007a Release and persistence of extracellular DNA in the open environment. Environ. Biosafety Res. 6: 37–53.[CrossRef][Medline]

NIELSEN, K. M., P. J. JOHNSEN and J. D. VAN ELSAS, 2007b Gene transfer and microevolution in soil, pp. 55–81 in Modern Soil Microbiology, 2nd Ed, edited by J. D. VAN ELSAS, J. K. JANSSON and J. T. TREVORS. CRC Press, Boca Raton.

OCHMAN, H., J. G. LAWRENCE and E. A. GROISMAN, 2000 Lateral gene transfer and the nature of bacterial innovation. Nature 405: 299–304.[CrossRef][Medline]

PALMEN, R., and K. J. HELLINGWERF, 1997 Uptake and processing of DNA by Acinetobacter calcoaceticus: a review. Gene 192: 179–190.[CrossRef][Medline]

PASTA, F., and M. A. SICARD, 1996 Exclusion of long heterologous insertions and deletions from the pairing synapsis in pneumococcal transformation. Microbiology 142: 695–705.[Abstract/Free Full Text]

PASTA, F., and M. A. SICARD, 1999 Polarity of recombination in transformation of Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 96: 2943–2948.[Abstract/Free Full Text]

PRUDHOMME, M., V. MEJEAN, B. MARTIN, O. HUMBERT and J. P. CLAVERYS, 1991 Generalized mismatch repair in Streptococcus pneumoniae, pp. 67–70 in Genetics and Molecular Biology of Streptococci, Lactococci, and Enterococci; 3rd International American Society for Microbiology Conference, Minneapolis, Minnesota, USA, June 6–9, 1990, edited by G. M. DUNNY, P. P. CLEARY and L. L. MCKAY. American Society for Microbiology, Washington, D.C.

PRUDHOMME, M., V. LIBANTE and J. P. CLAVERYS, 2002 Homologous recombination at the border: Insertion-deletions and the trapping of foreign DNA in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. USA 99: 2100–2105.[Abstract/Free Full Text]

RAVIN, A. W., and K.-C. CHEN, 1967 Heterospecific transformation of Pneumococcus and Streptococcus. III. Reduction of linkage. Genetics 37: 851–864.

RAVIN, A. W., and T. CHAKRABARTI, 1975 Genetic hybridization at the unlinked thy and str loci of Streptococcus. Genetics 81: 223–241.[Abstract/Free Full Text]

RAY, J., and K. M. NIELSEN, 2005 Experimental methods for assaying natural transformation and inferring horizontal gene transfer, pp. 491–520 in Molecular Evolution: Producing the Biochemical Data, Part B, edited by E. ZIMMER and E. ROALSON. Academic Press, San Diego.

RAYSSIGUIER, C., D. S. THALER and M. RADMAN, 1989 The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature 342: 396–401.[CrossRef][Medline]

REDFIELD, R. J., 1993 Genes for breakfast: the have-your-cake-and-eat-it-too of bacterial transformation. J. Hered. 84: 400–404.[Abstract/Free Full Text]

RETCHLESS, A. C., and J. G. LAWRENCE, 2007 Temporal fragmentation of speciation in bacteria. Science 317: 1093–1096.[Abstract/Free Full Text]

ROBERTS, M. S., and F. M. COHAN, 1993 The effect of DNA sequence divergence on sexual isolation in Bacillus. Genetics 134: 401–408.[Abstract]

ROZEN, D. E., L. MCGEE, B. R. LEVIN and K. P. KLUGMAN, 2007 Fitness costs of fluoroquinolone resistance in Streptococcus pneumonie. Antimicrob. Agents Chemother. 51: 412–416.[Abstract/Free Full Text]

SHEN, P., and H. HUANG, 1986 Homologous recombination in Escherichia coli: dependence on substrate length and homology. Genetics 112: 441–457.[Abstract/Free Full Text]

SIDDIQUI, A., and I. D. GOLDBERG, 1975 Intragenic transformation of Neisseria gonorrhoea and Neisseria perflava to streptomycin resistance and nutritional independence. J. Bacteriol. 124: 1359–1365.[Abstract/Free Full Text]

STRÄTZ, M., M. MAU and K. N. TIMMIS, 1996 System to study horizontal gene exchange among microorganisms without cultivation of recipients. Mol. Microbiol. 22: 207–215.[CrossRef][Medline]

SUVOROV, A. N., and J. J. FERRETTI, 2000 Replication origin of Streptococcus pyogenes, organization and cloning in heterologous systems. FEMS Microbiol. Lett. 189: 293–297.[CrossRef][Medline]

SZÖLLÖSI, G. J., I. DERÉNYI and T. VELLAI, 2006 The maintenance of sex in bacteria is ensured by its potential to reload genes. Genetics 174: 2173–2180.[Abstract/Free Full Text]

TEPFER, D., R. GARCIA-GONZALES, H. MANSOURI, M. SERUGA, B. MESSAGE et al., 2003 Homology-dependent DNA transfer from plants to a soil bacterium under laboratory conditions: implications in evolution and horizontal gene transfer. Transgenic Res. 12: 425–437.[CrossRef][Medline]

THOMAS, C. M., and K. M. NIELSEN, 2005 Mechanisms of, and barriers to, horizontal gene transfer between bacteria. Nat. Rev. Microbiol. 3: 711–721.[CrossRef][Medline]

TOWNSEND, J. P., K. M. NIELSEN, D. S. FISHER and D. L. HARTL, 2003 Horizontal acquisition of divergent chromosomal DNA in bacteria: effects of mutator phenotypes. Genetics 164: 13–21.[Abstract/Free Full Text]

VULIC, M., F. DIONISIO, F. TADDEI and M. RADMAN, 1997 Molecular keys to speciation: DNA polymorphism and the control of genetic exchange in enterobacteria. Proc. Natl. Acad. Sci. USA 94: 9763–9767.[Abstract/Free Full Text]

WANG, H., A. P. ROBERTS, D. LYRAS, J. I. ROOD, M. WILKS et al., 2000 Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX. J. Bacteriol. 182: 3775–3783.[Abstract/Free Full Text]

WILSON, G. A., and F. E. YOUNG, 1972 Intergenotic transformation of the Bacillus subtilis genospecies. J. Bacteriol. 111: 705–716.[Abstract/Free Full Text]

YOUNG, D. M., and L. N. ORNSTON, 2001 Functions of the mismatch repair gene mutS from Acinetobacter sp. strain ADP1. J. Bacteriol. 183: 6822–6831.[Abstract/Free Full Text]

ZAHRT, T. C., G. C. MORA and S. MALOY, 1994 Inactivation of mismatch repair overcomes the barrier to transduction between Salmonella typhimurium and Salmonella typhi. J. Bacteriol. 176: 1527–1529.[Abstract/Free Full Text]

ZAWADZKI, P., and F. M. COHAN, 1995 The size and continuity of DNA segments integrated in Bacillus transformation. Genetics 141: 1231–1243.[Abstract]

ZAWADZKI, P., M. S. ROBERTS and F. M. COHAN, 1995 The log-linear relationship between sexual isolation and sequence divergence in Bacillus transformation is robust. Genetics 140: 917–932.[Abstract]

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