Interspecies transformation of Acinetobacter: genetic evidence for a ubiquitous genus

The availability of a strain of Acinetobacter competent for transformation has made it possible to demonstrate the genetic relatedness of a large variety of gram-negative, oxidase-negative, nonmotile, and aerobic coccobacilli originally classified into eleven different genera. Deoxyribonucleic acid (DNA) species from 265 such strains are capable of transforming stable auxotrophs of the competent Acinetobacter to prototrophy. The compositions of these DNA species vary from 40 to 46.8% guanine plus cytosine. Strains with widely differing phenotypic properties are also included in this collection of acinetobacters. DNA species from all oxidase-positive strains of Moraxella and from a variety of common bacteria are unable to transform the competent Acinetobacter. Although acinetobacters are usually considered to be unable to reduce nitrate to nitrite, six strains known to carry out this reduction have been shown to be authentic acinetobacters since their DNA species readily transform the competent Acinetobacter auxotrophs to prototrophy. In contrast to previous findings that acinetobacters rarely grow with glucose as a sole carbon source, the results of the present study show that 17 of the 265 strains grow readily in a glucosemineral medium, and 48 other strains can mutate spontaneously to grow in such a medium. A second competent strain of Acinetobacter, originally unable to use glucose, d-xylose, or d-ribose as carbon sources, has been transformed for ability to dissimilate these compounds using DNA species from strains that normally grow on these sugars. Although most of the 265 Acinetobacter strains studied were originally grown on complex media when isolated from human sources, only nine of these strains require growth factors in order to grow in a mineral medium containing a single carbon and energy source. A simple transformation assay has been devised for rapid examination of large numbers of strains to determine whether or not they are acinetobacters. This assay, which is suitable for routine diagnostic work, includes a procedure for preparation of crude transforming DNA from a small quantity of bacterial paste. Samples of DNA prepared from Acinetobacter cultures that had died on slants and plates were still able to effect transformation of the competent auxotrophs to prototrophy.     

Natural transformation of Acinetobacter sp. strain BD413 with cell lysates of Acinetobacter sp., Pseudomonas fluorescens, and Burkholderia cepacia in soil microcosms

To elucidate the biological significance of dead bacterial cells in soil to the intra- and interspecies transfer of gene fragments by natural transformation, we have exposed the kanamycin-sensitive recipient Acinetobacter sp. strain BD413(pFG4) to lysates of the kanamycin-resistant donor bacteria Acinetobacter spp., Pseudomonas fluorescens, and Burkholderia cepacia. Detection of gene transfer was facilitated by the recombinational repair of a partially (317 bp) deleted kanamycin resistance gene in the recipient bacterium. The investigation revealed a significant potential of these DNA sources to transform Acinetobacter spp. residing both in sterile and in nonsterile silt loam soil. Heat-treated (80 degrees C, 15 min) cell lysates were capable of transforming strain BD413 after 4 days of incubation in sterile soil and for up to 8 h in nonsterile soil. Transformation efficiencies obtained in vitro and in situ with the various lysates were similar to or exceeded those obtained with conventionally purified DNA. The presence of cell debris did not inhibit transformation in soil, and the debris may protect DNA from rapid biological inactivation. Natural transformation thus provides Acinetobacter spp. with an efficient mechanism to access genetic information from different bacterial species in soil. The relatively short-term biological activity (e.g., transforming activity) of chromosomal DNA in soil contrasts the earlier reported long-term physical stability of DNA, where fractions have been found to persist for several weeks in soil. Thus, there seems to be a clear difference between the physical and the functional significance of chromosomal DNA in soil.     

Transformation and mobilization of cloning vectors in Acinetobacter spp.

R300B-, RSF1010-, and RK2-derived plasmids were introduced into Acinetobacter sp. strain HO1-N and Acinetobacter calcoaceticus BD413 by transformation and conjugal mobilization. The transformation frequencies of BD413 were 4.2 X 10(6) to 6.3 X 10(6) transformants per micrograms of DNA per 10(9) recipient cells. Conjugal mobilization frequencies were 1.1 X 10(-1) to 8.5 X 10(-1) per recipient. An improved method for the transformation of A. calcoaceticus BD413 is reported.     

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

The nptII(+) gene present in the genome of transgenic potato plants transforms naturally competent cells of the soil bacteria Pseudomonas stutzeri and Acinetobacter BD413 (both harboring a plasmid with an nptII gene containing a small deletion) with the same high efficiency as nptII(+) genes on plasmid DNA (3x10(-5)-1x10(-4) transformants per nptII(+)) despite the presence of a more than 10(6)-fold excess of plant DNA. However, in the absence of homologous sequences in the recipient cells the transformation by nptII(+) dropped by at least about 10(8)-fold in P. stutzeri and 10(9)-fold in Acinetobacter resulting in the latter strain in < or =1x10(-13) transformants per nptII(+). This indicated a very low probability of non-homologous DNA fragments to be integrated by illegitimate recombination events during transformation.     

Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater

The natural transformation of Acinetobacter calcoaceticus BD413 (trp E27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp ⁺) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca²⁺. With CaCl₂ solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl₂ and MgCl₂. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80°C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.     

Natural genetic transformation in monoculture Acinetobacter sp. strain BD413 biofilms

Horizontal gene transfer by natural genetic transformation in Acinetobacter sp. strain BD413 was investigated by using gfp carried by the autonomously replicating plasmid pGAR1 in a model monoculture biofilm. Biofilm age, DNA concentration, and biofilm mode of growth were evaluated to determine their effects on natural genetic transformation. The highest transfer frequencies were obtained in young and actively growing biofilms when high DNA concentrations were used and when the biofilm developed during continuous exposure to fresh medium without the presence of a significant amount of cells in the suspended fraction. Biofilms were highly amenable to natural transformation. They did not need to advance to an optimal growth phase which ensured the presence of optimally competent biofilm cells. An exposure time of only 15 min was adequate for transformation, and the addition of minute amounts of DNA (2.4 fg of pGAR1 per h) was enough to obtain detectable transfer frequencies. The transformability of biofilms lacking competent cells due to growth in the presence of cells in the bulk phase could be reestablished by starving the noncompetent biofilm prior to DNA exposure. Overall, the evidence suggests that biofilms offer no barrier against effective natural genetic transformation of Acinetobacter sp. strain BD413.     

Differential behavior of plasmids containing chromosomal DNA insertions of various sizes during transformation and conjugation in Haemophilus influenzae.

Plasmids with chromosomal insertions were constructed by removal of a 1.1-kilobase-pair piece from the 9.8-kilobase-pair vector plasmid pDM2 by EcoRI digestion and inserting in its place various lengths of chromosomal DNA (1.7, 3.4, and 9.0 kilobase pairs) coding for resistance to novobiocin. A fourth plasmid was constructed by insertion of the largest piece of chromosomal DNA into the SmaI site of pDM2. The plasmids without inserts were taken up poorly by competent cells and thus were considered not to contain specific DNA uptake sites. The presence of even the smallest insert of chromosomal DNA caused a large increase in transformation of Rec+ and Rec- strains. The frequency of plasmid establishment in Rec+ cells by transformation increased exponentially with increasing insert size, but in Rec- cells there was less transformation by the larger plasmids. Conjugal transfer of these plasmids was carried out with the 35-kilobase-pair mobilizing plasmid pHD147. The frequency of establishment of plasmids by this method not only was not markedly affected by the presence of the insertions, but also decreased somewhat with increase in insert size and was independent of rec-1 and rec-2 genes. Recombination between plasmid and chromosome was readily detected after transformation, but could not be detected after transconjugation even when the recipient cells were Rec+ and made competent. These data suggested that there is a special processing of plasmid DNA that enters the competent cells in transformation that makes possible recombination of homologous regions of the plasmid with the chromosome and pairing with the chromosome that aids plasmid establishment.     

Functions of the Mismatch Repair Gene mutS from Acinetobacter sp. Strain ADP1

The genus Acinetobacter encompasses a heterogeneous group of bacteria that are ubiquitous in the natural environment due in part to their ability to adapt genetically to novel challenges. Acinetobacter sp. strain ADP1 (also known as strain BD413) is naturally transformable and takes up DNA from any source. Donor DNA can be integrated into the chromosome by recombination provided it possesses sufficient levels of nucleotide sequence identity to the recipient's DNA. In other bacteria, the requirement for sequence identity during recombination is partly due to the actions of the mismatch repair system, a key component of which, MutS, recognizes mismatched bases in heteroduplex DNA and, along with MutL, blocks strand exchange. We have cloned mutS from strain ADP1 and examined its roles in preventing recombination between divergent DNA and in the repair of spontaneous replication errors. Inactivation of mutS resulted in 3- to 17-fold increases in transformation efficiencies with donor sequences that were 8 to 20% divergent relative to the strain ADP1. Strains lacking MutS exhibited increased spontaneous mutation frequencies, and reversion assays demonstrated that MutS preferentially recognized transition mismatches while having little effect on the repair of transversion mismatches. Inactivation of mutS also abolished the marker-specific variations in transforming efficiency seen in mutS(+) recipients where transition and frameshift alleles transformed at eightfold lower frequencies than transversions or large deletions. Comparison of the MutS homologs from five individual Acinetobacter strains with those of other gram-negative bacteria revealed that a number of unique indels are conserved among the Acinetobacter amino acid sequences.     

Recovery of DNA from the Acinetobacter calcoaceticus chromosome by gap repair.

A strain of Acinetobacter calcoaceticus that demonstrates unusually high competence for natural transformation by linear DNA has proven valuable for analysis of genes and gene clusters associated with aromatic catabolism. The transformation system allowed gap repair to be used to recover mutant chromosomal DNA within recombinant plasmids. The sizes of the recovered fragments, 5 and 7 kilobase pairs in length, indicate that gap repair will be a useful procedure for isolation of wild-type and modified gene clusters from the A. calcoaceticus chromosome.     

Natural Transformation of Acinetobacter calcoaceticus by Plasmid DNA Adsorbed on Sand and Groundwater Aquifer Material

It is known that plasmid DNA and linear duplex DNA molecules adsorb to chemically purified mineral grains of sand and to particles of several clay fractions. It seemed desirable to examine whether plasmid DNA would also adsorb to nonpurified mineral materials taken from the environment and, particularly, whether adsorbed plasmid DNA would be available for natural transformation of bacteria. Therefore, microcosms consisting of chemically pure sea sand plus buffered CaCl₂ solution were compared with microcosms consisting of material sampled directly from a groundwater aquifer (GWA) plus groundwater (GW) with respect to the natural transformation of Acinetobacter calcoaceticus by mineral-associated DNA. The GWA minerals were mostly sand with inorganic precipitates and organic material plus minor quantities of silt and clay (illite and kaolinite). The amount of plasmid DNA which adsorbed to GWA (in GW) was about 80% of the amount which adsorbed to purified sand (in buffered CaCl₂ solution). Plasmid DNA adsorbed on sand transformed A. calcoaceticus significantly less efficiently than did plasmid DNA in solution. In contrast, the transformation by sand-adsorbed chromosomal DNA was as high as that by DNA in solution. In GWA/GW microcosms, the efficiency of transformation by chromosomal DNA was similar to that in sand microcosms, whereas plasmid transformation was not detectable. However, plasmid transformants were found at a low frequency when GWA was loaded with both chromosomal and plasmid DNA. Reasons for the low transformation efficiency of plasmid DNA adsorbed to mineral surfaces are discussed. Control experiments showed that the amounts of plasmid and chromosomal DNA desorbing from sand during incubation with a cell-free filtrate of a competent cell suspension did not greatly contribute to transformation in sand microcosms, suggesting that transformation occurred by direct uptake of DNA from the mineral surfaces. Taken together, the observations suggest that plasmid DNA and chromosomal DNA fragments which are adsorbed on mineral surfaces in a sedimentary or soil habitat may be available (although with different efficiencies for the two DNA species) for transformation of a naturally competent gram-negative soil bacterium.     

Transformation of Acinetobacter sp. strain BD413(pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants

Here we show that horizontal transfer of DNA, extracted from transgenic sugar beets, to bacteria, based on homologous recombination, can occur in soil. Restoration of a 317-bp-deleted nptII gene in Acinetobacter sp. strain BD413(pFG4) cells incubated in sterile soil microcosms was detected after addition of nutrients and transgenic plant DNA encoding a functional nptII gene conferring bacterial kanamycin resistance. Selective effects of the addition of kanamycin on the population dynamics of Acinetobacter sp. cells in soil were found, and high concentrations of kanamycin reduced the CFU of Acinetobacter sp. cells from 10(9) CFU/g of soil to below detection. In contrast to a chromosomal nptII-encoded kanamycin resistance, the pFG4-generated resistance was found to be unstable over a 31-day incubation period in vitro.     

Transformation of Acinetobacter calco-aceticus (Bacterium anitratum)

A highly efficient transformation system has been demonstrated in a strain of Acinetobacter calco-aceticus (Bacterium anitratrum). During mixed growth of various stable, unencapsulated, mutant strains, deoxyribonucleic acid (DNA) is liberated and fully encapuslated transformants can be isolated. Purified DNA preparations have been used to transform suitable recipient mutant strains for ability to synthesize capsules, ability to dispense with a growth factor requirement, and resistance to streptomycin. When the wild-type strain is deprived of its capsule, either by mechanical stripping or by mutation, the unencapsulated cells tend to form large clumped masses. A nonclumping mutant of an unencapsulated strain has been isolated. When ability to synthesize capsules is transformed into this nonclumping strain, the resultant cells no longer form chains, unlike the wild-type encapsulated strain. It appears likely that the occurrence of transformation during growth of mixed cultures, with glucose or gluconate as the carbon source, may be the result of osmotic rupture resulting from the inability of unencapsulated strains to oxidize triose phosphates as fast as they are formed. The finding of transformation in Acinetobacter may provide an additional useful organism for the study of this mode of genetic transfer since this strain grows well in a simple mineral medium containing a single oxidizable source of carbon. Furthermore, no special supplementary factors seem to be required for transformation to take place.     

Effect of Genomic Location on Horizontal Transfer of a Recombinant Gene Cassette Between Pseudomonas Strains in the Rhizosphere and Spermosphere of Barley Seedlings

The use of genetically engineered bacteria in natural environments constitutes a risk of transfer of recombinant DNA to the indigenous bacteria. However, chromosomal genes are believed to be less likely to transfer than genes on mobilizable and conjugative plasmids. To study this assumption, horizontal transfer of a recombinant gene cassette inserted into the chromosome of a Pseudomonas stutzeri strain, into a mobilizable plasmid (pAGM42), and into a conjugative plasmid (pKJK5) isolated from barley rhizosphere was investigated. Horizontal transfer efficiencies of the gene cassette inserted into a conjugative plasmid was 8.20 × 10⁻³ transconjugants/(donors × recipients)^1/2 in the rhizosphere and 4.57 × 10⁻² transconjugants/(donors × recipients)^1/2 in the spermosphere. Mobilization of the plasmid pAGM42 by the plasmids RP4 and pKJK5 was also detected at high levels in the microcosms, transfer efficiencies were up to 4.36 × 10⁻³ transconjugants/(donors × recipients)^1/2. Transfer of chromosomal encoded genes could not be detected in the microcosms by conjugation or transformation. However, transformation did occur by using the same bacterial strains under laboratory conditions. The rhizosphere and especially the spermosphere thus proved to be hot spot environments providing favorable conditions for gene transfer by mobilization and conjugation, but these environments did not support transformation at a detectable level. Received: 21 July 2000 / Accepted: 21 August 2000     

Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone

Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ～9 kDa and ～30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5)-10(-6), suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.     

Transfer of genetic information within a colony of Haemophilus influenzae.

Different Haemophilus cultures were mixed and then spotted onto an agar plate. These mixed colonies were incubated at 37 degrees C and then scored for the presence of recombinants. It was found that conjugative plasmids transferred very efficiently and quickly under these conditions, but only between cells of the same species. Four small plasmids did not transfer at all, nor were they mobilized by the two conjugative plasmids studied. Chromosomal markers transferred very inefficiently. The evidence favored transfer by genetic transformation rather than by conjugation. When mixed cultures were inoculated into broth and then incubated, the transfer of conjugative plasmids was not observed. Transfer of chromosomal markers occurred only when the media used contained Eugonbroth in addition to brain heart infusion, and even then it was very inefficient. The addition of DNase completely eliminated such transfers. This and other evidence indicate that in cell suspensions, chromosomal marker transfer also occurs through transformation. A corrected map of several genetic markers is presented.     

Natural transformation in bacteria

Transformants may be formed by some bacterial species when the growing cultures are mixed. This phenomenon caused by the DNA release from bacterial cells is called natural transformation. DNA release is most likely to be mediated by cell autolysis. Both chromosomal markers and plasmids are transferred by natural transformation. The phenomenon is reproduced while growing bacteria together in sterile soil. The DNA adsorbed on sand and other soil solid particles was more resistant to DNAse action, than the free transforming DNA. Natural transformation seems to be one of the forms of the genetic exchange in bacteria in their habitats. An indirect argument for this suggestion is perfect coordination between the different steps of transformation process, at least, in some bacterial species.     

Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C. Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.     

Genetic transformation of Bacillus subtilis cells in the presence of clay minerals montmorillonite and kaolinite

The effect of the clay minerals montmorillonite and kaolinite on the transformation of competent Bacillus subtilis cells with chromosomal DNA was studied. Clay particles were found to substantially increase the transformation frequency of competent cells, as well as the rate of their spontaneous chromosomal and plasmid transformation. The effect was ascribed to the adsorption of bacterial cells on the surface of mineral particles.     

Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium

Acinetobacter sp. strain ADP1 is a nutritionally versatile soil bacterium closely related to representatives of the well-characterized Pseudomonas aeruginosa and Pseudomonas putida. Unlike these bacteria, the Acinetobacter ADP1 is highly competent for natural transformation which affords extraordinary convenience for genetic manipulation. The circular chromosome of the Acinetobacter ADP1, presented here, encodes 3325 predicted coding sequences, of which 60% have been classified based on sequence similarity to other documented proteins. The close evolutionary proximity of Acinetobacter and Pseudomonas species, as judged by the sequences of their 16S RNA genes and by the highest level of bidirectional best hits, contrasts with the extensive divergence in the GC content of their DNA (40 versus 62%). The chromosomes also differ significantly in size, with the Acinetobacter ADP1 chromosome <60% of the length of the Pseudomonas counterparts. Genome analysis of the Acinetobacter ADP1 revealed genes for metabolic pathways involved in utilization of a large variety of compounds. Almost all of these genes, with orthologs that are scattered in other species, are located in five major 'islands of catabolic diversity', now an apparent 'archipelago of catabolic diversity', within one-quarter of the overall genome. Acinetobacter ADP1 displays many features of other aerobic soil bacteria with metabolism oriented toward the degradation of organic compounds found in their natural habitat. A distinguishing feature of this genome is the absence of a gene corresponding to pyruvate kinase, the enzyme that generally catalyzes the terminal step in conversion of carbohydrates to pyruvate for respiration by the citric acid cycle. This finding supports the view that the cycle itself is centrally geared to the catabolic capabilities of this exceptionally versatile organism.