BACKGROUND
acinetobacter baumannii is a gram-negative coccobacillus that is increasingly recognized as a major pathogen causing nosocomial infections worldwide, particularly in patients admitted to intensive care units  <cit> .
a. baumannii can cause pneumonia, wound infections, urinary tract infections, bacteremia and meningitis  <cit> .
its clinical significance, especially in recent years, has increased because of the ability of the bacterium to acquire resistance determinants, making it one of the microorganisms threatening the current antibiotic era  <cit> .
the availability of the genome sequences of several strains of a. baumannii opens up new perspectives in the study of this bacterial species  <cit> .
the artificial introduction of mutations, by molecular techniques, is a useful way of advancing our understanding of the genetics of a. baumannii.
the method most commonly used to generate a. baumannii mutants involves integration of a plasmid into the chromosome by single crossover recombination.
this method requires an internal fragment homologous to the target gene cloned into a suicide vector carrying resistance cassettes  <cit> , which is a major limitation for systematic construction of mutants in post-genomic studies of a. baumannii.
the possibility that a second crossover event will return the mutant to a wild-type phenotype is another important inconvenience.
the gene replacement method is a useful way of overcoming these limitations.
gene replacement typically involves transformation of a non-replicating plasmid containing a deleted or modified gene, followed by low-frequency integration of a plasmid into the chromosome and selection for resolution events to identify gene replacement candidates.
in fact, in a. baumannii, the plasmids pssk <dig>  pex100t, and pjq <dig> are valuable tools for constructing mutants by this methodology  <cit> .
however, these gene replacement methodologies require several subcloning steps and phenotypic screenings.
as a means of circumventing these complicated approaches, we have developed a rapid and simple method of inactivating of chromosomal genes that does not require cloning steps.
moreover, the mutants grow directly on agar plates containing appropriate antibiotics and are confirmed by a simple pcr assay.
integration of a linear piece of foreign dna requires two recombination events, whereby the original genetic material is replaced by the recombinant dna  <cit> .
the methodology used in the present study is based on electroporation of a recipient a. baumannii strain with a linear pcr fragment carrying an antibiotic resistance cassette flanked by regions homologous to the target locus.
this method was used successfully to inactivate three chromosomal loci in a. baumannii .
moreover, the stability of the mutant, gene expression, and the efficiency of gene disruption and gene replacement methods were compared.
finally, the combination of both techniques was found to be an easy and useful method of obtaining double knockout mutants of a. baumannii.
RESULTS
replacement of the a. baumannii omp <dig> gene
a pcr product containing a kanamycin resistance cassette flanked by  <dig> bp of the regions surrounding the omp <dig> gene  was introduced into the a. baumannii atcc  <dig> strain by electroporation.
after selection on kanamycin-containing plates, the a. baumannii Δomp33::km mutant was obtained.
the frequency of generation of mutants by gene replacement was approximately 10- <dig>  the pcr tests with locus-specific primers revealed that  <dig> of  <dig> clones obtained had replaced the wild-type gene by the kanamycin cassette .
in addition, allelic replacement in mutant clones was further confirmed by sequencing the pcr products obtained .
aa.
baumannii atcc  <dig> chromosomal coordinates for each gene.
bthe length is expressed as number of amino acids.
cbased on national center for biotechnology information http://www.ncbi.nlm.nih.gov
disruption of the a. baumannii omp <dig> gene
the gene disruption method was also used to inactivate the omp <dig> gene.
gene disruption was carried out by cloning a 387-pb internal fragment of the omp <dig> gene into the pcr-bluntii-topo, to obtain the ptopo33int plasmid .
after transformation of the recombinant plasmid into the a. baumannii atcc  <dig> strain and selection on kanamycin-containing plates, the a. baumannii omp33::topo mutant was obtained.
the frequency of generation of mutants by gene disruption was approximately 10- <dig>  pcr tests with locus-specific primers revealed that all the clones analyzed  contained fragments of the expected size .
in addition, gene disruption in mutant clones was further confirmed by sequencing the pcr products obtained .
stable maintenance of plasmid insertion into the chromosome requires drugselection
gene knockout stability was tested by culturing both the Δomp33::km and omp33::topo a. baumannii mutants under nonselective conditions .
cultures of the mutant strains were initially grown in lb and at passages  <dig>   <dig>  and  <dig>  the cultures were dilution plated to obtain individual colonies, with replicate platings of  <dig> colonies for each strain on lb and lb supplemented with kanamycin.
the frequency of loss of kanamycin resistance in each passage after growth in non-selective conditions was 1% , 9% , and 37%  for the gene disrupted omp33::topo mutant.
by contrast, the gene-replaced Δomp33::km mutant was stable since no reversions were detected in any passage.
as expected, when the same experiment was carried out in the presence of selective pressure, both mutants remained stable .
complementation
taking advantage of the fact that the omp <dig> protein has been identified in the proteome of a. baumannii atcc  <dig> strain by 2-de and malditof/tof  <cit> , we observed the absence of the omp <dig> protein by 2-de analysis of the Δomp33::km mutant .
in order to complement the mutant phenotype, we constructed and tested the expression plasmid pet-ra.
the wild-type omp <dig> gene without its promoter region was cloned into this expression plasmid.
this construction was then introduced into the Δomp33::km mutant strain by electroporation.
the cell surface-associated proteins of the wild-type strain and the Δomp33::km mutant strain complemented with the pet-ra-omp <dig> plasmid were extracted and analyzed by 2de.
the omp <dig> protein was detected in the mutant complemented with the omp <dig> orf under the control of the β-lactamase ctx-m <dig> gene promoter of the pet-ra plasmid .
omp <dig> detection
western blot analysis was performed for further confirmation of the absence of omp <dig> in the a. baumannii mutants.
for this purpose, cell surface-associated proteins of wild-type strain, omp <dig> mutants, and pet-ra-omp33-complemented mutants were extracted and subjected to omp <dig> western blot analysis .
the omp <dig> protein was not detected in the cell surface-associated proteins of the mutants.
as expected, the omp <dig> protein was detected in the cell surface-associated proteins of both Δomp33::km and omp33::topo mutants containing the pet-raomp <dig> vector.
reproducibility of the gene replacement method
to ensure reproducibility of the gene replacement method, we produced the gene replacements of oxyr and soxr .
the same gene replacement method used to produce the Δomp33::km mutant was also used to construct the Δoxyr::km and Δsoxr::km mutants , with the primers listed in table  <dig>  the pcr tests with locus-specific primers revealed that  <dig> of the  <dig> clones obtained for the oxyr gene, and all clones  obtained for the soxr gene had replaced the wild-type gene with the kanamycin resistance cassette .
in addition, allelic replacement in mutant clones was further confirmed by sequencing the pcr products obtained .
transcriptional analyses demonstrated the lack of both oxyr and soxr gene expression in the Δoxyr::km and Δsoxr::km mutants, respectively .
*oligonucleotides including the indicated restriction site
construction of double knockout mutants
with the purpose of generating double knockout mutants, the recombinant plasmid ptopo33int was transformed into both Δoxyr::km and Δsoxr::km mutants.
after selection on zeocin- and kanamycin-containing plates, the Δoxyr::km-omp33::topo and Δsoxr::km-omp33::topo a. baumannii double knockout mutants were obtained.
pcr tests with locus-specific primers revealed that both mutants had fragments of the expected size .
in addition, gene disruption in mutant clones was further confirmed by sequencing the pcr products obtained, by transcriptional analyses to detect the oxyr and soxr genes , and by western blot analyses to detect the omp <dig> gene .
discussion
allelic mutation experiments enable investigation of the functions of many unknown genes identified during the sequencing of entire genomes.
a number of methods can be used to inactivate bacterial chromosomal genes.
as mentioned above, disruption of the a. baumannii chromosome can be achieved by integration of a plasmid into the chromosome by single crossover recombination  <cit> .
for this purpose, an internal fragment that is homologous to the target gene must be cloned into a non-replicating plasmid carrying at least one antibiotic resistance cassette.
however, the stability of this type of mutant must be taken into account, because if the gene-disrupted mutant cells are grown in a medium lacking antibiotic pressure, the integrated sequence could be removed, and the disrupted gene could revert to the original wild-type  <cit> .
we tested this possibility, and found that this is indeed the case, as also found in similar studies with e. coli  <cit> .
therefore, one limitation of the method is that the resulting mutants should always be maintained in an appropriate medium containing selective antibiotics.
another disadvantage of the method is that further manipulations of the mutant strain are restricted, because the same vector cannot be used , thus making it impossible to construct multiple gene knockout mutants.
the gene replacement method has recently been used to generate stable a. baumannii mutants  <cit> .
this method is based on integration of a plasmid containing the inactivated gene of interest into the bacterial chromosome by single crossover recombination, followed by resolution  of the integrated dna by a second recombination event, resulting in replacement of the original wild-type gene by the inactivated gene.
the key step in this procedure, in a. baumannii, is counter-selection  by the detection of sucrose sensitivity in the presence of the bacillus subtilis sacb gene coding for levansucrase  <cit> .
this method of counter-selection has been found to be useful for several other environmental bacteria  <cit> .
plasmids pssk <dig>  pex100t, and pjq <dig> have been successfully used to obtain a. baumannii mutants by this method  <cit> .
however, most bacteria subjected to homologous recombination, even under negative selection for the sacb gene, are wild-type and it is not possible to isolate the desired mutant directly  <cit> .
another disadvantage of this method is that integration of the dna may not always provide the desired replacement, since foreign dna with low or no sequence homology would rely on illegitimate recombination events, as previously reported for acinetobacter and other species  <cit> .
in addition, all of these gene replacement methodologies are time-consuming, and require several steps involving subcloning into a suicide delivery vector followed by electroporation into e. coli and subsequent transfer into a. baumannii by electroporation or conjugation.
to avoid such situations, we propose a method based on the electroporation of a. baumannii electrocompetent cells with linear dna, a pcr product including an antibiotic resistance cassette flanked by regions homologous to the target locus.
however, as expected, we noted an important disadvantage of the replacement method , with respect to the gene disruption method , i.e.
the low efficiency with regard to obtaining mutants .
in addition, we observed more illegitimate recombination events with the new method than with the gene disruption technique, since several colonies acquired the resistance antibiotic cassette , although the wild-type target gene was not replaced .
nevertheless, the new method is a useful genetic tool for systematic generation of knockouts.
moreover, to our knowledge, there are no previous reports of double knockout mutant strains of a. baumannii.
however, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene-knockout mutants in a. baumannii.
taking into account the results presented here, it intuitively appears that the gene replacement method would be successful with any strain of a. baumannii, including clinical strains, with the only limitation being the use of an appropriate antibiotic resistance marker.
although the kanamycin resistance cassette cannot be used in clinical strains , use of another antibiotic resistance marker such as rifampicin , would be appropriate for generating mutants by gene replacement in these problematic strains.
CONCLUSIONS
we report mutagenesis of three a. baumannii genes by use of a simple and rapid method.
the method offers advantages such as no cloning steps, stability even in the absence of selective pressure, and the possibility of constructing multiple gene knockout mutants.
the method may therefore facilitate the understanding of the genetics of a. baumannii.
although not tested, it is also possible that this novel method may also work with other pathogenic bacteria, in which genetic manipulation techniques are generally less well established than for e. coli and other bacterial species.
finally, the gene disruption method is recommended when only one a. baumannii gene must be inactivated, and when it is possible to maintain selective pressure, since it is the fastest and most efficient method of producing a. baumannii mutants described so far.

 acinetobacter baumannii is a gram-negative coccobacillus that is increasingly recognized as a major pathogen causing nosocomial infections worldwide, particularly in patients admitted to intensive care units. a. baumannii can cause pneumonia, wound infections, urinary tract infections, bacteremia and meningitis. its clinical significance, especially in recent years, has increased because of the ability of the bacterium to acquire resistance determinants, making it one of the microorganisms threatening the current antibiotic era. the availability of the genome sequences of several strains of a. baumannii opens up new perspectives in the study of this bacterial species. the artificial introduction of mutations, by molecular techniques, is a useful way of advancing our understanding of the genetics of a. baumannii. the method most commonly used to generate a. baumannii mutants involves integration of a plasmid into the chromosome by single crossover recombination. this method requires an internal fragment homologous to the target gene cloned into a suicide vector carrying resistance cassettes, which is a major limitation for systematic construction of mutants in post-genomic studies of a. baumannii. the possibility that a second crossover event will return the mutant to a wild-type phenotype is another important inconvenience. the gene replacement method is a useful way of overcoming these limitations. gene replacement typically involves transformation of a non-replicating plasmid containing a deleted or modified gene, followed by low-frequency integration of a plasmid into the chromosome and selection for resolution events to identify gene replacement candidates. in fact, in a. baumannii, the plasmids pssk pex100t, and pjq are valuable tools for constructing mutants by this methodology. however, these gene replacement methodologies require several subcloning steps and phenotypic screenings. as a means of circumventing these complicated approaches, we have developed a rapid and simple method of inactivating of chromosomal genes that does not require cloning steps. moreover, the mutants grow directly on agar plates containing appropriate antibiotics and are confirmed by a simple pcr assay. integration of a linear piece of foreign dna requires two recombination events, whereby the original genetic material is replaced by the recombinant dna. the methodology used in the present study is based on electroporation of a recipient a. baumannii strain with a linear pcr fragment carrying an antibiotic resistance cassette flanked by regions homologous to the target locus. this method was used successfully to inactivate three chromosomal loci in a. baumannii. moreover, the stability of the mutant, gene expression, and the efficiency of gene disruption and gene replacement methods were compared. finally, the combination of both techniques was found to be an easy and useful method of obtaining double knockout mutants of a. baumannii.  replacement of the a. baumannii omp gene a pcr product containing a kanamycin resistance cassette flanked by bp of the regions surrounding the omp gene was introduced into the a. baumannii atcc strain by electroporation. after selection on kanamycin-containing plates, the a. baumannii Δomp33::km mutant was obtained. the frequency of generation of mutants by gene replacement was approximately 10- the pcr tests with locus-specific primers revealed that of clones obtained had replaced the wild-type gene by the kanamycin cassette. in addition, allelic replacement in mutant clones was further confirmed by sequencing the pcr products obtained. aa. baumannii atcc chromosomal coordinates for each gene. bthe length is expressed as number of amino acids. cbased on national center for biotechnology information http://www.ncbi.nlm.nih.gov disruption of the a. baumannii omp gene the gene disruption method was also used to inactivate the omp gene. gene disruption was carried out by cloning a 387-pb internal fragment of the omp gene into the pcr-bluntii-topo, to obtain the ptopo33int plasmid. after transformation of the recombinant plasmid into the a. baumannii atcc strain and selection on kanamycin-containing plates, the a. baumannii omp33::topo mutant was obtained. the frequency of generation of mutants by gene disruption was approximately 10- pcr tests with locus-specific primers revealed that all the clones analyzed contained fragments of the expected size. in addition, gene disruption in mutant clones was further confirmed by sequencing the pcr products obtained. stable maintenance of plasmid insertion into the chromosome requires drugselection gene knockout stability was tested by culturing both the Δomp33::km and omp33::topo a. baumannii mutants under nonselective conditions. cultures of the mutant strains were initially grown in lb and at passages and the cultures were dilution plated to obtain individual colonies, with replicate platings of colonies for each strain on lb and lb supplemented with kanamycin. the frequency of loss of kanamycin resistance in each passage after growth in non-selective conditions was 1%, 9%, and 37% for the gene disrupted omp33::topo mutant. by contrast, the gene-replaced Δomp33::km mutant was stable since no reversions were detected in any passage. as expected, when the same experiment was carried out in the presence of selective pressure, both mutants remained stable. complementation taking advantage of the fact that the omp protein has been identified in the proteome of a. baumannii atcc strain by 2-de and malditof/tof, we observed the absence of the omp protein by 2-de analysis of the Δomp33::km mutant. in order to complement the mutant phenotype, we constructed and tested the expression plasmid pet-ra. the wild-type omp gene without its promoter region was cloned into this expression plasmid. this construction was then introduced into the Δomp33::km mutant strain by electroporation. the cell surface-associated proteins of the wild-type strain and the Δomp33::km mutant strain complemented with the pet-ra-omp plasmid were extracted and analyzed by 2de. the omp protein was detected in the mutant complemented with the omp orf under the control of the β-lactamase ctx-m gene promoter of the pet-ra plasmid. omp detection western blot analysis was performed for further confirmation of the absence of omp in the a. baumannii mutants. for this purpose, cell surface-associated proteins of wild-type strain, omp mutants, and pet-ra-omp33-complemented mutants were extracted and subjected to omp western blot analysis. the omp protein was not detected in the cell surface-associated proteins of the mutants. as expected, the omp protein was detected in the cell surface-associated proteins of both Δomp33::km and omp33::topo mutants containing the pet-raomp vector. reproducibility of the gene replacement method to ensure reproducibility of the gene replacement method, we produced the gene replacements of oxyr and soxr. the same gene replacement method used to produce the Δomp33::km mutant was also used to construct the Δoxyr::km and Δsoxr::km mutants, with the primers listed in table the pcr tests with locus-specific primers revealed that of the clones obtained for the oxyr gene, and all clones obtained for the soxr gene had replaced the wild-type gene with the kanamycin resistance cassette. in addition, allelic replacement in mutant clones was further confirmed by sequencing the pcr products obtained. transcriptional analyses demonstrated the lack of both oxyr and soxr gene expression in the Δoxyr::km and Δsoxr::km mutants, respectively. *oligonucleotides including the indicated restriction site construction of double knockout mutants with the purpose of generating double knockout mutants, the recombinant plasmid ptopo33int was transformed into both Δoxyr::km and Δsoxr::km mutants. after selection on zeocin- and kanamycin-containing plates, the Δoxyr::km-omp33::topo and Δsoxr::km-omp33::topo a. baumannii double knockout mutants were obtained. pcr tests with locus-specific primers revealed that both mutants had fragments of the expected size. in addition, gene disruption in mutant clones was further confirmed by sequencing the pcr products obtained, by transcriptional analyses to detect the oxyr and soxr genes, and by western blot analyses to detect the omp gene. discussion allelic mutation experiments enable investigation of the functions of many unknown genes identified during the sequencing of entire genomes. a number of methods can be used to inactivate bacterial chromosomal genes. as mentioned above, disruption of the a. baumannii chromosome can be achieved by integration of a plasmid into the chromosome by single crossover recombination. for this purpose, an internal fragment that is homologous to the target gene must be cloned into a non-replicating plasmid carrying at least one antibiotic resistance cassette. however, the stability of this type of mutant must be taken into account, because if the gene-disrupted mutant cells are grown in a medium lacking antibiotic pressure, the integrated sequence could be removed, and the disrupted gene could revert to the original wild-type. we tested this possibility, and found that this is indeed the case, as also found in similar studies with e. coli. therefore, one limitation of the method is that the resulting mutants should always be maintained in an appropriate medium containing selective antibiotics. another disadvantage of the method is that further manipulations of the mutant strain are restricted, because the same vector cannot be used, thus making it impossible to construct multiple gene knockout mutants. the gene replacement method has recently been used to generate stable a. baumannii mutants. this method is based on integration of a plasmid containing the inactivated gene of interest into the bacterial chromosome by single crossover recombination, followed by resolution of the integrated dna by a second recombination event, resulting in replacement of the original wild-type gene by the inactivated gene. the key step in this procedure, in a. baumannii, is counter-selection by the detection of sucrose sensitivity in the presence of the bacillus subtilis sacb gene coding for levansucrase. this method of counter-selection has been found to be useful for several other environmental bacteria. plasmids pssk pex100t, and pjq have been successfully used to obtain a. baumannii mutants by this method. however, most bacteria subjected to homologous recombination, even under negative selection for the sacb gene, are wild-type and it is not possible to isolate the desired mutant directly. another disadvantage of this method is that integration of the dna may not always provide the desired replacement, since foreign dna with low or no sequence homology would rely on illegitimate recombination events, as previously reported for acinetobacter and other species. in addition, all of these gene replacement methodologies are time-consuming, and require several steps involving subcloning into a suicide delivery vector followed by electroporation into e. coli and subsequent transfer into a. baumannii by electroporation or conjugation. to avoid such situations, we propose a method based on the electroporation of a. baumannii electrocompetent cells with linear dna, a pcr product including an antibiotic resistance cassette flanked by regions homologous to the target locus. however, as expected, we noted an important disadvantage of the replacement method, with respect to the gene disruption method, i.e. the low efficiency with regard to obtaining mutants. in addition, we observed more illegitimate recombination events with the new method than with the gene disruption technique, since several colonies acquired the resistance antibiotic cassette, although the wild-type target gene was not replaced. nevertheless, the new method is a useful genetic tool for systematic generation of knockouts. moreover, to our knowledge, there are no previous reports of double knockout mutant strains of a. baumannii. however, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene-knockout mutants in a. baumannii. taking into account the results presented here, it intuitively appears that the gene replacement method would be successful with any strain of a. baumannii, including clinical strains, with the only limitation being the use of an appropriate antibiotic resistance marker. although the kanamycin resistance cassette cannot be used in clinical strains, use of another antibiotic resistance marker such as rifampicin, would be appropriate for generating mutants by gene replacement in these problematic strains.  we report mutagenesis of three a. baumannii genes by use of a simple and rapid method. the method offers advantages such as no cloning steps, stability even in the absence of selective pressure, and the possibility of constructing multiple gene knockout mutants. the method may therefore facilitate the understanding of the genetics of a. baumannii. although not tested, it is also possible that this novel method may also work with other pathogenic bacteria, in which genetic manipulation techniques are generally less well established than for e. coli and other bacterial species. finally, the gene disruption method is recommended when only one a. baumannii gene must be inactivated, and when it is possible to maintain selective pressure, since it is the fastest and most efficient method of producing a. baumannii mutants described so far. 
moreover, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene knockout mutants in a. baumannii. we describe here a rapid and simple method of obtaining a. baumannii mutants by gene replacement via double crossover recombination, by use of a pcr product that carries an antibiotic resistance cassette flanked by regions homologous to the target locus. acinetobacter baumannii is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. this study provides a rapid and simple method of obtaining stable mutants of a. baumannii free of foreign plasmidic dna, which does not require cloning steps, and enables construction of multiple gene knockout mutants. the methodologies developed to inactivate a. baumannii genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double gene knockout mutant strains of a. baumannii. in addition, we disrupted one of these genes by integration of a plasmid into the chromosome by single crossover recombination, the most widely used method of obtaining a. baumannii mutants. comparison of the different techniques revealed absolute stability when the gene was replaced by a double recombination event, whereas up to 40% of the population reverted to wild-type when the plasmid was disrupting the target gene after passages in broth without selective pressure.
 acinetobacter baumannii is a multidrug-resistant bacterium responsible for nosocomial infections in hospitals worldwide. study of mutant phenotypes is fundamental for understanding gene function. the methodologies developed to inactivate a. baumannii genes are complicated and time-consuming; sometimes result in unstable mutants, and do not enable construction of double gene knockout mutant strains of a. baumannii.  we describe here a rapid and simple method of obtaining a. baumannii mutants by gene replacement via double crossover recombination, by use of a pcr product that carries an antibiotic resistance cassette flanked by regions homologous to the target locus. to demonstrate the reproducibility of the approach, we produced mutants of three different chromosomal genes by this method. in addition, we disrupted one of these genes by integration of a plasmid into the chromosome by single crossover recombination, the most widely used method of obtaining a. baumannii mutants. comparison of the different techniques revealed absolute stability when the gene was replaced by a double recombination event, whereas up to 40% of the population reverted to wild-type when the plasmid was disrupting the target gene after passages in broth without selective pressure. moreover, we demonstrate that the combination of both gene disruption and gene replacement techniques is an easy and useful procedure for obtaining double gene knockout mutants in a. baumannii.  this study provides a rapid and simple method of obtaining stable mutants of a. baumannii free of foreign plasmidic dna, which does not require cloning steps, and enables construction of multiple gene knockout mutants. 