the rapid exchange of genetic information mediated by hgt enables bacteria to adapt to new environmental niches , to spread harmful traits such as antibiotic resistance cassettes or pathogenicity islands and to maintain genome integrity . hgt by means of conjugation and transduction relies on cell - cell contacts and/or mobile genetic elements , whereas natural transformation consists of the uptake of naked dna from the environment . the ability to acquire exogenous dna is called natural competence , a physiological state that is of transient nature for most bacterial species . the development of natural competence is often a highly regulated process , which frequently requires environmental cues such as nutrient availability or species - specific competence pheromones . initiation of natural competence results from the expression of so - called competence genes , which in part encode specialized proteins that constitute the so - called dna - uptake machinery . contrary to the different regulatory networks controlling the onset of natural competence , the components of the dna - uptake machinery are often conserved in gram - positive and gram - negative bacteria . moreover , many of these proteins share homology with type iv pili ( tfp ) and type ii secretion systems ( t2ss ) . thus , it is tempting to speculate that dna uptake by naturally competent bacteria occurs via nearly universal machineries . however , dna - uptake complexes , especially their composition and cellular localization , have so far been poorly characterized . moreover , most of the existing information is based on data that were acquired from the naturally competent gram - positive bacterium bacillus subtilis and , more recently , also from streptococcus pneumoniae . however , the situation is different and less well studied for gram - negative bacteria , where external dna must be first translocated across the outer membrane ( and the periplasmic space ) . in our recent study , we aimed to gain insight into the process of dna uptake and to visualize the dna - uptake machinery in the human pathogen vibrio cholerae using a cell - biology based approach . in this gram - negative bacterium , natural competence is induced during growth on chitinous surfaces , which the bacterium encounters in its natural reservoir . substantial information concerning the regulatory circuit of natural competence and transformation of this organism has been gathered in less than a decade ( first reviewed by seitz and blokesch and later also by sun et al . ) . however , the dna - uptake machinery of v. cholerae has never been investigated and has remained a mystery until recently . as a first step , seitz and blokesch identified the minimum competence regulon of v. cholerae based on previously unpublished and published expression data ( and blokesch , unpublished ) , locus organization , homology to other naturally competent bacteria and conservation in other naturally transformable vibrio species . a minimal ( most likely still incomplete ) competence set of 19 genes was identified with the majority of genes encoding proteins with homology to biogenesis or structural components of tfp . to verify the importance of these candidate genes with respect to dna uptake and natural transformation , each gene was deleted from the parental v. cholerae o1 el tor strain , and the mutant was assessed for its transformability . notably , pild was excluded in this study as the deletion mutant displayed a strong growth defect ( both in liquid culture and on solidified agar plates ) . furthermore , the gene coding for the single - strand binding protein ( ssb ) could not be deleted . the latter finding is in agreement with recent studies that report the essentiality of ssb in v. cholerae . all of the other mutants were impaired for transformation even though low numbers of transformants were consistently obtained for strains lacking components of the tfp portion of the dna - uptake machinery . such residual transformability was never observed for those mutants devoid of the competence genes comea , comec , and comf as well as the reca mutant , which served as a control in this assay . the encoded competence proteins were further categorized ( using a recently developed whole - cell duplex pcr dna - uptake assay ) as required for transport across the outer membrane or inner membrane . all of the tfp - related proteins and comea were required for dna - uptake across the outer membrane , whereas comec , comf , and reca were necessary only for inner membrane dna translocation or recombination . moreover , the results demonstrated that all of the steps involving tfp - related components or comea function upstream of the inner - membrane channel comec , whereas the cytoplasmic protein reca was confirmed as acting downstream of comec ( similar to dpra ; seitz and blokesch , data not shown ) . finally , as the comec and comf mutant of v. cholerae consistently displayed the same phenotype with respect to the absence of transformability and the accumulation of transforming dna within the periplasmic space , it was hypothesized that the two proteins comec and comf work in concert in mediating the translocation of the incoming dna across the inner membrane ( fig .  1 ) . the comec and comf proteins might drive dna import into the cytoplasm . based on the similar phenotypes of both the comec and comf mutants , it was suggested that comf works in concert with comec in the translocation of the dna across the inner membrane . as a second step , seitz and blokesch imaged a variety of competence - related proteins ( fig .  2 ) . first , the authors used immunofluorescence labeling against an affinity tag that was genetically added to the major pilin subunit pila . using this approach , notably , this structure extended well beyond the outer membrane , thereby contradicting the previously hypothesized pseudopilus model for v. cholerae . for the majority of piliated cells ( 95% ) in the absence of each of the tfp - related components encoded by the minimum competence gene set , the cells appeared non - piliated . however , a pilus structure was still observable in the mutants lacking either a tfp - unrelated competence gene ( comea , comec , comf , and reca ; all non - transformable ) or the potential retraction atpase ( pilt ) . interestingly , the latter mutant population contained a significantly higher fraction of cells with two or more tfps . model of the localization of the dna - uptake machinery ( including the tfp ) in v. cholerae . lower part : schematic representation of a v. cholerae cell ( b ) . upon induction of natural competence , several pilq protein foci were observed ( in blue ; image : mcherry - pilq translational fusion false - colored in blue [ c ] ) . a similar multi - foci pattern also became evident for the atpase pilb ( in red ; image : pilb - dsred translational fusion [ d ] ) . nonetheless , the majority of cells only displayed a single tfp ( in green ; image : immunofluorescence image of pila - strep [ e ] ) . it was therefore hypothesized that a few pre - assembled tfp complexes containing , among other components , pilq ( blue ) but lacking the major pilin subunit pila exist within the cells and that pila polymerization is only initiated after the elongation atpase pilb ( in red ) co - localizes with one of the pre - assembled complexes . next , the authors sought to also localize other competence proteins that were produced as translational fusions ( with fluorescent proteins ) and replaced the native proteins . all of the competence - induced cells displayed several distinct foci for the secretin pilq as well as for the putative traffic atpase pilb ( fig . however , how can such a discrepancy between a single pilus and several outer membrane secretins ( pilq ) and elongation atpases ( pilb ) be explained ? to address this question , seitz and blokesch demonstrated that the tfp consistently co - localized with one of the pilq foci . moreover , the pilb atpase foci were dynamic within the cell ( which was not the case for a pilb variant with a mutated walker b motif ) and sometimes aggregated in close proximity of the pilq protein complexes . the authors therefore hypothesized that each cell might contain several pre - assembled tfp complexes that might solely lack the major pilin subunit and that pilus elongation one at a time  occurs upon stimulation of pilin polymerization through pilb ( fig .  2 ) . such a model would suggest that a higher percentage of cells contain more than one pilus during pilb overproduction , which was exactly what the authors described . another important finding of this study was that a gene cluster of five genes ( e.g. , vc0857 to vc0861 ) encoding hypothetical proteins or putative type iv pilins contributed significantly to efficient transformation . interestingly , a minor pilin ( comp ) was recently shown to directly interact with the species - specific dna of neisseria thereby answering one of the many open questions in the field of natural transformation , namely  which , if any , protein acts as a receptor for transforming dna  in neisseria . however , the comp mutants of n. gonorrhoeae , although impaired for natural transformation , are properly piliated and exert a normal tfp function . we recently showed that in contrast to neisseria and haemophilus influenzae , v. cholerae does not differentiate between species - specific and species - non - specific dna at the level of the dna - uptake process , thus excluding a role of the vc0857 - 0861 operon - encoded proteins in species - specific dna recognition . notably , no pili were observed in any of the five mutant strains , consistent with the absence of transforming dna in the periplasm . a putative function of these proteins could therefore be to activate the tfp assembly machinery , which could potentially work in a similar manner as recently demonstrated for three minor pseudopilins involved in t2ss assembly in klebsiella oxytoca . another competence gene that was part of this study but not further investigated was vc1612 . in previous studies , the vc1612 gene was slightly upregulated when v. cholerae was grown on chitin surfaces and was inducible by the transformation regulatory protein tfox . furthermore , a blast analysis against the neisseria gonorrhoeae fa1090 genome indicated that the vc1612 protein was homologous to jcvi locus nt03ng0804 (  type iv pilus biogenesis - stability protein pilw ( pilf )  ) ( supplementary data in ref . as a v. cholerae vc1612 knockout strain behaved similar to other tfp - related mutants ( e.g. , lack of both piliation and dna uptake into the periplasm ) , it was suggested that this protein might participate in the tfp portion of the dna - uptake complex . next , seitz and blokesch aimed to predict the subcellular localization of vc1612 using the web - based psortb 3.0 algorithm . however , as the scores were similar for cytoplasmic and outer - membrane localization , the program proposed that the protein might have multiple localization sites . given that a signal peptide was also not predicted by the signalp server , the authors suggested in their model that the localization of vc1612 is cytoplasmic ( fig . interestingly , the vc1612 protein shows 34% identity ( and 51% similarity ) to pilf of pseudomonas aeruginosa strain pao1 ( ncbi reference sequence : np_252494.1 ) . for p. aeruginosa , it was initially suggested that pilf might be localized to the inner face of the cytoplasmic membrane , although a more recent study by koo et al . challenged this notion and demonstrated that pilf is an outer membrane lipoprotein involved in the insertion and polymerization of the secretin pilq ( in a chaperone - like manner ) . thus , given that future studies will also demonstrate outer membrane localization for the vc1612 protein of v. cholerae , it would be tempting to speculate that the protein also acts as pilotin , which could foster pilq secretin assembly ( fig .  3 ) . the pilf protein of v. cholerae might act as pilotin . in our previous study , however , blast comparisons with pilf of p. aeruginosa indicate that the vc1612 protein of v. cholerae ( suggested annotation : pilf ) might act as a pilotin , thereby assisting the pilq secretin in its assembly . while comparing the competence / tfp - related genes of different organisms , it became obvious that there were many variations with respect to the organization of the genes ( e.g. , whether they clustered together in operons or not ) . for example , whereas the major pilin - encoding gene pila and the gene coding for the elongation atpase pilb exist in a single operon in v. cholerae ( supplementary table of ref . 17 ) , this is not the case for many other tfp - containing organisms ( for an example , see the genetic map of the tfp genes of myxococcus xanthus ) . interestingly the pilmnopq operon ( fig . 4 ) is conserved in many proteobacteria , including for example p. aeruginosa strain pao1 , pseudomonas stutzeri a1501 , escherichia coli k12 , legionella pneumophila strain paris , legionella longbeachae strain nsw150 , and m. xanthus ( according to the genolist database ; see also review ) . the conserved pilmnopq operon encoding components of the tfp - part of the dna - uptake machinery . for details , see text . what is so special about these five genes that warrants such a conserved operon structure ? indeed , many studies have recently addressed potential protein - protein interactions between these five proteins , and all of these studies suggested that the encoded proteins form a complex that connects the inner and outer membrane ( including the outer membrane secretin pilq ) . interestingly , friedrich et al . recently demonstrated that this protein complex is assembled in a sequential outside - in pathway , which therefore occurs opposite to the gene succession ( pilq - p - o - n - m ) . it is tempting to speculate that the genetic organization aims to avoid the toxic effects of the gene products that are encoded toward the end of the operon . thus , the conserved gene sequence would ensure that a given stoichiometry is preserved and that the assembly is only initiated after all of the proteins are synthesized ( e.g. , full - length mrna is available in the cell ) . notably , the enhanced expression of pilq and pilp in trans results in severe toxicity in both v. cholerae and e. coli . in summary , we conclude that we are only beginning to understand the basic components of the dna - uptake machinery of v. cholerae and its functionality . moreover , the composition and mechanistic aspects of type iv pili and the dna - uptake machineries are generally not well understood , even though many hypothetical models of these multiprotein complexes exist . notably , many of those models have not changed dramatically over the last decade ( compare , for example , refs .
natural competence for transformation is a developmental program that allows certain bacteria to take up free extracellular dna from the environment and integrate this dna into their genome . 
 thereby , natural transformation acts as mode of horizontal gene transfer and impacts bacterial evolution . 
 the number of genes induced upon competence induction varies significantly between organisms . 
 however , all of the naturally competent bacteria possess competence genes that encode so - called dna - uptake machineries . 
 some components of these multi - protein complexes resemble subunits of type iv pili and type ii secretion systems . 
 however , knowledge on the mechanistic aspects of such dna - uptake complexes is still very limited . here , we discuss some new findings regarding the dna - uptake machinery of the naturally transformable human pathogen vibrio cholerae . 
 the potential of this organism to initiate the competence program was discovered less than a decade ago . 
 however , recent studies have provided new insight into both the regulatory pathways of competence induction and into the dna uptake dynamics .