@article{smeets_natural_2002,
	title = {Natural transformation in Helicobacter pylori: {DNA} transport in an unexpected way},
	volume = {10},
	url = {http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD0-45CC7MN-2&_user=108429&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059713&_version=1&_urlVersion=0&_userid=108429&md5=f2b1fdb78b9205089de1f768c2d7f121},
	doi = {{10.1016/S0966-842X(02)02314-4}},
	abstract = {a Dept of Medical Microbiology and Infection Control, {VU} University Medical Center, {PO} box 7057, 1007 {MB} Amsterdam, The Netherlands b Dept of Gastroenterology and Hepatology, Dijkzigt Hospital, Room L481, Dr. Molewaterplein 40, 3015 {GD} Rotterdam, The Netherlands Like other bacterial species with a high frequency of inter-strain recombination, the human gastric pathogen Helicobacter pylori is competent for natural transformation. Recent data, however, indicate that its {DNA-uptake} system differs significantly from that in other species that contain {DNA-uptake} systems related to type {IV} pili. Instead, in H. pylori it has been suggested that the five proteins that form the transmembrane channel of the transformation system are closely related to subunits of type {IV} secretion systems. It has been proposed that in Helicobacter pylori, the system involved in natural transformation of {DNA} is similar to that involved in type {IV} secretion. Author Keywords: Type {IV} secretion; Helicobacter pylori; {DNA} uptake/natural transformation; Cag {PAI} Subject-index terms: Microbiology; Cell biology Helicobacter pylori colonizes the gastric mucosa of humans and, if left untreated, this will result in a lifelong gastritis. Although infection is usually asymptomatic, it can manifest clinically as peptic or duodenal ulcers. Additionally, colonization with H. pylori is believed to induce gastric atrophy and increase the risk of gastric cancer. New genotypes of H. pylori are generated by recombination fast enough to eliminate, essentially, the effect of clonal descent on the population structure [1]. The main mechanism of horizontal gene transfer used by H. pylori is natural transformation. The transformation systems of several Gram-positive and Gram-negative species have been studied in some detail and are either dependent on the expression of pili or consist of proteins that resemble pilin subunits [2]. Recent data from Hofreuter et al., however, indicate that the transformation system of H. pylori is fundamentally different: rather than resembling pili, the structural core of the H. pylori {DNA-translocation} apparatus is related to type {IV} secretion systems [3 and 4]. Type {IV} secretion systems translocate bacterial macromolecules into the extracellular compartment or into other cells. The translocated molecules range from proteins that are injected into mammalian host cells to plasmid {DNA} that is transferred during conjugation. Although the particular set of protein components involved varies between type {IV} systems, they generally consist of a pilus-like mating system and a mating channel that spans the bacterial cell wall. The archetypal type {IV} secretion system is the Vir system, encoded by the Agrobacterium tumefaciens Ti-plasmid. Ti stands for ‘tumor inducing’, and describes the neoplastic root nodules that are typical of crown gall, a plant disease caused by A. tumefaciens. These nodules are induced following the introduction of Ti-plasmid {DNA} into the plant cells by the Vir system. The total set of proteins involved in Vir-mediated {DNA} transfer includes {VirB1–VirB11} and {VirD4,} which are encoded by the {virB} operon. The transmembrane transport pore is proposed to consist of four proteins: {VirB7,} {VirB8,} {VirB9} and {VirB10} {(Fig.} 1). The H. pylori {comB} transformation operon, as described by Hofreuter et al., carries homologs of core components of a type {IV} secretion system: {comB7,} {comB8,} {comB9} and {comB10} (formerly designated orf2, {comB1,} {comB2} and {comB3).} Like most type {IV} secretion systems, the {comB} genes have the same co-linear organization as their {virB} counterparts. Perhaps more important, the predicted cellular topology of the {ComB} protein products strongly suggests that they resemble the building blocks of the {VirB} transport pore on a structural level {(Fig.} 2). {comB7,} {comB8,} {comB9} and {comB10} are organized in a tandem arrangement with little overlap in their reading frames. The first reading frame of the operon, {comB7,} is predicted to code for a peptide with a putative lipoprotein signal sequence for export to the periplasm and lipid modification, like {virB7.} {VirB7} binds {VirB9} by disulfide bonding between two cysteine residues [5] and this bond is necessary for the assembly of a stable transport apparatus [6]. Hofreuter et al. have shown that the cysteine residues involved in this bonding are conserved in {ComB7} and {ComB9.} Moreover, their experiments show that {ComB9} and {ComB10,} when overproduced in H. pylori, are degraded faster in the absence of {ComB7} and {ComB8,} an indication there is a functional analogy between {VirB7} and {ComB7.} {ComB8,} like {VirB8,} is a membrane-anchored protein without a signal sequence for export. {ComB9} and {ComB10} do have cleavable leader sequences but, as in the Vir system, only {ComB10} appears to remain anchored in the cytoplasmic membrane. Together, the {ComB} proteins have all the characteristics of a pore-forming transmembrane complex, suitable for the translocation of {DNA} through the cellular envelope. In addition to the {ComB} system, H. pylori also contains a ‘classical’ type {IV} secretion system that is encoded by genes on the cag pathogenicity island {(cag-PAI).} The {cag-PAI} is a non-conserved chromosomal locus with the characteristics of a horizontally acquired cassette (e.g. aberrant {GC-content} and codon usage), which translocates the {CagA} protein into gastric epithelial cells [7]. The {cag-PAI} carries genes coding for homologs of {VirD4,} {VirB4,} {VirB7,} {VirB9,} {VirB10} and {VirB11} [8]. The conclusion that the Cag type {IV} system and the {ComB} system are functionally unrelated comes from various lines of evidence. First, mutations in the {comB} operon do not affect the function of cag [4]. Second, almost half of all H. pylori strains lack the {cag-PAI,} and these strains are also capable of transformation. Additionally, artificial deletion of the {cag-PAI} in other strains does not result in a decreased transformation efficiency [4 and 9]. The energy for molecular transfer by type {IV} systems is provided by homologs of the membrane-bound {ATPases} {VirD4,} {VirB4} and {VirB11.} In addition to the {cag-PAI-encoded} {VirD4} {(HP0524),} {VirB4} {(CagE)} and {VirB11} {(HP525)} homologs, H. pylori contains three chromosomal {VirB4} homologs {(Hp0017,} {HP0441} and {HP0459),} one {VirB11} homolog {(HP1421)} and one {VirD4} {(HP1006)} homolog [10]. The {VirD4} homolog was shown not to be required for the transformation of H. pylori [11]. In A. tumefaciens, {VirD4} is thought to be involved in the coupling of the {Ti-DNA–protein} complex to the transport system [12], and such a function is probably dispensable in the reversed {DNA} transport of transformation. {VirB4} and {VirB11} both have a proposed function in the transport system itself. An earlier study of H. pylori protein–protein interactions indicated that a {virB4} homolog 23 kb upstream of the {comB} locus interacts with {comB10} [13]. Hofreuter et al. show that a null mutant in this protein is indeed affected in its transformation [4] and designated this {virB4} homolog {comB4.} The H. pylori {virB11} homolog might also be part of the {comB} system. In A. tumefaciens, {VirB11} interacts with {VirB9} and {VirB10} [14] and is involved in the assembly of the transport system. A similar function might be carried out by the H. pylori {virB11} homolog, but its contribution to the transformation of H. pylori remains to be proven. The {VirB1,} {VirB2,} {VirB3,} {VirB5} and {VirB6} proteins of A. tumefaciens are involved in Ti-mediated {DNA} translocation, but there are no obvious homologs of these proteins in H. pylori. The H. pylori genomic sequence [10] contains 1590 predicted {ORFs.} Many of these lack a known function, and some might serve analogous purposes. The apparent absence of homologs, however, can also be explained by the differences between vir-mediated secretion and {comB-mediated} {DNA} uptake. The first protein encoded by the {virB} locus of A. tumefaciens is {VirB1.} {VirB1} is targeted to the periplasm where it is spliced into a carboxy-terminal and an amino-terminal half, which both have a different function [15]. The amino-terminal half remains in the periplasm and is involved in murein hydrolysis to provide the channel for the Vir transport system; the carboxy-terminal half is secreted and its function is not exactly clear [16]. The cell wall lysis performed in A. tumefaciens by {VirB1} in H. pylori might be replaced by {ComL,} a homolog of a murein hydrolase involved in natural transformation of Neisseria gonorrhoeae [17]. A null mutant in H. pylori {comL,} however, could not be obtained and is supposed to be lethal [11]. Therefore, its role in transformation is as yet uncertain. {VirB2,} {VirB3,} {VirB5} and {VirB6} serve as structural components of the A. tumefaciens T-pilus {(VirB2} and {VirB5)} [18 and 19] or are involved in its assembly {(VirB3} and {VirB6)} [20]. There is no obvious reason why a natural transformation system should be dependent on a structure similar to the T-pilus, and this could explain the absence of similar proteins in H. pylori. The differences between secretion and {DNA} import also creates new demands for the {ComB} system. The first step in natural transformation is the binding of the extracellular double-stranded {DNA.} Next, transforming species usually restrict the {DNA} into fragments of a suitable length before transport into the cytoplasm. The imported {DNA} is normally single stranded (ss) [2]. Vir and other type {IV} secretion systems involved in conjugation also transfer ss {DNA.} Therefore, at least a {DNA-binding} function and probably also endonuclease and ss {DNA-degrading} functions are required in the {comB} system. It is tempting to speculate these tasks are fulfilled by one or more proteins that interact with {ComB7,} in analogy to the T-pilus association with {VirB7} in A. tumefaciens [21]. A candidate protein for {DNA} binding or processing is {ComH,} a putatively exported protein necessary for {DNA} translocation in H. pylori, with homologs neither in other naturally transformable bacteria nor in type {IV} secretion systems [22]. With the analogy to type {IV} secretion systems in mind, an outline of a minimal H. pylori {DNA-uptake} system is shown in Fig. 2. Usually, being the first of a kind means being the only one for a limited time. In the case of the {comB} transformation system, the runner-up is located on the Campylobacter jejuni plasmid {pVir.} The partial sequence of this plasmid revealed a locus with the same organization as the H. pylori {comB8,} {comB9} and {comB10} genes, with 55\% sequence similarity [23]. Bacon et al. showed that a null mutant in the {comB10} homolog attenuated adherence to and invasion of {INT407} cells of the host strain, but also reduced its transformation competence by 80\% and the authors concluded that this operon is also involved in natural transformation. Thus, {pVir} appears to contain the first described plasmid-encoded transformation system. Mutants in this plasmid-borne {comB} system still have significant transformation activity. In addition, some plasmid-less C. jejuni strains such as {NCTC11168} are highly competent for transformation. An alternative {DNA-uptake} system must therefore be present in C. jejuni, although the chromosomal sequence of C. jejuni {NCTC11168} does not contain homologs of {comB8,} {comB9} and {comB10} [24]. As plasmids are more prone to transfer between bacterial species, the plasmid-encoded {comB} homologs implicated in transformation of C. jejuni by Bacon et al. might also be present in other bacteria, and thus could serve in natural transformation of a wide range of bacterial species.  },
	number = {4},
	journal = {Trends in Microbiology},
	author = {Leonard C. Smeets and Johannes G. Kusters},
	month = apr,
	year = {2002},
	pages = {159--162}
},

@article{hofreuter_natural_1998,
	title = {Natural competence for {DNA} transformation in Helicobacter pylori: identification and genetic characterization of the {comB} locus},
	volume = {28},
	issn = {{0950-382X}},
	url = {http://www.ncbi.nlm.nih.gov/pubmed/9663688},
	abstract = {The gram-negative bacterial pathogen Helicobacter pylori, an important aetiological agent of gastroduodenal disease in humans, belongs to a group of bacterial species displaying competence for genetic transformation. Here, we describe the {comB} gene locus of H. pylori involved in {DNA} transformation competence. It consists of a cluster of four tandemly arranged genes with partially overlapping open reading frames, orf2, {comB1,} {comB2} and {comB3,} constituting a single transcriptional unit. Orf2 encodes a 37-amino-acid peptide carrying a signal sequence, whereas {comB1,} {comB2} and {comB3} produce 29 {kDa,} 38 {kDa} and 42 {kDa} proteins, respectively, as demonstrated by immunoblotting with specific antisera. For Orf2 and {ComB1,} no homologous proteins were identified in the database. For {ComB3,} the best homologies were found with {TraS/TraB} from the Pseudomonas aeruginosa conjugative plasmid {RP1} and {TrbI} of plasmid {RP4,} {VirB10} from the Ti plasmid of Agrobacterium tumefaciens and {PtlG,} a protein involved in secretion of pertussis toxin of Bordetella pertussis. Defined transposon knock-out mutants in individual {comB} genes resulted in transformation-defective phenotypes ranging from a 90\% reduction to a complete loss of the natural transformation efficiency. The {comB2} and {comB3} genes show homology to {HP0528} and {HP0527,} respectively, located on the {cagII} pathogenicity island of H. pylori strain 26695.},
	number = {5},
	journal = {Molecular Microbiology},
	author = {D Hofreuter and S Odenbreit and G Henke and R Haas},
	month = jun,
	year = {1998},
	note = {{PMID:} 9663688},
	keywords = {Amino Acid {Sequence,Bacterial} {Proteins,Base} {Sequence,Chromosome} {Mapping,Chromosomes,} {Bacterial,DNA,} {Bacterial,DNA-Binding} {Proteins,Gene} {Expression,Genes,} {Bacterial,Helicobacter} {pylori,Molecular} Sequence {Data,Mutagenesis,} {Insertional,Operon,Sequence} Analysis, {DNA,Transformation,} Bacterial},
	pages = {1027--38}
},

@article{banta_stability_1998,
	title = {Stability of the Agrobacterium tumefaciens {VirB10} Protein Is Modulated by Growth Temperature and Periplasmic Osmoadaption},
	volume = {180},
	url = {http://jb.asm.org/cgi/content/abstract/180/24/6597},
	abstract = {Export of oncogenic {T-DNA} from the phytopathogen Agrobacterium tumefaciens is mediated by the products of the {virB} operon. It has recently been reported {(K.} J. Fullner and E. W. Nester, J. Bacteriol. 178:1498-1504, 1996) that {DNA} transfer does not occur at elevated temperatures; these observations correlate well with much earlier studies on the temperature sensitivity of crown gall tumor development on plants. In testing the hypothesis that this loss of {DNA} movement reflects a defect in assembly or maintenance of a stable {DNA} transfer machinery at high temperature, we have found that steady-state levels of {VirB10} are sensitive to growth temperature while levels of several other {VirB} proteins are considerably less affected. This temperature-dependent failure to accumulate {VirB10} is exacerbated in an attachment-deficient mutant strain {(chvB)} which exhibits pleiotropic defects in periplasmic osmoadaption, and virulence of a {chvB} mutant can be partially restored by lowering the temperature at which the bacteria and the plant tissue are cocultivated. Furthermore, the stability of {VirB10} is diminished in cells lacking functional {VirB9,} but only under conditions of low osmolarity. We propose that newly synthesized {VirB10} is inherently labile in the presence of a large osmotic gradient across the inner membrane and is rapidly degraded unless it is stabilized by {VirB9-dependent} assembly into oligomeric complexes. The possibility that {VirB10-containing} complexes are not assembled properly at elevated temperatures suggests an explanation for the decades-old observation that tumor formation is exquisitely sensitive to ambient temperature.
},
	number = {24},
	journal = {J. Bacteriol.},
	author = {Lois M. Banta and Jutta Bohne and S. Dawn Lovejoy and Kathleen Dostal},
	month = dec,
	year = {1998},
	pages = {6597--6606}
},

@article{solomon_whos_1996,
	title = {Who's competent and when: regulation of natural genetic competence in bacteria},
	volume = {12},
	url = {http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCY-3W25BGJ-4N&_user=108429&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000059713&_version=1&_urlVersion=0&_userid=108429&md5=aeaa5112d286ac2b1733005cd683f1b2},
	doi = {10.1016/0168-9525(96)10014-7},
	abstract = {Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, {MA} 02139, {UK} Natural genetic competence, the ability of cells to bind to and to take up exogenous {DNA,} is widespread among bacteria and might be an important mechanism for the horizontal transfer of genes. Competent cells express specialized proteins that assemble into a {DNA-uptake} complex. In many organisms, the development of competence and expression of the uptake machinery is regulated in response to cell-cell signaling and/or nutritional conditions. Exciting new progress has been made in characterizing the signals and pathways that regulate the development of competence. },
	number = {4},
	journal = {Trends in Genetics},
	author = {Jonathan M. Solomon and Alan D. Grossman},
	month = apr,
	year = {1996},
	pages = {150--155}
},

@article{hofreuter_natural_2001,
	title = {Natural transformation competence in {{\textless}i{\textgreater}Helicobacter} pylori{\textless}/i{\textgreater} is mediated by the basic components of a type {IV} secretion system},
	volume = {41},
	url = {http://dx.doi.org/10.1046/j.1365-2958.2001.02502.x},
	doi = {10.1046/j.1365-2958.2001.02502.x},
	abstract = {Helicobacter pylori {(Hp),} a Gram-negative bacterial pathogen and aetiologic agent of gastroduodenal disease in humans, is naturally competent for genetic transformation. Natural competence in bacteria is usually correlated with the presence of type {IV} pili or type {IV} pilin-like proteins, which are absent in Hp. Instead, we recently identified the {comB} operon in Hp, carrying four genes tentatively designated as orf2, {comB1,} {comB2} and {comB3.} We show here that all {ComB} proteins and the 37-amino-acid Orf2 peptide display significant primary sequence and structural homology/identity to the basic components of a type {IV} secretion apparatus. {ComB1,} {ComB2} and {ComB3,} now renamed {ComB8,} {ComB9} and {ComB10,} correspond to the Agrobacterium tumefaciens {VirB8,} {VirB9} and {VirB10} proteins respectively. The peptide Orf2 carries a lipoprotein motif and a second cysteine residue homologous to {VirB7,} and was thus designated {ComB7.} The putative {ATPase} {ComB4,} encoded by the open reading frame hp0017 of strain 26695, corresponds to {virB4} of the A. tumefaciens type {IV} secretion system. A Hp {comB4} transposon insertion mutant was totally defective in natural transformation. By complementation of a {Hp0394comB} deletion mutant, we demonstrate that each of the proteins from {ComB8} to {ComB10} is absolutely essential for the development of natural transformation competence. The putative lipoprotein {ComB7} is not essential, but apparently stabilizes the apparatus and modulates the transformation efficiency. Thus, pathogenic type I Hp strains contain two functional independent type {IV} transport systems, one for protein translocation encoded by the cag pathogenicity island and one for uptake of {DNA} by natural transformation. The latter system indicates a possible novel mechanism for natural {DNA} transformation in bacteria.},
	number = {2},
	journal = {Molecular Microbiology},
	author = {Dirk Hofreuter and Stefan Odenbreit and Rainer Haas},
	year = {2001},
	pages = {379--391}
},

@article{chen_ins_2005,
	title = {The Ins and Outs of {DNA} Transfer in Bacteria},
	volume = {310},
	url = {http://www.sciencemag.org/cgi/content/abstract/310/5753/1456},
	doi = {10.1126/science.1114021},
	abstract = {Transformation and conjugation permit the passage of {DNA} through the bacterial membranes and represent dominant modes for the transfer of genetic information between bacterial cells or between bacterial and eukaryotic cells. As such, they are responsible for the spread of fitness-enhancing traits, including antibiotic resistance. Both processes usually involve the recognition of double-stranded {DNA,} followed by the transfer of single strands. Elaborate molecular machines are responsible for negotiating the passage of macromolecular {DNA} through the layers of the cell surface. All or nearly all the machine components involved in transformation and conjugation have been identified, and here we present models for their roles in {DNA} transport.
},
	number = {5753},
	journal = {Science},
	author = {Ines Chen and Peter J. Christie and David Dubnau},
	month = dec,
	year = {2005},
	pages = {1456--1460}
},

@article{das_agrobacterium_2000,
	title = {The Agrobacterium {T-DNA} Transport Pore Proteins {VirB8,} {VirB9,} and {VirB10} Interact with One Another},
	volume = {182},
	url = {http://jb.asm.org/cgi/content/abstract/182/3/758},
	doi = {{10.1128/JB.182.3.758-763.2000}},
	abstract = {The {VirB} proteins of Agrobacterium tumefaciens form a transport pore to transfer {DNA} from bacteria to plants. The assembly of the transport pore will require interaction among the constituent proteins. The identification of proteins that interact with one another can provide clues to the assembly of the transport pore. We studied interaction among four putative transport pore proteins, {VirB7,} {VirB8,} {VirB9} and {VirB10.} Using the yeast two-hybrid assay, we observed that {VirB8,} {VirB9,} and {VirB10} interact with one another. In vitro studies using protein fusions demonstrated that {VirB10} interacts with {VirB9} and itself. These results suggest that the outer membrane {VirB7-VirB9} complex interacts with the inner membrane proteins {VirB8} and {VirB10} for the assembly of the transport pore. Fusions that contain small, defined segments of the proteins were used to define the interaction domains of {VirB8} and {VirB9.} All interaction domains of both proteins mapped to the N-terminal half of the proteins. Two separate domains at the N- and C-terminal ends of {VirB9} are involved in its homotypic interaction, suggesting that {VirB9} forms a higher oligomer. We observed that the alteration of serine at position 87 of {VirB8} to leucine abolished its {DNA} transfer function. Studies on the interaction of the mutant protein with the other {VirB} proteins showed that the {VirB8S87L} mutant is defective in interaction with {VirB9.} The mutant, however, interacted efficiently with {VirB8} and {VirB10,} suggesting that the {VirB8-VirB9} interaction is essential for {DNA} transfer.
},
	number = {3},
	journal = {J. Bacteriol.},
	author = {Anath Das and {Yong-Hong} Xie},
	month = feb,
	year = {2000},
	pages = {758--763}
},

@article{sikorski_natural_1998,
	title = {Natural genetic transformation of Pseudomonas stutzeri in a non-sterile soil},
	volume = {144},
	journal = {Microbiology},
	author = {J. Sikorski and S. Graupner and M. G. Lorenz and W. Wackernagel},
	year = {1998},
	pages = {569--576}
},

@article{finkel_dna_2001,
	title = {{DNA} as a Nutrient: Novel Role for Bacterial Competence Gene Homologs},
	volume = {183},
	url = {http://jb.asm.org/cgi/content/abstract/183/21/6288},
	doi = {{10.1128/JB.183.21.6288-6293.2001}},
	abstract = {The uptake and stable maintenance of extracellular {DNA,} genetic transformation, is universally recognized as a major force in microbial evolution. We show here that extracellular {DNA,} both homospecific and heterospecific, can also serve as the sole source of carbon and energy supporting microbial growth. Mutants unable to consume {DNA} suffer a significant loss of fitness during stationary-phase competition. In Escherichia coli, the use of {DNA} as a nutrient depends on homologs of proteins involved in natural genetic competence and transformation in Haemophilus influenzae and Neisseria gonorrhoeae. Homologs of these E. coli genes are present in many members of the [gamma] subclass of Proteobacteria, suggesting that the mechanisms for consumption of {DNA} may have been widely conserved during evolution.
},
	number = {21},
	journal = {J. Bacteriol.},
	author = {Steven E. Finkel and Roberto Kolter},
	month = nov,
	year = {2001},
	pages = {6288--6293}
},

@article{hvarstein_natural_????,
	title = {Natural Competence in the Genus Streptococcus: Evidence that Streptococci Can Change Pherotype by Interspecies Recombinational Exchanges},
	author = {{LS} {HÅVARSTEIN} and R. {HAKENBECK} and P. {GAUSTAD}}
},

@article{averhoff_type_2003,
	title = {Type {IV} pili-related natural transformation systems: {DNA} transport in mesophilic and thermophilic bacteria},
	volume = {180},
	url = {http://dx.doi.org/10.1007/s00203-003-0616-6},
	doi = {10.1007/s00203-003-0616-6},
	abstract = {Horizontal gene flow is a driving force for bacterial adaptation. Among the three distinct mechanisms of gene transfer in bacteria, conjugation, transduction, and transformation, the latter, which includes competence induction, {DNA} binding, and {DNA} uptake, is perhaps the most versatile mechanism and allows the incorporation of free {DNA} from diverse bacterial species. Here we review {DNA} transport machineries mediating uptake of naked {DNA} in gram-positive and gram-negative bacteria. Different putative models of transformation machineries comprising components similar to proteins of type {IV} pili are presented. Emphasis is placed on a comparative discussion of the underlying mechanisms of {DNA} transfer in mesophilic and extremely thermophilic bacteria, highlighting conserved and distinctive features of these transformation machineries.},
	number = {6},
	journal = {Archives of Microbiology},
	author = {Beate Averhoff and Alexandra Friedrich},
	month = dec,
	year = {2003},
	pages = {385--393}
},

@article{lorenz_plasmid_1992,
	title = {Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater},
	volume = {157},
	url = {http://dx.doi.org/10.1007/BF00248681},
	doi = {{10.1007/BF00248681}},
	abstract = {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.},
	number = {4},
	journal = {Archives of Microbiology},
	author = {Michael G. Lorenz and Karin Reipschläger and Wilfried Wackernagel},
	month = apr,
	year = {1992},
	pages = {355--360}
},

@article{thorstenson_essential_1994,
	title = {The essential virulence protein {VirB8} localizes to the inner membrane of Agrobacterium tumefaciens.},
	volume = {176},
	url = {http://jb.asm.org/cgi/content/abstract/176/6/1711},
	abstract = {Agrobacterium tumefaciens genetically transforms plant cells by transferring a specific {DNA} fragment from the bacterium through several biological membranes to the plant nucleus where the {DNA} is integrated. This complex {DNA} transport process likely involves membrane-localized proteins in both the plant and the bacterium. The 11 hydrophobic or membrane-localized proteins of the {virB} operon are excellent candidates to have a role in {DNA} export from agrobacteria. Here, we show by {TnphoA} mutagenesis and immunogold electron microscopy that one of the {VirB} proteins, {VirB8,} is located at the inner membrane. The observation that a {virB8::TnphoA} fusion restores export of alkaline phosphatase to the periplasm suggests that {VirB8} spans the inner membrane. Immunogold labeling of {VirB8} was detected on the inner membrane of vir-induced A. tumefaciens by transmission electron microscopy. Compared with that of the controls, {VirB8} labeling was significantly greater on the inner membrane than on the other cell compartments. These results confirm the inner membrane localization of {VirB8} and strengthen the hypothesis that {VirB} proteins help form a transfer {DNA} export channel or gate.
},
	number = {6},
	journal = {J. Bacteriol.},
	author = {Y R Thorstenson and P C Zambryski},
	month = mar,
	year = {1994},
	pages = {1711--1717}
},

@article{hofreuter_topology_2003,
	title = {Topology and membrane interaction of Helicobacter pylori {ComB} proteins involved in natural transformation competence},
	volume = {293},
	issn = {1438-4221},
	url = {http://www.sciencedirect.com/science/article/B7GW0-4DS36M3-1W/2/67b37abc02cc072e46965ff4d81c563b},
	doi = {10.1078/1438-4221-00258},
	abstract = {Abstract
The human gastric pathogen Helicobacter pylori is naturally competent for genetic transformation. The H. pylori {comB} gene cluster encodes the {VirB4-homologous} {ATPase} {ComB4} and the structural proteins {ComB7} - {ComB10,} which share significant sequence identity to the Agrobacterium tumefaciens {virB-encoded} type {IV} secretion system. To study the topology of the {ComB7} - 10 proteins, we applied {TnMax} transposon mutagenesis by generating fusions of {ComB} proteins with mature [beta]-lactamase {(BlaM)} or alkaline phosphatase {(PhoA).} Our data show that the putative lipoprotein {ComB7} is secreted and is found membrane-attached, probably by its lipid anchor. According to our topology mapping {ComB8} is a bitopic membrane protein with a short N-terminal portion in the cytoplasm and the remainder of the protein expanding into the periplasmic space. {ComB9} was verified as a periplasmic protein, tightly attached to the membrane. The N-terminus of {ComB10} is anchored in the cytoplasmic membrane and the major portion of the protein, including a putative coiled-coil domain, is located in the periplasm. Limited protease digestion and protein extraction under different salt and {pH} conditions confirmed the periplasmic localization and the tight membrane association of the {ComB} protein complex. A hypothetical model of the {ComB} {DNA} transformation pore in H. pylori is presented.},
	number = {2-3},
	journal = {International Journal of Medical Microbiology},
	author = {Dirk Hofreuter and Arno Karnholz and Rainer Haas},
	year = {2003},
	keywords = {H. pylori,protease digestion,shuttle {mutagenesis,TnMax6,TnMax9,type} {IV} secretion system},
	pages = {153--165}
}