3.A.11 The Bacterial Competence-related DNA Transformation Transporter (DNA-T) Family
Many Gram-negative bacteria (species of Haemophilus, Neisseria, Helicobacter and Acinetobacter) as well as Gram-positive bacteria (species of Bacillus, Mycobacterium and Streptomyces) are capable of natural competence (uptake of DNA under normal physiological conditions). The mechanism appears to be fairly similar in the best studied Gram-negative and Gram-positive bacteria (Chen et al., 2005; Dubnau and Losick, 2006). However one of the three primary constituents involved in DNA uptake in Gram-positive bacteria, ComFA, an ATP-dependent helicase, has not been identified in Gram-negative bacteria, even though the other two constituents, ComEA and ComEC, have been identified and shown to be necessary for competence. In both types of organisms, three steps appear to be involved (Dubnau, 1999). (1) DNA binds to the cell surface, probably via a type IV pilus structure, (2) the DNA may or may not be cleaved, generating double strand breaks, and (3) one of the two DNA strands is taken up while the other strand is degraded on the external surface of the cell. The extracellular nuclease that is believed to digest the 2nd DNA strand is EndA (O32150) in Gram-positive bacteria (Berge et al., 2002). The best characterized uptake system is that in Bacillus subtilis. It consists of three proteins designated ComEA, ComEC and ComFA. The N-terminus of ComEA traverses the cytoplasmic membrane, and the remainder of the protein is localized to the external surface. It is a double-stranded DNA-binding receptor that extracts the DNA molecule from the type IV pilus and feeds it into the transmembrane channel. It exhibits no apparent base sequence specificity. The central hydrophobic domain of ComEC spans the membrane 8-10 times and is flanked by large N-terminal and C-terminal hydrophilic domains (Bergé et al. 2002).
Both ComEA and EC are required for DNA transport into the cytosol, but while ComEC is not required for binding, ComEA is partially required. ComFA resembles PriA of E. coli, an ATP-driven DNA translocase with homology to DNA/RNA helicases. It has Walker A- and B-type ATP-binding consensus motifs that are essential for normal rates of transport and transformation. Loss of ComFA results in a 1000x decrease in transformation, suggesting that it is not absolutely essential. The ComEA-EC-FA complex thus probably drives single-stranded DNA through the ComEC channel in a process energized by ComFA-catalyzed ATP hydrolysis. The energy source for transport has not been rigorously established, but the rate of transport is about 200 nucleotides/sec. A role for the pmf has been established both in B. subtilis and in H. influenzae (Dubnau, 1999).
The oligomeric ComEC channel protein in B. subtilis has been reported to have 7 TMSs and possibly one laterally inserted amphipathic helix (Draskovic and Dubnau, 2005). In other bacteria, ComEC homologues have 7-12 putative TMSs (see TC entries). The Bacillus protein contains an intramolecular disulfide bond in its N-terminal extracellular loop which provides stability (Draskovic and Dubnau, 2005).
ComEA and ComEC homologues are found in species of Neisseria, Haemophilus, Acinetobacter, Vibrio, Xylella, Deinococcus, Pseudomonas, Synecocystis, and E. coli. The ComEC proteins are large (about 740-840 amino acyl residues) but ComA homologues vary much more in size (from ~90 to ~250 residues). Shorter homologues of ComEC (400-570 aas) are found in Helicobacter, Treponema, Borrelia and Halobacterium species. One of the H. pylori competence proteins is homologous to a VirB DNA transfer protein of A. tumefaciens (see the IVSP family; TC #3.A.7). Moreover, some of the competence proteins of Acinetobacter do not seem to resemble those of other bacteria. Thus, although these bacteria possess homologous proteins, the mechanism of DNA uptake may not be general for all naturally transformable bacteria.
Recent evidence suggests that the DNA uptake machinery assembles and disassembles dynamically, but only at the cell poles (Hahn et al., 2005). This dynamic assembly system for single stranded DNA uptake may be coupled to processing for recombination with chromosomal DNA (Kidane and Graumann, 2005). Proteins required for transformation of Bacillus subtilis and other competent bacteria are associated with the membrane or reside in the cytosol. RecA, ComGA, ComFA and SsbB are directed to the cell poles in competent cells, and the uptake of transforming DNA occurs preferentially at the poles. In fact, ComGA, ComFA, DprA (Smf), SsbB (YwpH), RecA and YjbF (CoiA) are located at the cell poles where they colocalize. These six competence (Com) proteins reside in close proximity to one another. The com gene knockouts reduce the stabilities of Com proteins. Proteins in the transformation complex include ComEC and ComEA. Because ComGA and ComFA are membrane-associated, while DprA, SsbB, RecA and YjbF are soluble, a picture emerges of a large multiprotein polar complex, involving both cytosolic and membrane proteins. This complex mediates the binding and uptake of single-stranded DNA, the protection of this DNA from cellular nucleases and its recombination with the recipient chromosome (Kramer et al., 2007).
In competent Bacillus subtilis, seven ComG proteins are required to allow exogenous DNA to access to membrane-bound receptor ComEA during transformation. A multimeric complex called the 'competence pseudopilus' or the ComGC multimer, has a heterogeneous size, estimated to range between 40 and 100 monomers, covalently linked by disulfide bonds (Chen et al., 2006). The prepilin peptidase ComC, the thiol-disulfide oxidoreductase pair BdbDC, and all seven ComG proteins are necessary and sufficient to form the pseudopilus complex, i.e., to facilitate binding of exogenous DNA to ComEA. The initial steps of pseudopilus biogenesis include the processing of ComGC in the cytoplasmic membrane and consist of two independent events, proteolytic cleavage by ComC and formation of an intramolecular disulfide bond by BdbDC. The other ComG proteins are required to assemble the mature ComGC monomers in the membrane into a multimeric complex proposed to span the cell envelope. A role of the competence pseudopilus in DNA binding and uptake during transformation has been proposed (Chen et al., 2006).
Random cell-to-cell variations in gene expression within an isogenic population can lead to transitions between alternative states of gene expression. Little is known about how these variations (noise) in natural systems affect such transitions. In Bacillus subtilis, noise in ComK, the protein that regulates competence for DNA uptake, is thought to cause cells to transition to the competent state in which genes encoding DNA uptake proteins are expressed. Maamar et al. (2007), have demonstrated that noise in comK expression selects cells for competence and that experimental reduction of this noise decreases the number of competent cells. They show that transitions are limited temporally by a reduction in comK transcription. These results illustrate how such stochastic transitions are regulated in a natural system and suggest that noise characteristics are subject to evolutionary forces.
The generalized transport reaction catalyzed by DNA-T family members is thus believed to be:
ssDNA (out) + ATP → ssDNA (in) + ADP + Pi
DNA translocase. The three-layered DNA uptake machinery at the cell pole in competent Bacillus subtilis cells is a stable complex (Kaufenstein et al., 2011).
ComEA-ComEC-ComFA of Bacillus subtilis
ComFA (463 aas; OTMSs; P39145)
ComEA (205 aas; 1 TMS; P39694)
ComeEC (776 aas; 12 TMSs; P39695)
The single stranded DNA uptake competence transporter, ComEA/ComEC/ComFA (Cvitkovitch 2001; Claverys et al. 2006).
ComEA/EC/FA of Streptococcus pneumoniae
ComEA (CelA) (216 aas; 1 TMS; Q8DQ41)
ComEC (CelB) (746 aas; ~10 TMSS; Q8DQ40)
ComFA (Competence DNA/RNA helicase protein)
(432 aas; 0-2 TMSs; Q8CWM9)
The multicomponent DNA uptake competence transporter involving both competence and pilus biogenesis proteins (Averhoff, 2009; Karuppiah et al. 2013).
Competence system of Thermus thermophilus (16 components)
The phagosome escape and virulence competence system, ComEA-EC, FA, FC. Functions in conjunction with the pseudopilus described under TC# 3.A.14.1.2 (Rabinovich et al., 2012).
ComEA - EC; ComFA; ComFC of Listeria monocytogenes
ComEA; DNA receptor (G2JTB5)
ComEB; unknown function (G2JTB4)
ComEC; channel (G2JNR0)
ComFA; DNA helicase (G2JSZ9)
ComFC; unknown function (G2JSZ8)
Putative competence system ComE1, ComE3, ComF1, ComFC plus pilin and additional coompetence-related proteins. It is not sure that all of these proteins comprise a single system. Their encoding genes are scattered thoughout the genome with only a few gene clusters.
Competince system of Candidatus Beckwithbacteria bacterium
The competence channel protein, ComEC. It might be energized by the helicase, PriA instead of the ComFA protein in B. subtilis. No ComEA homologue has been identified in H. pylori (Stingl et al., 2010; K. Stingl, personal communication).
ComEC (ComE3) and PriA of Helicobacter pylori