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 fitnessenhancing 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.In bacteria, transformation and conjugation usually mediate the transport of single-stranded DNA (ssDNA) across one or more membranes. Transformation involves the uptake of environmental DNA, whereas conjugation permits the direct transfer of DNA between cells (Fig. 1). Other DNA-transport phenomena in bacteria, such as the passage of DNA through the bacterial division septa and those carried out by many bacteriophages (1), involve the movement of double-stranded DNA (dsDNA) and will not be discussed here. Transformation and conjugation probably evolved for the acquisition of fitness-enhancing genetic information, but other mutually nonexclusive theories posit that transformation might have evolved to provide templates for DNA repair or to supply nutrition for bacteria (2). Today, both processes are recognized as important mechanisms for horizontal gene transfer and genome plasticity over evolutionary history, and they are largely responsible for the rapid spread of antibiotic resistance among pathogenic bacteria (3, 4). Bacterial TransformationNaturally transformable bacteria acquire a physiological state known as "competence" through the regulated expression of genes for protein components of the uptake machinery. Natural transformation has been most studied in Bacillus subtilis, Streptococcus pneumoniae, Neisseria gonorrhoeae, and Haemophilus influenzae. These and other competent bacteria use similar proteins for DNA uptake, with few differences between species. An interesting exception is Helicobacter pylori, which uses a conjugation-like system for transformation (5). Here, we will discuss the DNA uptake systems of B. subtilis and N. gonorrhoeae as representative of those in Gram-positive and -negative bacteria, respectively (Fig. 1A). The main distinction between these cell types is that Gram-negative bacteria are enclosed by cytoplasmic and outer membranes, with an intervening periplasmic space and thin layer of peptidoglycan (~3 to 7 nm) (6). Gram-positive bacteria lack an outer membrane, and their cytoplasmic membrane is surrounded by a ~22-nm periplasmic space and a thick layer of peptidoglycan (~33 nm) (7).* To whom correspondence should be addressed. Peter.J.Christie@uth.tmc.edu (P.J.C.); dubnau@phri.org (D.D.). (8,9). In the absence of ComEA, 20% residual DN...
In competent Bacillus subtilis, the ComG proteins are required to allow exogenous DNA to access to membranebound receptor ComEA during transformation. Here we describe a multimeric complex containing the pilin-like protein ComGC. Due to similarities to the type 4 pilus and the type 2 secretion system pseudopilus, we have tentatively named it the "competence pseudopilus." The ComGC multimer is released from cells upon digestion of the cell wall with lysozyme and has a heterogeneous size, estimated to range between 40 and 100 monomers, covalently linked by disulfide bonds. We determined that the prepilin peptidase ComC, the thiol-disulfide oxidoreductase pair BdbDC, and all seven ComG proteins are necessary to form the pseudopilus. Furthermore, these proteins are also sufficient to form a functional complex, i.e. able 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. We discuss the possible role of the competence pseudopilus in DNA binding and uptake during transformation.Genetically competent bacteria are able to take up exogenous DNA and undergo transformation. In the Gram-positive Bacillus subtilis, the development of competence is a finely regulated process, requiring the expression of the transcriptional regulator ComK, which leads to the expression of the late competence proteins, involved in the binding, uptake, and processing of transforming DNA (1, 2).Several late competence proteins from B. subtilis have been identified and characterized (3). ComEA is a membrane-bound DNA-binding protein (4), required both for DNA binding and transport (5). In addition, all seven genes contained in the comG operon are necessary for DNA binding (6), and it is thought that the ComG proteins allow exogenous DNA to contact its receptor ComEA (4). The ComG proteins share similarities with those involved in the formation of type 4 pili and in type 2 secretion from Gram-negative organisms. ComGA belongs to the family of traffic NTPases; ComGB is a conserved protein with several membrane-spanning domains; ComGF is predicted to be an integral membrane protein; ComGC, -GD, -GE, and -GG are similar to type 4 pilins. Pilin-like proteins are required for type 2 secretion, but because they were not known to form a pilus, they have been called "pseudopilins" (7), a term we have adopted here for ComGC, -GD, -GE, and -GG. The competence pseudopilins are made as precursors and undergo proteolytic cleavage by the prepilin peptidase ComC, also required for translocation of ComGC across the membrane (8). Finally, the protein disulfide oxidoreductase pair BdbDC is needed for the stability of the pilin-like protein ComGC in the membrane (9), probably by catalyzing th...
Bacteria can acquire genetic diversity, including antibiotic resistance and virulence traits, by horizontal gene transfer. In particular, many bacteria are naturally competent for uptake of naked DNA from the environment in a process called transformation. Here, we used optical tweezers to demonstrate that the DNA transport machinery in Bacillus subtilis is a force-generating motor.Single DNA molecules were processively transported in a linear fashion without observable pausing events. Uncouplers inhibited DNA uptake immediately, suggesting that the transmembrane proton motive force is needed for DNA translocation. We found an uptake rate of 80 ± 10 bp s −1 that was force-independent at external forces <40 pN, indicating that a powerful molecular machine supports DNA transport.Natural competence for DNA transformation is a genetically programmed, physiological state. Competent cells express specialized proteins that assemble into a DNA transport complex that moves macromolecular DNA through the bacterial cell wall and membrane 1,2 . These competence proteins are most likely surface-exposed DNA receptors, which facilitate DNA translocation through the cell wall, membrane pores and motor molecules that power DNA transport (reviewed in ref. 3; Fig. 1). The first characterized step in the process of transformation of B. subtilis is the irreversible binding of DNA to the cell surface receptor, ComEA. For DNA to gain access to this receptor, the products of the comG operon must be present, although these proteins do not themselves seem to bind DNA. The ComG proteins resemble those involved in the assembly of type IV pili and in type II secretion, and may form a 'pseudopilus' that traverses the thick cell wall to permit access to ComEA. However, no structure resembling a true pilus (in the sense of a fiber that extends beyond the cell surface) has been described in B. subtilis. After binding, DNA is cleaved by the membranelocalized nuclease NucA. The newly introduced end of one strand then initiates transport across the membrane. In addition to ComEA, which seems to present the DNA end to the internalization machinery, transport requires the putative channel proteins ComEC and the ATPase ComFA. Genetic tests have demonstrated that DNA crosses the membrane in linear fashion, as the uptake of a linked pair of markers is delayed compared to that of either single marker.
The development of genetic competence in the Grampositive eubacterium Bacillus subtilis is a complex postexponential process. Here we describe a new bicistronic operon, bdbDC, required for competence development, which was identified by the B. subtilis Systematic Gene Function Analysis program. Inactivation of either the bdbC or bdbD genes of this operon results in the loss of transformability without affecting recombination or the synthesis of ComK, the competence transcription factor. BdbC and BdbD are orthologs of enzymes known to be involved in extracytoplasmic disulfide bond formation. Consistent with this, BdbC and BdbD are needed for the secretion of the Escherichia coli disulfide bond-containing alkaline phosphatase, PhoA, by B. subtilis. Similarly, the amount of the disulfide bond-containing competence protein ComGC is severely reduced in bdbC or bdbD mutants. In contrast, the amounts of the competence proteins ComGA and ComEA remain unaffected by bdbDC mutations. Taken together, these observations imply that in the absence of either BdbC or BdbD, ComGC is unstable and that BdbC and BdbD catalyze the formation of disulfide bonds that are essential for the DNA binding and uptake machinery.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.