Competent Bacillus Subtilis Research Articles

Recombination proteins differently control the acquisition of homeologous DNA during Bacillus subtilis natural chromosomal transformation.

Natural chromosomal transformation (CT) plays a major role in prokaryote evolution, yet factors that govern the integration of DNA from related species remain poorly understood. We show that in naturally competent Bacillus subtilis cells the acquisition of homeologous sequences is governed by sequence divergence (SD). Integration initiates in a minimal efficient processing segment via homology-directed CT, and its frequency decreases log-linearly with increased SD up to 15%. Beyond this and up to 23% SD the interspecies boundaries prevail, the CT frequency marginally decreases, and short (<10-nucleotides) segments are integrated via homology-facilitated micro-homologous integration. Both mechanisms are RecA dependent. We identify the other recombination proteins required for the acquisition of homeologous DNA. The absence of AddAB, RecF, RecO, RuvAB or RecU, crucial for repair-by-recombination, did not affect CT. However, dprA, radA, recJ, recX or recD2 inactivation strongly decreased intraspecies and interspecies CT. Interspecies CT was not detected beyond ~8% SD in ΔdprA, ~10% in ΔrecJ, ΔradA, ΔrecX and ~14% in ΔrecD2 cells. We propose that DprA, RecX, RadA/Sms, RecJ and RecD2 accessory proteins are important for the generation of genetic diversity. Together with RecA, they facilitate gene acquisition from bacteria of related species.

Viral SPP1 DNA is infectious in naturally competent Bacillus subtilis cells: inter- and intramolecular recombination pathways.

A proteolyzed bacteriophage (phage) might release its DNA into the environment. Here, we define the recombination functions required to resurrect an infective lytic phage from inactive environmental viral DNA in naturally competent Bacillus subtilis cells. Using phage SPP1 DNA, a model that accounts for the obtained data is proposed (i) the DNA uptake apparatus takes up environmental SPP1 DNA, fragments it, and incorporates into the cytosol different linear single-stranded (ss) DNA molecules shorter than genome-length; (ii) the SsbA-DprA mediator loads RecA onto any fragmented linear SPP1 ssDNA, but negative modulators (RecX and RecU) promote a net RecA disassembly from these ssDNAs not homologous to the host genome; (iii) single strand annealing (SSA) proteins, DprA and RecO, anneal the SsbA- or SsbB-coated complementary strands, yielding tailed SPP1 duplex intermediates; (iv) RecA polymerized on these tailed intermediates invades a homologous region in another incomplete molecule, and in concert with RecD2 helicase, reconstitutes a complete linear phage genome with redundant regions at the ends of the molecule; and (v) DprA, RecO or viral G35P SSA, may catalyze the annealing of these terminally redundant regions, alone or with the help of an exonuclease, to produce a circular unit-length duplex viral genome ready to initiate replication.

Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase (MenE) is an essential enzyme in bacterial vitamin K biosynthesis and an important target in the development of new antibiotics. It is a member of the adenylating enzymes (ANL) family, which reconfigure their active site in two different active conformations, one for the adenylation half-reaction and the other for a thioesterification half-reaction, in a domain-alternation catalytic mechanism. Although several aspects of the adenylating mechanism in MenE have recently been uncovered, its thioesterification conformation remains elusive. Here, using a catalytically competent Bacillus subtilis mutant protein complexed with an OSB-CoA analogue, we determined MenE high-resolution structures to 1.76 and 1.90 Å resolution in a thioester-forming conformation. By comparison with the adenylation conformation, we found that MenE's C-domain rotates around the Ser-384 hinge by 139.5° during domain-alternation catalysis. The structures also revealed a thioesterification active site specifically conserved among MenE orthologues and a substrate-binding mode distinct from those of many other acyl/aryl-CoA synthetases. Of note, using site-directed mutagenesis, we identified several residues that specifically contribute to the thioesterification half-reaction without affecting the adenylation half-reaction. Moreover, we observed a substantial movement of the activated succinyl group in the thioesterification half-reaction. These findings provide new insights into the domain-alternation catalysis of a bacterial enzyme essential for vitamin K biosynthesis and of its adenylating homologues in the ANL enzyme family.

An optimized technique for rapid genome modifications of Bacillus subtilis

The transformation efficiency of naturally competent Bacillus subtilis cells can be significantly increased using β recombinase binding sequences, as revealed by the results of this study. Plasmids containing different variations of these so called six-site-marker-cassettes were investigated. Furthermore, an optimized protocol for knock-out or knock-in mutations combining the Cre–lox-system and the six-sites is presented, which can be used for multiple genome modifications of B. subtilis.

A simple method to provide a shuttling plasmid for delivery to other host ascertained by prolonged stability of extracellular plasmid DNA released from Escherichia coli K12 endA mutant, deficient in major endonuclease

Escherichia coli lyses by lambda phage propagation. Circular plasmid DNA was present during E. coli lysis as an extracellular plasmid DNA (excpDNA) that was stable enough to transform coexisting competent Bacillus subtilis cells. Detailed investigations unveiled that excpDNA is transient in both quality and quantity, with stability lasting no more than several hours. A survey using E. coli lambda lysogens with various genetic backgrounds demonstrated that the loss of Endonuclease I (ΔendA::kan) conferred extraordinary stability upon excpDNA for as long as 48 h. Studies on endA mutants suggested that excpDNA remained localized in cell debris, in contrast to E. coli genome DNA, which diffused into medium at an early point in lysis. Lambda lysogens constructed on endA recA mutants are presented for potential pipelines in delivery to other competent proficient microbes.

Plasmid transformation of competent Bacillus subtilis by lysed protoplast DNA

Transformation of competent Bacillus subtilis with DNA obtained from lysed protoplasts (LP transformation) was analyzed using several different plasmid vectors: pC194, pUB110, pCB1 (consisting of pC194 and pBluescript II SK+), and pAC32R2 (consisting of pUB110 derivative and pUC19). LP transformation of B.subtilis QB936 with pCB1 was 6500-fold higher than that achieved using conventional transformation with purified DNA. Greater transformation efficiencies were also obtained using pAC32R2. However, transformation frequencies using both protoplast-derived and purified pC194 were very low (1.4-2.0×10(2) transformants per μg DNA). Hence, the efficiency of transformation depends on the nucleotide sequence of the donor plasmid. The LP transformation frequency using pC194 obtained from an add5 mutant was remarkably enhanced (1.6×10(8) transformants per μg DNA), indicating that this unique form of high molecular weight DNA is likely responsible for part of the stimulatory effect. Chromosomal DNA inhibited plasmid transformation using pC194 and pUB110, but had little effect on pCB1 transformation. Conversely, pCB1 DNA did not inhibit transformation with protoplast-derived chromosomal DNA. Competence proteins under the control of transcription factor ComK were likely required for LP plasmid transformation. The DNA concentration-dependence of plasmid transformation was first order and the slope value was one.

Role of ComEA in DNA uptake during transformation of competent Bacillus subtilis

The role of the competence protein ComEA in DNA uptake during transformation of competent Bacillus subtilis was analyzed by lysed-protoplast transformation (LP transformation). A comEA deletion mutant was constructed by a fusion polymerase chain reaction. Transformants of the mutant were obtained by LP transformation at a frequency of 1.1 × 10(2) transformants per μg DNA, representing a low relative efficiency of transformation [RET (mutant/wild type)] of 2.7 × 10(-6). This implied an important role of the protein during DNA uptake. When analyzing LP transformation of comEA with a plasmid (5.7 kb), a similar RET (mutant/wild type) of 5.6 × 10(-5) was obtained. Following addition of DNA into the comEA mutant culture, the number of transformants increased at a rate of 0.5 transformants/min, which was very low compared with the wild-type (6.9×10(4) transformants/min). However, even in the comEA mutant, DNA uptake began immediately after addition of DNA. Using co-transformation analysis of the comEA mutant, short linkages at distances of 2-156 kb could be detected, but not long linkages at distances of 671-1662 kb. Taken together, the results indicate that ComEA plays an important role in the transfer of transforming DNA into the DNA channel and in controlling the rate of DNA uptake.

Subcellular Positioning: Unstable Filaments On the Move

A key question in cell biology is how proteins and entire protein complexes localize to defined subcellular positions in non-compartmentalized cells or within cell compartments. A recent report involving computational modeling and live-cell imaging suggests that dynamically unstable protein filaments provide an adaptable and versatile positioning system.

Role of ComFA in controlling the DNA uptake rate during transformation of competent Bacillus subtilis

The roles of ComFA and ComEC in DNA uptake by competent Bacillus subtilis were analyzed by transformation with DNA in protoplast lysates (LP transformation). Deletion mutants of comFA and comEC and putative Walker A mutants (K152N, K152Q, K152E) of comFA were constructed by fusion polymerase chain reaction. Transformants of comEC mutant with purified DNA and DNA in protoplast lysate were not obtained, which shows a lack of transformation ability and backwards recombination of the mutant. Transformants of the comFA mutant were obtained by LP transformation (1.8 × 10(4) transformants/μg DNA). Low relative efficiency of transformation (RET) of comFA compared to wild type (4.3 × 10(-4)) showed an important role for comFA in DNA uptake. Walker A mutants showed 1.8-19 × 10(-4) RET, suggesting a dependence on ATPase activity for transformation. Co-transformation between short linkages was only detected in comFA mutants. The results demonstrated that ComFA controlled the DNA uptake rate. The interpretation was further supported by analyzing the plasmid used in LP transformation of the comFA mutant. The RET of comFA compared to the wild type was 2.7 × 10(-2), 60-fold higher than that with chromosomal DNA (4.3 × 10(-4)). Following addition of DNA into comFA culture, transformants were obtained after 15 min, with the number of transformants increasing over time. The kinetics strongly suggested that in comFA mutants, formation of another DNA uptake complex without ComFA would be a lengthy process.

Integration of stable extracellular DNA released from Escherichia coli into the Bacillus subtilis genome vector by culture mix method

The stable cloning of giant DNA is a necessary process in the production of recombinant/synthetic genomes. Handling DNA molecules in test tubes becomes increasingly difficult as their size increases, particularly above 100 kb. The need to prepare such large DNA molecules in a regular manner has limited giant DNA cloning to certain laboratories. Recently, we found stable plasmid DNA of up to 100 kb in Escherichia coli culture medium during the infection and propagation of lambda phage. The extracellular plasmid DNA (excpDNA) released from lysed E. coli was demonstrably stable enough to be taken up by competent Bacillus subtilis also present in the medium. ExcpDNA transfer, induced by simply mixing E. coli lysate with recipient B. subtilis, required no biochemical purification of the DNA. Here, this simple protocol was used to integrate excpDNA into a B. subtilis genome, designated the ‘BGM vector’. A slightly modified protocol for DNA cloning in BGM is presented for DNA fragments >100 kb. This technique should facilitate giant DNA cloning in the BGM vector and allow its application to other hosts that can undergo natural transformation.

Designed horizontal transfer of stable giant DNA released from Escherichia coli

DNA in the environment is a source to mediate horizontal gene transfer (HGT). Present molecular cloning methods are based on this HGT principle. However, DNA in the extracellular environment, particularly with high molecular-weight, is thought to be prone to shearing or digestion by nucleases. Here we discovered that extracellular plasmid DNA released from lysed Escherichia coli remained intact and stable. Furthermore, it was demonstrated that plasmids up to 100 kb in size were taken up by co-present competent Bacillus subtilis cells. The detailed kinetics of the process together with sensitivity to added DNase I indicated that plasmid DNA released from lysed E. coli into the culture medium was stable enough for quantitative efficacy in the transformation of B. subtilis. Our results will be useful for the development of methods to transfer giant DNAs from general host E. coli without their biochemical purification.

DprA/Smf protein localizes at the DNA uptake machinery in competent Bacillus subtilis cells

BackgroundDprA is a widely conserved bacterial protein and has been shown to confer an important function during transformation in competent cells, possibly through protection of incoming DNA. B. subtilis DprA (called Smf) and has been shown to play an important role during transformation with chromosomal DNA, but its mode of action is unknown.ResultsWe show that B. subtilis DprA/Smf is more important for transformation with plasmid DNA than with chromosomal DNA. A functional Smf-YFP fusion localized as discrete foci to the cell pole in a subset of cells grown to competence, dependent on the ComK master transcription factor. Smf-YFP foci colocalized with ComGA-CFP. However, a considerable number of cells having high ComK activity contained Smf dispersed throughout the cytosol and lacked a polar Smf assembly. The absence of polar Smf-YFP foci in these cells strongly correlated with the absence of ComGA-CFP foci, and comGA mutant cells mostly lacked polar Smf-YFP foci. Smf formed polar assemblies in the absence of RecA, and RecA formed dynamic threads after addition of DNA in a smf deletion strain. Upon addition of DNA, Smf-YFP foci relocalized from the poles to the cell centre, dependent on the presence of RecA protein.ConclusionOur data show that Smf is recruited to the polar competence machinery, and that polar Smf assembly requires a functional DNA uptake complex. High ComK levels drive expression of Smf in 20% of all cells grown to competence, but not all competent cells contain a polar DNA uptake machinery, showing that ComK activity is necessary but not sufficient to achieve assembly of the uptake machinery in all cells. Smf and RecA localize independently of each other, in agreement with our finding that Smf is much more important for plasmid transformation than RecA, but RecA influences the dynamic localization pattern of Smf. Our data show that DprA/Smf acts downstream of the DNA uptake machinery, and support the idea that Smf protects incoming ssDNA, possibly in conjunction with RecA.

A Macromolecular Complex Formed by a Pilin-like Protein in Competent Bacillus subtilis

In competent Bacillus subtilis, the ComG proteins are required to allow exogenous DNA to access to membrane-bound 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.

DNA taken into Bacillus subtilis competent cells by lysed-protoplast transformation is not ssDNA but dsDNA

Competent Bacillus subtilis incorporates whole-genome DNA (4215 kb) from the protoplast lysate of B. subtilis subtilis [Akamatsu, T. and Taguchi, H., Biosci. Biotechnol. Biochem., 65, 823-829 (2001)]. A continuous incorporated DNA is longer than 1500 kb [J. Biosci. Bioeng., 101, 257-262 (2006)]. Whether the incorporated DNA is single-stranded (ssDNA) or double-stranded DNA (dsDNA) has been studied by examining the transforming activity of the incorporated DNA. B. subtilis BEST7027 was used as the donor strain, which has a heterologous region consisting of the 145 kb region of the Synechocystis sp. PCC6803 genome and erm gene. The donor DNA was transferred to a wild-type or a recA recipient strain (AYG2 or SYN9), and protoplast lysate was prepared from the transformants and used as the donor DNA source for the second recipient strain (AU1 or AV1). The intergenote region showed a significant transforming activity. When DNase I was added to both cells collected from the first transformation mixture and the following protoplastization, the result was similar to that obtained without DNase I. All of the observations strongly suggest that the incorporated DNA is dsDNA, and the transformation of competent B. subtilis by DNA in protoplast lysate is different from that by purified DNA taken up conventionally.

Intracellular Protein and DNA Dynamics in Competent Bacillus subtilis Cells

We have found that two DNA repair/recombination proteins localize differentially to the cell poles in competent Bacillus subtilis cells. RecA protein colocalized with competence protein ComGA, and its polar localization largely depended on ComGA and ComK activity, while RecN oscillated between the poles in a minute time frame, independent of any competence factor. Oscillation of RecN arrested upon addition of external DNA, suggesting that an interaction with incoming single-stranded (ss) DNA favors the localization of RecN at the pole containing the competence machinery. In agreement with this model, purified RecN protein showed ATP-dependent binding to ssDNA. Addition of DNA resulted in the formation of RecA threads emanating from the competence machinery. Our data show that in competent bacteria there exists a specifically positioned and dynamic ssDNA binding apparatus that accepts ssDNA taken up through the polar competence machinery and processes ssDNA for recombination with chromosomal DNA via extended RecA filaments.

ComE, a competence protein from Neisseria gonorrhoeae with DNA-binding activity.

Neisseria gonorrhoeae is naturally able to take up exogenous DNA and undergo genetic transformation. This ability correlates with the presence of functional type IV pili, and uptake of DNA is dependent on the presence of a specific 10-bp sequence. Among the known competence factors in N. gonorrhoeae, none has been shown to interact with the incoming DNA. Here we describe ComE, a DNA-binding protein involved in neisserial competence. The gene comE was identified through similarity searches in the gonococcal genome sequence, using as the query ComEA, the DNA receptor in competent Bacillus subtilis. The gene comE is present in four identical copies in the genomes of both N. gonorrhoeae and Neisseria meningitidis, located downstream of each of the rRNA operons. Single-copy deletion of comE in N. gonorrhoeae did not have a measurable effect on competence, whereas serial deletions led to gradual decrease in transformation frequencies, reaching a 4 x 10(4)-fold reduction when all copies were deleted. Transformation deficiency correlated with impaired ability to take up exogenous DNA; however, the mutants presented normal piliation and twitching motility phenotype. The product of comE has 99 amino acids, with a predicted signal peptide; by immunodetection, a 8-kDa protein corresponding to processed ComE was observed in different strains of N. gonorrhoeae and N. meningitidis. Recombinant His-tagged ComE showed DNA binding activity, without any detectable sequence specificity. Thus, we identified a novel gonococcal DNA-binding competence factor which is necessary for DNA uptake and does not affect pilus biogenesis or function.

ComEA is a DNA receptor for transformation of competent Bacillus subtilis.

Competent cells of Bacillus subtilis efficiently bind and internalize DNA. ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step. We show here that ComEA, a bitopic membrane protein, is itself capable of high-affinity DNA binding. A domain necessary for DNA binding is located at the C-terminus of ComEA. Proteins with similar 60-80 amino acid residue domains are widespread among bacteria and higher organisms. ComEA shows a marked preference for double-stranded DNA and can bind to oligomers as small as 22 bp in length. DNA binding by ComEA exhibits no apparent base sequence specificity. Using a membrane vesicle DNA-binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed. We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA. Possible specific roles for the ComG proteins are discussed.

Characterization of comE, a late competence operon of Bacillus subtilis required for the binding and uptake of transforming DNA.

The binding and transport of DNA by competent Bacillus subtilis requires the assembly of a specialized apparatus. We present here the characterization of comE, an operon under competence control that is required for both DNA binding to the competent cell surface, and for uptake. comE contains three open reading frames (ORF1-3) read in the forward direction, preceded by a long untranslated leader sequence and an apparent E sigma A promoter. A minor promoter also is responsible for transcription of ORF2 and -3. A transcript containing a single ORF is produced in the reverse direction. The reverse ORF overlaps ORF1 and the untranslated comE leader. The comE transcript is present at a very low level during growth and at an elevated level in stationary-phase cells. Conversely, the reverse transcript is present during exponential growth and disappears during stationary phase. The reverse ORF resembles prokaryotic and eukaryotic pyrroline-5'-carboxylate reductases, while ORF2 is similar to several dCMP deaminases. ORF1 and ORF3 are predicted to be integral membrane proteins. The latter is specifically required for DNA uptake but not for binding.

A membrane protein with similarity to N-methylphenylalanine pilins is essential for DNA binding by competent Bacillus subtilis

In a cloned copy of comG open reading frame 3 (ORF3), an in-frame deletion was generated by site-directed in vitro mutagenesis, removing the coding sequence for 15 amino acids from the central portion of this pilin-related protein. The mutagenized ORF3 was incorporated into the Bacillus subtilis chromosome, replacing the wild-type ORF3. The presence of the deleted ORF3 in the chromosome, as confirmed by Southern analysis, was associated with the complete loss of competence by the mutant strain. The ability of the mutant cells to bind exogenous radiolabeled DNA was reduced to the level of nonspecific binding of DNA by noncompetent cells. The chromosomal ORF3 mutation was partially complemented in trans by a plasmid-encoded wild-type ORF3 copy under PSPAC control upon induction of the PSPAC promoter. Using antiserum raised against a synthetic 14-mer oligopeptide deduced from the ORF3 sequence, an immunoreactive band of approximately the expected molecular size was obtained in Western blot (immunoblot) experiments with extracts of cells containing the plasmid-encoded inducible gene. A signal was also detected when cells harboring the chromosomal wild-type or mutant ORF3 in single copy were grown in competence medium. This signal was detected only in the light-buoyant-density (competent) cell fraction and only after the transition from the exponential to the stationary growth phase. In cell fractionation experiments with competent cell extracts, the immunoreactive protein was found in both the NaOH-insoluble and -soluble membrane fractions and was sensitive to proteinase K treatment of either protoplasts or whole cells.

A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity.

The virB operon of the Agrobacterium tume-faciens pTiA6NC plasmid likely plays a role in directing T-DNA transfer events at the bacterial membrane, as determined previously by mutagenesis and cellular fractionation studies and by DNA sequence analysis of the approximately 12-kilobase-pair operon. The DNA sequence analysis also revealed consensus mononucleotide binding domains in the deduced virB5 and virB11 gene products, suggesting that one or both of these proteins couple energy, by means of nucleotide triphosphate (NTP) hydrolysis, to T-DNA transport. In this report, the product of virB11, an essential virulence gene, was overproduced in Escherichia coli and purified by using immunoaffinity chromatography. The immunoaffinity purified protein, as well as NaDodSO4/polyacrylamide gel-eluted protein, bound and hydrolyzed ATP in the absence of DNA effectors. VirB11 protein also demonstrated in vitro autophosphorylation activity. VirB11 protein was localized primarily to the cytoplasmic membrane by immunoblot analysis of membrane fractions. The deduced VirB11 protein exhibits sequence similarity to comG ORF1, a protein required for uptake of DNA by competent Bacillus subtilis cells. These findings suggest that phosphorylation may serve to activate a component(s) of the A. tumefaciens T-DNA transport apparatus and may also represent a general activation mechanism of other bacterial DNA transport systems.