Polar positioning of a conjugation protein from the integrative and 2 conjugative element ICEBs1 of Bacillus subtilis

27 ICEBs1 is an integrative and conjugative element found in the chromosome of Bacillus 28 subtilis. ICEBs1 encodes functions needed for its excision and transfer to recipient cells. We 29 found that the ICEBs1 gene conE (formerly yddE) is required for conjugation and that 30 conjugative transfer of ICEBs1 requires a conserved ATPase domain of ConE. ConE belongs to 31 the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, 32 SpoIIIE, VirB4, and VirD4. We found that a ConE-GFP (green fluorescent protein) fusion 33 associated with the membrane predominantly at the cell poles in ICEBs1 donor cells. At least one 34 ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles, as ConE35 GFP was dispersed throughout the cytoplasm in a strain lacking ICEBs1. We also visualized the 36 subcellular location of ICEBs1. When integrated in the chromosome, ICEBs1 was located near 37 midcell along the length of the cell, a position characteristic of that chromosomal region. 38 Following excision, ICEBs1 was more frequently found near a cell pole. Excision of ICEBs1 also 39 caused altered positioning of at least one component of the replisome. Taken together, our 40 findings indicate that ConE is a critical component of the ICEBs1 conjugation machinery, that 41 conjugative transfer of ICEBs1 from B. subtilis likely initiates at a donor cell pole, and that 42 ICEBs1 affects the subcellular position of the replisome. 43 44 Berkmen, Lee, Loveday, and Grossman 3 Introduction 45 Integrative and conjugative elements (also known as conjugative transposons) and 46 conjugative plasmids are key elements in horizontal gene transfer and are capable of mediating 47 their own transfer from donor to recipient cells. ICEBs1 is an integrative and conjugative 48 element found in some Bacillus subtilis strains. Where found, ICEBs1 is integrated into the 49 leucine tRNA gene trnS-leu2 (Fig. 1) (7, 14, 21). 50 ICEBs1 gene expression, excision, and potential mating are induced by activation of RecA 51 during the SOS response following DNA damage (7). In addition, ICEBs1 is induced by 52 increased production or activation of the ICEBs1-encoded regulatory protein RapI. Production 53 and activity of RapI are indicative of the presence of potential mating partners that do not contain 54 a copy of ICEBs1 (7). Under inducing conditions, the ICEBs1 repressor ImmR (6) is inactivated 55 by proteolytic cleavage mediated by the anti-repressor and protease ImmA (12). Most ICEBs1 56 genes then become highly expressed (7). One of these genes (xis) encodes an excisionase, which 57 in combination with the element’s integrase causes efficient excision and formation of a double58 stranded circle (7, 38). The circular form is nicked at the origin of transfer, oriT, by a DNA 59 relaxase, the product of nicK (39). Under appropriate conditions, ICEBs1 can then mate into B. 60 subtilis and other species, including the pathogens Listeria monocytogenes and B. anthracis (7). 61 Once transferred to a recipient, ICEBs1 can be stably integrated into the genome at its attachment 62 site in trnS-leu2 by the ICEBs1-encoded integrase (38). 63 In contrast to what is known about ICEBs1 genes and proteins involved in excision, 64 integration, and gene regulation, less is known about the components that make up the Gram65 positive mating machinery, defined as the conjugation proteins involved in DNA transfer (18, 66 24). The well-characterized Gram-negative mating machinery can serve as a preliminary model 67 Berkmen, Lee, Loveday, and Grossman 4 (15, 16, 37, 48). The Gram-negative mating machinery is a Type IV secretion (T4S) system 68 composed of at least eight conserved proteins that span the cell envelope. For example, the 69 conjugation apparatus of the Agrobacterium tumefaciens Ti plasmid (pTi) is composed of 11 70 proteins (VirB1 through VirB11) including the ATPase VirB4 (16). VirB4 family members 71 interact with several components of their cognate secretion systems and may energize machine 72 assembly and/or substrate transfer (16, 48). The secretion substrate is targeted to the conjugation 73 machinery by a “coupling protein”. Coupling proteins, such as VirD4 of pTi, interact with a 74 protein attached to the end of the DNA substrate and couple the substrate to other components of 75 the conjugation machinery. Coupling proteins might also energize the translocation of DNA 76 through the machinery. Both VirB4 and VirD4 belong to the large HerA/FtsK superfamily of 77 ATPases (29). Two other characterized members of this superfamily are the chromosome 78 partitioning proteins FtsK and SpoIIIE (29), which are ATP-dependent DNA pumps {reviewed 79 in (2)}. 80 Some of the proteins encoded by Gram-positive conjugative elements are homologous to 81 components of the conjugation machinery from Gram-negative organisms (1, 9, 14, 29) 82 indicating that some aspects of conjugative DNA transfer may be similar in Gram-positives and 83 Gram-negatives. For example, ConE (formerly YddE) of ICEBs1 has sequence similarities to 84 VirB4 (29). YdcQ may be the ICEBs1-encoded coupling protein as it is phylogenetically related 85 to other coupling proteins (29, 44). Despite some similarities, the cell envelopes and many of the 86 genes encoding the conjugation machinery are different between Gram-positive and Gram87 negative organisms, indicating that there are likely to be significant structural and mechanistic 88 differences as well. 89 To begin to define the conjugation machinery of ICEBs1 and to understand spatial aspects of 90 Berkmen, Lee, Loveday, and Grossman 5 conjugation, we examined the function and subcellular location of ConE of ICEBs1. Our results 91 indicate that ConE is likely a crucial ATPase component of the ICEBs1 conjugation machinery. 92 We found that ConE and excised ICEBs1 DNA were located at or near the cell poles. We 93 propose that the conjugation machinery is likely located at the cell poles and that mating might 94 occur from a donor cell pole. 95 96 Materials and Methods 97 Media and growth conditions 98 For B. subtilis and E. coli strains, routine growth and strain constructions were done on LB 99 medium. For all reported experiments with B. subtilis, cells were grown at 37 o C in S7 defined 100 minimal medium (54) with MOPS buffer at 50 mM rather than 100 mM, with 0.1% glutamate 101 and supplemented with auxotrophic requirements (40 μg/ml tryptophan; 40 μg/ml phenylalanine; 102 200 μg/ml threonine) as needed. Either 1% glucose or succinate was used as a carbon source, as 103 indicated. Antibiotics were used at standard concentrations (27). 104 Strains, alleles, and plasmids 105 E. coli strains used for routine cloning were AG115 (MC1061 F’lacI q lacZ::Tn5) and 106 AG1111 (MC1061 F’lacI q lacZM15 Tn10). B. subtilis strains used in experiments and their 107 relevant genotypes are listed in Table 1 and are derivatives of JH642 containing the trpC2 and 108 pheA1 mutations (45). B. subtilis strains were constructed by natural transformation (27) or 109 conjugation (7). Strains cured of ICEBs1 (ICEBs1 0 ), the spontaneous streptomycin (str) resistant 110 allele, ∆(rapIphrI)342::kan, and ICEBs1::kan were described previously (7). The unmarked 111 deletions ∆nicK306 (39) and ∆xis190 (38) and the tau-YFP (dnaX-yfp) fusion (42) have also 112 been described. All cloned fragments into newly constructed plasmids were verified by 113 Berkmen, Lee, Loveday, and Grossman 6 sequencing. 114 (i) Unmarked conE mutations. The basic strategy for constructing unmarked alleles of conE 115 was similar to that previously described for construction of ∆nicK306 (39). conE∆(88-808) is an 116 unmarked, in-frame deletion of codons 88 through 808 of conE, resulting in the fusion of codons 117 1 through 87 to codons 809 through 831. This deletion keeps the upstream and overlapping yddD 118 gene intact. The splice-overlap-extension PCR method (28) was used to generate a 1.9 kb DNA 119 fragment containing the conE∆(88-808) allele. This fragment was cloned into the 120 chloramphenicol resistance vector pEX44 (19)), upstream of lacZ. The resulting plasmid, 121 pMMB941, was used to replace conE with conE∆(88-808) in strain JMA168. 122 Mutations in the Walker A and B motifs of conE were made using a strategy similar to that 123 for construction of conE∆(88-808). conE(K476E) contains an unmarked missense mutation in 124 conE, converting a lysine at codon 476 to a glutamic acid. conE(D703A/E704A) contains two 125 missense mutations, converting the aspartate and glutamate at 703 and 704 in conE to alanines. 126 DNA fragments (3 kb) containing the conE alleles were generated by PCR and cloned into pKG1 127 (7). The resulting plasmids, pMMB1083 and pMMB1231, were used to introduce conE(K476E) 128 and conE(D703A/E704A), respectively, into the chromosome. 129 (ii) Constructs for complementation of conE alleles. The thrC::{(Pxis-(conE-lacZ)) mls} 130 allele was constructed to express conE from its presumed native promoter (Pxis) of ICEBs1. 131 conE was cloned into pKG1, downstream of Pxis and upstream of lacZ, creating plasmid 132 pMMB943. pMMB943 was transformed into JH642 to create the thrC::{(Pxis-(conE-lacZ)) mls} 133 allele. A similar strategy was used to produce thrC::{(Pxis-(yddD conE-lacZ)) mls} from plasmid 134 pMMB942, thrC::{(Pxis-(yddD-lacZ)) mls} from plasmid pMMB1004, and thrC::{(Pxis-(yddD 135 conE(K476E)-lacZ)) mls} from pMMB1083. thrC325::{(ICEBs1-311 (∆attR::tet)) mls} (strain 136 Berkmen, Lee, Loveday, and Grossman 7 MMB1218) contains ICEBs1 inserted at thrC. It is incapable of excision due to deletion of the 137 right-side attachment site attR as described previously (39). 138 (iii) Overexpression of RapI. rapI was overexpressed from Pspank(hy) in single copy in the 139 chromosome at amyE (amyE::{(Pspank(hy)-rapI) spc}) as described (7), or from Pxyl, also at 140 amyE. To construct amyE::{(Pxyl-rapI) spc}, rapI was cloned downstream of Pxyl in vector 141 pDR160, (from D. Rudner, Harvard Medical School, Boston). The resulting plasmid, pMMB856, 142 was integrated at amyE in B. subtilis by homologous recombination. 143 (iv) Construction of a vector for double integration at lacA. We constructed the vector 144 pMMB752 for introducing DNA via double crossover at lacA. First, an 891 bp PCR fragment of 145 the 5’ end of lacA was cloned into the tetracycline-resistance vector pDG1513 to generate 146 pMMB739. Second, a 1042 bp PCR fragment of the 3’ end of lacA was cloned into pMMB739 147 to generate pMMB752. 148 (v) GFP fusions to ConE, ConE∆(88-808), and ConE(K476E). The vector pMMB759 was 149 derived from pMMB752. It allows fusion of the C-terminus of a protein to a 23 amino acid 150 linker followed by monomeric GFPmut2 (mGFPmut2). A fragment (containing the 23 amino 151 acid linker and mGFPmut2) was digested from pLS31 (49) with XhoI and SphI and ligated into 152 pMMB752 to generate pMMB759. 153 lacA::{(Pxis-yddD conE-mgfpmut2) tet} expresses yddD and conE-mgfpmut2 from the 154 presumed native promoter (Pxis) of ICEBs1. We inserted a 363 bp PCR fragment containing the 155 Pxis promoter into pMMB759, upstream of mgfpmut2, generating pMMB762. A 2.9 kb PCR 156 fragment of yddD and conE missing its stop codon was cloned into the KpnI and XhoI sites of 157 pMMB762, downstream of Pxis and upstream of mgfpmut2, creating plasmid pMMB786. 158 pMMB786 was transformed into JH642 to create the lacA::{(Pxis-yddD conE-mgfpmut2) tet} 159 Berkmen, Lee, Loveday, and Grossman 8 allele. lacA::{(Pxis-yddD conE∆(88-808)-mgfpmut2) tet} and lacA::{(Pxis-yddD conE(K476E)160 mgfpmut2) tet} were generated using a similar strategy but using PCR fragments synthesized 161 from templates pMMB1082 for conE∆(88-808) and pMMB1083 for conE(K476E). 162 ConE-GFP was partially functional in mating. Expression of yddD and conE-gfp from their 163 presumed native promoter (Pxis) at the heterologous site (lacA) in conE (K476E) donors 164 increased the frequency of mating at least 250-fold (0.001% mating efficiency for strain 165 MMB1134 compared to <0.000004% for MMB1118). In addition, expression of conE-gfp at 166 lacA in conE + donors had no effect on mating frequency (8% mating efficiency for strain 167 MMB968 compared to 7% for JMA168). 168 (vi) Visualization of chromosomal regions using the lac operator/lac repressor system. The 169 lac operator/lac repressor system has been used previously to visualize chromosome regions in 170 B. subtilis (e.g., (42, 50, 56)). To mark the 47° (in ICEBs1) and 48° (outside of ICEBs1) regions, 171 we inserted a plasmid containing a tandem array of lac operators near yddM (pMMB779) and 172 ydeDE (pMMB854), respectively, by single crossover. yddM (47°) and ydeDE (48°) are not 173 disrupted in these constructs. We inserted a 466 bp PCR fragment of the 3’ end of yddM into the 174 NheI and EcoRI sites of pPSL44a to generate pMMB779. pPSL44a is pGEMcat containing an 175 XhoI fragment from pLAU43 that includes a 4.5 kb array of lac operators (11). Ten base pairs of 176 random sequence intersperses each lacO site of pLAU43, leading to greater genetic stability by 177 reducing the frequency of recombination (35). We inserted a 728 bp PCR fragment including the 178 3’ ends and intergenic region between ydeD and ydeE into the NheI and EcoRI sites of pPSL44a 179 to generate pMMB845. The lac operator arrays were amplified in vivo by selecting for resistance 180 to chloramphenicol (25 μg/ml) as described previously (56). 181 Berkmen, Lee, Loveday, and Grossman 9 Mating Assays 182 We assayed ICEBs1 DNA transfer as described previously (7). We used donor B. subtilis 183 cells in which ICEBs1 contained a kanamycin resistance gene. Recipient cells lacked ICEBs1 184 (ICEBs1 0 ) and were distinguishable from donors as they were streptomycin resistant. Donors and 185 recipient cells were grown separately in minimal glucose medium for at least four generations. 186 ICEBs1 was induced in the donors in mid-exponential phase (optical density at 600 nm to ~0.4) 187 by addition of IPTG (1 mM) for 1 hr to induce expression of rapI (from Pspank(hy)-rapI). 188 Donors and ICEBs1 0 recipient cells (CAL419) were mixed and filtered on sterile cellulose nitrate 189 membrane filters (0.2 μm pore size). Filters were placed in Petri dishes containing Spizizen’s 190 minimal salts (27) and 1.5% agar and incubated at 37oC for 3 hours. Cells were washed off the 191 filter and the number of transconjugants (recipients that received ICEBs1) per ml was measured 192 by determining the number of kan R strep R colony forming units (CFUs) after the mating. Percent 193 mating is the (number of transconjugant CFUs per donor CFU) x 100%. 194 Live cell fluorescence microscopy 195 Microscopy was performed as described (10). Cells were grown at least four generations to 196 mid-exponential phase (optical density at 600 nm to ~0.4) in minimal medium. RapI 197 overexpression was induced with either 1 mM IPTG for 1 hour for strains containing 198 amyE::{(Pspank(hy)-rapI) spc} or with 1% xylose for ~2 hours for strains containing 199 amyE::{(Pxyl-rapI) spc}. Cells were stained with FM4-64 (1 μg/ml; Molecular Probes) to 200 visualize membranes. Live cells were immobilized on pads of 1% agarose containing Spizizen’s 201 minimal salts. All images were captured at room temperature with a Nikon E800 microscope 202 equipped with a 100x DIC objective and a Hamamatsu digital camera. We used the Chroma filter 203 sets 41002b (TRITC) for FM4-64, 31044 for CFP, 41012 for GFP, and 41028 for YFP. 204 Berkmen, Lee, Loveday, and Grossman 10 Improvision Openlabs 4.0 Software was used to process images. Cell length and focus position 205 was measured and plotted as described previously (10, 40). Each strain was examined in at least 206 two independent experiments with similar results. 207