a blunt end ligation strategy with smai for vector digestion and alui for fragment preparation was used . gdna digestion was optimized as above and fragments were prepared as described with a reaction time of 40 min at 37 c using 0.5 u alui per mg gdna . put18 was digested at 40 ng ml with 2 u ml 
 smai for 2 h at 25 c and the enzyme was inactivated for 20 min at 65 c. subsequently , the vector was dephosphorylated with 5 u antarctic phosphatase for 1 h at 37 c to prevent self - ligation . approximately 100,000 transformations were plated on large dishes at approximately 5,000 clones per plate and colonies were harvested by scraping each plate with 25 ml lb . pooled cells were mixed for 2 h at 4 c on a rolling mixer to obtain a homogenous cell suspension . finally , a fraction corresponding to 1 ml of a e600 = 8.0 culture was used for plasmid preparation . for two - hybrid assays , bth101 cells were double transformed with plasmid combinations using 96-well plates and spotted on lb ( 50 mg ml carbenicillin , 25 mg ml kanamycin , 0.5 mm iptg , 160 mg l x - gal ) . positive control was a double transformation of put18c_zip and pkt25_zip ( euromedex ) , encoding an interacting leucine zipper . for the two hybrid screen , the put18 library was transformed into bth101 aiming at 200,000 transformants . cells were regenerated in prewarmed lb for 15 min at 37 c , then washed four times in m63 and plated on m63 with 50 mg ml carbenicillin , 25 mg ml kanamycin , 0.5 mm iptg , 40 mg ml x - gal and 0.2% d(+)-maltose at 100,000 clones per plate . clones appeared after approximaltely 60 h at 30 c. c - terminal gfp fusions were constructed using the plasmid psg1154 . ynek , ymda and ywpg were pcr amplified from the genome of b.  subtilis 168 with specific primers using phusion polymerase ( thermo scientific ) . t4 dna ligase ( 1 u l ) from fermentas was used to ligate digested plasmid and inserts . null alleles of dyna ( fbb002 ) or minj ( rd021 ) were transformed with chromosomal dna of strains psb001 - 003 , respectively and selected on spectinomycin plates . correct integration into the amye locus was checked on starch ( 0.1% ) plates that where subsequently stained with iodine . localization of gfp tagged ynek , ymda and ywpg in b.  subtilis was studied using a zeiss axioimager m1 equipped with a zeiss axiocam hrm camera . after 60 minutes induction 2 l of cell culture ( around od600 1 ) were placed on agarose bed on a glass slide , covered with a glass slip , and observed under light microscope . a blunt end ligation strategy with smai for vector digestion and alui for fragment preparation was used . gdna digestion was optimized as above and fragments were prepared as described with a reaction time of 40 min at 37 c using 0.5 u alui per mg gdna . put18 was digested at 40 ng ml with 2 u ml 
 smai for 2 h at 25 c and the enzyme was inactivated for 20 min at 65 c. subsequently , the vector was dephosphorylated with 5 u antarctic phosphatase for 1 h at 37 c to prevent self - ligation . approximately 100,000 transformations were plated on large dishes at approximately 5,000 clones per plate and colonies were harvested by scraping each plate with 25 ml lb . pooled cells were mixed for 2 h at 4 c on a rolling mixer to obtain a homogenous cell suspension . finally , a fraction corresponding to 1 ml of a e600 = 8.0 culture was used for plasmid preparation . for two - hybrid assays , bth101 cells were double transformed with plasmid combinations using 96-well plates and spotted on lb ( 50 mg ml carbenicillin , 25 mg ml kanamycin , 0.5 mm iptg , 160 mg positive control was a double transformation of put18c_zip and pkt25_zip ( euromedex ) , encoding an interacting leucine zipper . for the two hybrid screen , the put18 library was transformed into bth101 aiming at 200,000 transformants . cells were regenerated in prewarmed lb for 15 min at 37 c , then washed four times in m63 and plated on m63 with 50 mg ml carbenicillin , 25 mg ml kanamycin , 0.5 mm iptg , 40 mg ml x - gal and 0.2% d(+)-maltose at 100,000 clones per plate . ynek , ymda and ywpg were pcr amplified from the genome of b.  subtilis 168 with specific primers using phusion polymerase ( thermo scientific ) . t4 dna ligase ( 1 u l ) from fermentas was used to ligate digested plasmid and inserts . null alleles of dyna ( fbb002 ) or minj ( rd021 ) were transformed with chromosomal dna of strains psb001 - 003 , respectively and selected on spectinomycin plates . correct integration into the amye locus was checked on starch ( 0.1% ) plates that where subsequently stained with iodine . localization of gfp tagged ynek , ymda and ywpg in b.  subtilis was studied using a zeiss axioimager m1 equipped with a zeiss axiocam hrm camera . after 60 minutes induction 2 l of cell culture ( around od600 1 ) were placed on agarose bed on a glass slide , covered with a glass slip , and observed under light microscope .
membrane dynamics are involved in crucial processes in eukaryotic and prokaryotic cells . 
 membrane fusion and fission events are often catalyzed by proteins that belong to the dynamin family of large gtpases . 
 it has recently been shown that members of the dynamin superfamily are also present in many bacterial species . 
 although structural information about full length bacterial dynamin - like proteins is available , their molecular role remains unclear . 
 we have shown previously that dyna , a dynamin - like protein found in the firmicute bacillus subtilis is able to fuse membranes in vitro . 
 in contrast to other members of the dynamin family this membrane remodeling activity was not dependent on guanosine nucleotides , but required magnesium . 
 dyna assemblies localize in foci that are often enriched at sites of septation and hence a potential role during bacterial cytokinesis was discussed . in order to identify potential interaction partners we constructed a bacterial - two hybrid ( b2h ) library and screened for dyna interacting proteins . 
 three potential interaction partner have been identified , ynek , rnasey ( ymda ) , and ywpg . 
 localization of these proteins phenocopies that of dyna , supporting the potential interaction in vivo .