Table 1. Propagation phenotypes of deletion mutants Deletion of a DNA relaxase gene in the D(118,397–124,982) Bacteriophagea (D)b cm (I)b pfu/ml mutant. The genes deleted in D(118,397–124,982) included Wild Type 1.43 ± 0.05 5.5 ± 1.9 x 108 orf200, which was found via multi-iteration PsiBlast and reverse D(118397–124982) 1.37 ± 0.04 4.4 ± 1.3 x 108 PsiBlast searches to be related to the DNA relaxase from the DNA translocation operon of Enterobacter cloacae plasmid D(181412–189531) 0.85 ± 0.04 7.7 ± 1.5 x 108 CloDF13 (mobC).20 The weakest link in this association was aThe Results section describes the bacteriophages. bThe Materials and challenged using HHpred as described in the Materials and Methods section describes procedures for determining D and I. Methods Section. Alignments of proteins clearly related to either orf200 or CloDF13 mobC were developed using SAM with a stringent inclusion threshold of E = 1.0 1029. Each was picked fragment. Details are in the Materials and Methods Section. As out of the library with the other as queried by HHpred, with an described below, the genes deleted included a DNA translocation E value of 4.4 10222 (Table 2, row 1). However, both families operon. also matched a variety of helix-turn-helix domains in their The propagation phenotype of D(118,397–124,982). Next, N-terminal domains (Table 2, row 2). we determined whether the D(118,397–124,982) mutant had To test that orf200 and CloDF13 mobC were related beyond a propagation defect in relation to the wild type phage. The both simply having helix-turn-helix domains, the entire operation following characteristics were compared: average plaque diameter was repeated with their C-terminal domains only. The result was (D) and average infectious particle number per ml of a plaque (I). finding of significant homology (Table 2, row 3). Thus, it is clear The value of I was assayable because the plaque-supporting, 0.1% that 0305w8-36 orf200 contains a DNA relaxase domain, with ©No agarose signi2ficant gel was weak 0differen1cebe2tween enough so that wiLldtypaeand a 0.2 ml portion nD(11d8,397–eto of a plaque homologs saDNA Btransloicase that include othat CloDF13 ascts as cacoiupliengprontein mobC, the latter described focrtraensfer in . (about 25% of the total cleared region) was pipeted for titering. reference 21. Conjugation relaxases are usually encoded adjacent 124,982) deletion mutant was observed for either D or I of the nicked DNA into the conjugation system.21,22 (Table 1). That is to say, no growth defect was detected for the D Deletion of a coupling protein/translocase gene in the D (118,397–124,982) mutant. (118,397–124,982) mutant. The candidate for coupling protein Table 2. Homologs of the D0305w8o-36relaxase, ncoupolingATtPase and dmembirane sbindting prroteiinbute. Querya Family Membersb Target of interest Target Family Membersc Relaxase Similarities E valued 4.4e-22 3.4e-5 to 1e-3 3.9e-7 3.0e-10 0.009 4.2e-7 2e-9 0.005 4e-22 4.5e-5 0.006 2e-5 1 2 3 4 5 6 7 8 9 10 11 12 0305w8-36 orf200 0305w8-36 orf200 N-ter. domain 0305w8-36 C-ter. domain orf200 0305w8-36 orf200 0305w8-36 C-ter. domain orf200 0305w8-36 C-ter. domain orf200 Gamma-77020183 C-ter. domain Gamma-77020183 C-ter. domain 0305w8-36 orf201 0305w8-36 orf201 0305w8-36 orf201 N-ter. domain 0305w8-36 orf207 165 165 166 72 1 1 1 1 1 1 1 151 CloDF13mobC Various HTH families CloDF13mobC SPO1 gp27.3 SPO1 gp27.3 GBSV1 Bacillus Virus 1 gp40 CloDF13 mobC SPO1 gp27.4 ING plasmid traG NP_858410 ING plasmid traG SPO1 gp27.9 Coupling ATPase Similarities Membrane Binding Protein Similarities 66 53 1 1 43 1 85 1 15 15 1 aSource sequence for a protein query family. bNumber of query family members for each source sequence. cNumber of target family members. dValues for HHpred matches, without secondary structure prediction, between the families of the proteins indicated. The context is an 80,000 family library, except for 1 sequence to 1 sequence matches, which were blastP results in nr plus env nr. www.landesbioscience.com Bacteriophage 27