RESEARCH PAPER Bacteriophage 2:1, 25–35; January/February/March 2012; G 2012 Landes Bioscience Islands of non-essential genes, including a DNA translocation operon, in the genome of bacteriophage 0305w8-36 Saurav Pathria, Mandy Rolando, Karen Lieman, Shirley J. Hayes, Stephen C. Hardies and Philip Serwer* Department of Biochemistry; The University of Texas Health Science Center; San Antonio, TX USA Keywords: bacteriophage, long-genome, deletion mutant, DNA sequencing, electron microscopy, fluorescence microscopy, informatics, microbial biofilm © We investigate genes of lytic, Bacillus thuringiensis bacteriophage 0305w8-36 that are non-essential for laboratory propagation, but might have a function in the wild. We isolate deletion mutants to identify these genes. The non- permutation of the genome (218.948 Kb, with a 6.479 Kb terminal repeat and 247 identified orfs) simplifies isolation of deletion mutants. We find two islands of non-essential genes. The first island (3.01% of the genomic DNA) has an informatically identified DNA translocation operon. Deletion causes no detectable growth defect during propagation in a t2ransloc0aseand 1mem2brane-bindLing aanchor nproteinds.The erelaxasseis in a Bfamilyi,first oidenstified chere, iwith ehomnologs cine. dilute agarose overlay. Identification of the DNA translocation operon begins with a DNA relaxase and continues with a other bacteriophages. The second deleted island (3.71% of the genome) has genes for two metallo-protein chaperonins and two tRNAs. Deletion causes a significant growth defect. In addition, (1) we find by “in situ” (in-plaque) single-particle fluorescence microscopy that adsorption to the host occurs at the tip of the 486 nm long tail, (2) we develop a procedure of 0305w8-36 purification that does not cause tail contraction, and (3) we then find by electron microscopy that 0305w8-36 undergoes tail tip-tail tip dimerization that potentially blocks adsorption to host cells, presumably with effectiveness that observation thDat0305ow8-36 does nnot loyseliqtuid cultdures, eiven sthougth03r05wi8-36 bisgenuomicatllylyetic. . increases as the bacteriophage particle concentration increases. These observations provide an explanation of the previous Introduction Bacteriophages isolated in the past were selected for characteristics useful either for studies of molecular biology or for applications in the management of infectious disease-causing bacteria.1,2 The methods of bacteriophage isolation included both liquid culture in various broths and single-plaque propagation in broth-containing agar gels. A plaque-supporting agar gel typically had an agar concentration between 0.4 and 1.0%, as described in references recently reviewed.1 However, we have found that some bacterio- phages do not propagate sufficiently to clear hosts during use of either of these methods. These latter bacteriophages include two aggregating RNA bacteriophages3 and an aggregating double- stranded DNA bacteriophage, 0305w8-36 of Bacillus thuringien- sis.4 The aggregation characteristic of these bacteriophages included the propagation-associated formation of aggregates that varied in size, and often had hundreds to thousands of bacterio- phage particles. Subsequently, eukaryotic RNA virus, HTLV-1, was found to form similar aggregates during propagation.5,6 The aggregating bacteriophages were propagated in agarose gels with unusually low concentrations of 0.05–0.15% (reviewed in ref. 7). *Correspondence to: Philip Serwer; Email address: serwer@uthscsa.edu Submitted: 11/09/11; Revised: 01/27/12; Accepted: 01/31/12 http://dx.doi.org/10.4161/bact.19546 Aggregating bacteriophages are potentially useful either for studies of ecology or for management of bacteria that have not been successfully managed with either antibiotics or the lytic bacteriophages traditionally used. Bacteriophage 0305w8-36 is genomically lytic8 and is also phenotypically lytic when pro- pagated in dilute agarose gels. The dilute agarose apparently contributes to the degree of host cell lysis because liquid cultures do not visibly clear during infection in the same broth, although the bacteriophage does propagate in liquid culture to a limited extent.9 These observations suggest a new paradigm in which difficult-to-detect, aggregating bacteriophages constitute a signifi- cant percentage of environmental bacteriophages. As previously reviewed, bacteriophages are present in biofilms10-12 and aggre- gating bacteriophages are potentially also a component of environmental biofilms. Indeed, electron microscopy of thin sections provides compel- ling evidence that bacteriophage aggregates are at least sometimes a major structural component of human dental plaque, a bio- film.13 The biofilm-forming, aggregating bacteriophage of refer- ence 13 has a nucleic acid (presumably DNA) that is contained in a shell that is indistinguishable in radius from that of 0305w8-36. www.landesbioscience.com Bacteriophage 25