Transduction by phiBB-1, a bacteriophage of Borrelia burgdorferi.

We previously described a bacteriophage of the Lyme disease agent Borrelia burgdorferi designated phiBB-1. This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus Borrelia. To demonstrate the ability of phiBB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into B. burgdorferi CA-11.2A cells. The kan cassette recombined into a resident cp32 and was stably maintained. The cp32 containing the kan cassette was packaged by phiBB-1 released from this B. burgdorferi strain. phiBB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of B. burgdorferi. This is the first direct evidence of a mechanism for lateral gene transfer in B. burgdorferi.

Characterization of Borrelia burgdorferi BlyA and BlyB proteins: a prophage-encoded holin-like system.

The conserved cp32 plasmid family of Borrelia burgdorferi was recently shown to be packaged into a bacteriophage particle (C. H. Eggers and D. S. Samuels, J. Bacteriol. 181:7308-7313, 1999). This plasmid encodes BlyA, a 7.4-kDa membrane-interactive protein, and BlyB, an accessory protein, which were previously proposed to comprise a hemolysis system. Our genetic and biochemical evidence suggests that this hypothesis is incorrect and that BlyA and BlyB function instead as a prophage-encoded holin or holin-like system for this newly described bacteriophage. An Escherichia coli mutant containing the blyAB locus that was defective for the normally cryptic host hemolysin SheA was found to be nonhemolytic, suggesting that induction of sheA by blyAB expression was responsible for the hemolytic activity observed previously. Analysis of the structural features of BlyA indicated greater structural similarity to bacteriophage-encoded holins than to hemolysins. Consistent with holin characteristics, subcellular localization studies with E. coli and B. burgdorferi indicated that BlyA is solely membrane associated and that BlyB is a soluble protein. Furthermore, BlyA exhibited a holin-like function by promoting the endolysin-dependent lysis of an induced lambda lysogen that was defective in the holin gene. Finally, induction of the cp32 prophage in B. burgdorferi dramatically stimulated blyAB expression. Our results provide the first evidence of a prophage-encoded holin within Borrelia.

Bacteriophages of spirochetes.

Historically, a number of bacteriophage-like particles have been observed in association with members of the bacterial order Spirochetales, the spirochetes. In the last decade, several spirochete bacteriophages have been isolated and characterized at the molecular level. We have recently characterized a bacteriophage of the Lyme disease agent, Borrelia burgdorferi, which we have designated phiBB-1. Here we review the history of the association between the spirochetes and their bacteriophages, with a particular emphasis on phiBB-1 and its prophage, the 32-kb circular plasmid family of B. burgdorferi.

Disruption of the Borrelia burgdorferi gac gene, encoding the naturally synthesized GyrA C-terminal domain.

The C-terminal domain of the A subunit of DNA gyrase, which we term Gac, is naturally synthesized in Borrelia burgdorferi as an abundant DNA-binding protein. Full-length GyrA, which includes the C-terminal domain, is also synthesized by the spirochete and functions as a subunit of DNA gyrase. We have disrupted synthesis of Gac as an independent protein and demonstrated that it is not essential for growth in a coumarin-resistant background. We detected no alterations in DNA maintenance, condensation, or topology in B. burgdorferi lacking this small DNA-binding protein.

Molecular evidence for a new bacteriophage of Borrelia burgdorferi.

We have recovered a DNase-protected, chloroform-resistant molecule of DNA from the cell-free supernatant of a Borrelia burgdorferi culture. The DNA is a 32-kb double-stranded linear molecule that is derived from the 32-kb circular plasmids (cp32s) of the B. burgdorferi genome. Electron microscopy of samples from which the 32-kb DNA molecule was purified revealed bacteriophage particles. The bacteriophage has a polyhedral head with a diameter of 55 nm and appears to have a simple 100-nm-long tail. The phage is produced constitutively at low levels from growing cultures of some B. burgdorferi strains and is inducible to higher levels with 10 microg of 1-methyl-3-nitroso-nitroguanidine (MNNG) ml(-1). In addition, the prophage can be induced with MNNG from some Borrelia isolates that do not naturally produce phage. We have isolated and partially characterized the phage associated with B. burgdorferi CA-11.2A. To our knowledge, this is the first molecular characterization of a bacteriophage of B. burgdorferi.

Cloning and expression of the Borrelia burgdorferi lon gene.

The ATP-dependent protease Lon (La) of Escherichia coli degrades abnormal proteins and is involved in the regulation of capsular polysaccharide synthesis. In addition, mutations in the E. coli lon gene suppress temperature-sensitive mutations in other genes. The lon gene of Borrelia burgdorferi, encoding a homolog of the Lon protease, has been cloned and sequenced. The gene encodes a protein of 806 amino acids. The deduced amino acid sequence of the B. burgdorferi Lon protease shares substantial sequence identity with those of other known Lon proteases. The transcription start point of the B. burgdorferi lon gene was identified by primer extension analysis and the potential promoter did not show similarities to the consensus heat-shock promoter in E. coli. The 5'-end of the B. burgdorferi lon gene appears to suppress the temperature-sensitive phenotype of an E. coli lpxA mutant.