We have identified and characterized an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive antigenic variation of a surface-exposed lipoprotein, VlsE. A 28 kb linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. Portions of several of the 15 nonexpressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. This combinatorial variation could potentially produce millions of antigenic variants in the mammalian host.

Francisella tularensis is a gram-negative pathogen that causes life-threatening infections in humans and has potential for use as a biological weapon. The genetic basis of the F. tularensis virulence is poorly understood. This study screened a total of 3,936 transposon mutants of the live vaccine strain for infection in a mouse model of respiratory tularemia by signature-tagged mutagenesis. We identified 341 mutants attenuated for infection in the lungs. The transposon disruptions were mapped to 95 different genes, virtually all of which are also present in the genomes of other F. tularensis strains, including human pathogenic F. tularensis strain Schu S4. A small subset of these attenuated mutants carried insertions in the genes encoding previously known virulence factors, but the majority of the identified genes have not been previously linked to F. tularensis virulence. Among these are genes encoding putative membrane proteins, proteins associated with stress responses, metabolic proteins, transporter proteins, and proteins with unknown functions. Several attenuated mutants contained disruptions in a putative capsule locus which partially resembles the poly-␥-glutamate capsule biosynthesis locus of Bacillus anthracis, the anthrax agent. Deletional mutation analysis confirmed that this locus is essential for F. tularensis virulence.

DNA methylation is an important epigenetic mechanism for phenotypic diversification in all forms of life. We previously described remarkable cell-to-cell heterogeneity in epigenetic pattern within a clonal population of Streptococcus pneumoniae, a leading human pathogen. We here report that the epigenetic diversity is caused by extensive DNA inversions among hsdS A, hsdS B, and hsdS C, three methyltransferase hsdS genes in the Spn556II type-I restriction modification (R-M) locus. Because hsdS A encodes the sequence recognition subunit of this type-I R-M DNA methyltransferase, these site-specific recombinations generate pneumococcal cells with variable HsdSA alleles and thereby diverse genome methylation patterns. Most importantly, the DNA methylation pattern specified by the HsdSA1 allele leads to the formation of opaque colonies, whereas the pneumococci lacking HsdSA1 produce transparent colonies. Furthermore, this HsdSA1-dependent phase variation requires intact DNA methylase activity encoded by hsdM in the Spn556II (renamed colony opacity determinant or cod) locus. Thus, the DNA inversion-driven ON/OFF switch of the hsdS A1 allele in the cod locus and resulting epigenetic switch dictate the phase variation between the opaque and transparent phenotypes. Phase variation has been well documented for its importance in pneumococcal carriage and invasive infection, but its molecular basis remains unclear. Our work has discovered a novel epigenetic cause for this significant pathobiology phenomenon in S. pneumoniae. Lastly, our findings broadly represents a significant advancement in our understanding of bacterial R-M systems and their potential in shaping epigenetic and phenotypic diversity of the prokaryotic organisms because similar site-specific recombination systems widely exist in many archaeal and bacterial species.

The Lyme disease spirochete Borrelia burgdorferipossesses 15 silent vls cassettes and a vlsexpression site (vlsE) encoding a surface-exposed lipoprotein. Segments of the silent vls cassettes have been shown to recombine with the vlsE cassette region in the mammalian host, resulting in combinatorial antigenic variation. Despite promiscuous recombination within the vlsE cassette region, the 5′ and 3′ coding sequences of vlsE that flank the cassette region are not subject to sequence variation during these recombination events. The segments of the silent vlscassettes recombine in the vlsE cassette region through a unidirectional process such that the sequence and organization of the silent vls loci are not affected. As a result of recombination, the previously expressed segments are replaced by incoming segments and apparently degraded. These results provide evidence for a gene conversion mechanism in VlsE antigenic variation.