Tyrosine site-specific recombinases mediate DNA inversions affecting the expression of outer surface proteins of Bacteroides fragilis

The chromosome of Bacteroides fragilis has been shown to undergo 13 distinct DNA inversions affecting the expression of capsular polysaccharides and mediated by a serine site-specific recombinase designated Mpi. In this study, we demonstrate that members of the tyrosine site-specific recombinase family, conserved in B. fragilis, mediate additional DNA inversions of the B. fragilis genome. These DNA invertases flip promoter regions in their immediate downstream region. The genetic organization of the genes regulated by these invertible promoter regions suggests that they are operons and many of the products are predicted to be outer membrane proteins. Phenotypic analysis of a deletion mutant of one of these DNA invertases, tsr15 (aapI), which resulted in the promoter region for the downstream genes being locked ON, confirmed the synthesis of multiple surface proteins by this operon. In addition, this deletion mutant demonstrated an autoaggregative phenotype and showed significantly greater adherence than wild-type organisms in a biofilm assay, suggesting a possible functional role for these phase-variable outer surface proteins. This study demonstrates that DNA inversion is a universal mechanism used by this commensal microorganism to phase vary expression of its surface molecules and involves at least three conserved DNA invertases from two evolutionarily distinct families.     

Complete nucleotide sequence of the mycoplasma virus P1 genome

Mycoplasma virus P1 is one of only four viruses isolated from the genus Mycoplasma. The host for P1, Mycoplasma pulmonis, possesses complex, phase-variable restriction and modification enzymes and the Vsa family of phase-variable surface proteins. The ability of P1 virus to infect host cells is influenced by these phase-variable systems, rendering P1 a valuable tool for assessing host properties. The double-stranded P1 DNA genome was sequenced (11,660 bp) and 11 ORFs were identified. The predicted P1 DNA polymerase is similar to that of phages that are known to have terminal protein (TP) attached to the 5' end of their genome, consistent with previous studies indicating that P1 DNA has covalently attached TP. Most of the other predicted P1 proteins have little sequence similarity to known proteins, and P1 virus is unrelated to the other mycoplasma virus, MAV1, for which the genome sequence is known. One of the predicted P1 proteins, the ORF 8 gene product, contains a repetitive collagen-like motif characteristic of some bacteriophage tail fiber proteins and is a candidate for interacting with the Vsa proteins.     

Variations in the surface proteins and restriction enzyme systems of Mycoplasma pulmonis in the respiratory tract of infected rats

Restriction and modification (R-M) systems are generally thought to protect bacteria from invasion by foreign DNA. This paper proposes the existence of an alternative role for the phase-variable R-M systems encoded by the hsd loci of Mycoplasma pulmonis. Populations of M. pulmonis cells that arose during growth in different environments were compared with respect to R-M activity and surface antigen production. When M. pulmonis strain X1048 was propagated in laboratory culture medium, > 95% of colony-forming units (cfu) lacked R-M activity and produced the variable surface protein VsaA. Mycoplasmas isolated from the nose of experimentally infected rats also lacked R-M activity and produced VsaA. In contrast, the cell population of mycoplasmas isolated from the lower respiratory tract of the infected rats was more complex. The most dramatic results were obtained for mycoplasmas isolated from the trachea. At 14 days postinfection, 38% of mycoplasma isolates produced a Vsa protein other than VsaA, and 34% of isolates had active restriction systems. These data suggest that differences in selection pressures in animal tissues affect the surface proteins and the R-M activity of the mycoplasmal cell population. We propose that variations in the production of R-M activity and cell surface proteins are important for the survival of the mycoplasma within the host.     

High-frequency rearrangements in the chromosome of Mycoplasma pulmonis correlate with phenotypic switching

Mycoplasma pulmonis is a murine pathogen that causes chronic respiratory disease in laboratory rats and mice. Several examples of high-frequency phenotypic switching have been reported for M. pulmonis, the molecular basis of which is unknown. We report here that during growth the M. pulmonis chromosome undergoes DNA rearrangements at a high frequency. Some of the rearrangements we examined correlated with changes in the susceptibility of the cells to mycoplasma virus P1, an example of phenotypic switching involving changes in surface antigen structure. Other rearrangements, unrelated to phenotypic switching, involved a DNA element present in the chromosome in multiple copies. The high level of DNA recombination that occurred in M. pulmonis indicates that this may be one of the most variable genomes studied to date. High levels of DNA recombination may contribute to the unusually high rate of evolution that mycoplasmas are thought to be undergoing. Understanding the molecular basis for this phenomenon may provide an insight into the chronic nature of many mycoplasmal infections.     

Gene rearrangements in the vsa locus of Mycoplasma pulmonis

The vsa genes of Mycoplasma pulmonis encode the V-1 lipoproteins. Most V-1 proteins contain repetitive domains and are thought to be involved in mycoplasma-host cell interactions. Previously, we have reported the isolation and characterization of six vsa genes comprising a 10-kb region of the genome of M. pulmonis strain KD735-15. In the current study, vsa-specific probes were used to clone several fragments from a genomic library of KD735-15 DNA and assemble a single 20-kb contig containing 11 vsa genes. The middle region of the vsa locus contains a large open reading frame (ORF) that is not a vsa gene and has undergone an internal deletion in some strains. The ORF is predicted to encode a membrane protein that may have a role in disease pathogenesis. To examine vsa genes in a strain of M. pulmonis that is unrelated to KD735-15, strain CT was studied. Through Southern hybridization and genomic cloning analyses, CT was found to possess homologs of the KD735-15 vsaA, -C, -E, and -F genes and two unique genes (vsaG and vsaH) that were not found in KD735-15. High-frequency, site-specific DNA inversions serve to regulate the phase-variable production of individual V-1 proteins. As a result of the sequence analysis of vsa recombination products, a model in which DNA inversion arises from strand exchange involving at least six nucleotides of the vrs box is proposed.     

Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis

An invertible DNA element of 6.8 kb, designated the hsd1 locus, was identified in the chromosome of Mycoplasma pulmonis. Infection of host cells with mycoplasma virus P1 revealed that the organism's restriction and modification (R-M) properties are controlled by inversion of hsd1. The nucleotide sequence of hsd1 revealed several genes, the predicted amino acids of which bear striking similarity to the subunits of the type I R-M enzymes previously found only in enteric bacteria.     

Two-dimensional display and whole genome comparison of bacterial pathogen genomes of high G+C DNA content

Three highly mutable loci of the wall-less pathogens Mycoplasma bovis, Mycoplasma pulmonis and Mycoplasma agalactiae undergo high-frequency genomic rearrangements and generate extensive antigenic variation of major surface lipoproteins. Adjacent to each locus, an open reading frame exists as a single chromosomal copy and is predicted to encode a site-specific DNA recombinase exhibiting high homology to the recombinases XerD of Escherichia coli and CodV of Bacillus subtilis. Each of the mycoplasmal proteins are members of the lambda integrase family of tyrosine site-specific recombinases and likely mediates site-specific DNA inversions observed within the adjacent, variable loci.     

Identification of MrpI as the sole recombinase that regulates the phase variation of MR/P fimbria,a bladder colonization factor of uropathogenic Proteus mirabilis

Proteus mirabilis is a common cause of urinary tract infection (UTI) in individuals with structural abnormalities or long-term catheterization. The expression of mannose-resistant/Proteus-like (MR/P) fimbria is phase variable because of the inversion of a 251 bp DNA fragment that carries the promoter for the mrp operon. Previous studies have shown that mrpI, which is transcribed divergently from the mrp operon, encodes a recombinase capable of switching the orientation of this invertible element. In this study, we constructed isogenic mrpI null mutants from a clinical isolate of P. mirabilis, HI4320. A polymerase chain reaction (PCR)-based invertible element assay revealed that the isogenic mrpI null mutants were locked in one phase, either expressing (locked on) MR/P fimbriae or not (locked off), which indicated that MrpI was the sole recombinase that regulated the phase variation of MR/P fimbria. The locked-on and locked-off mutants were evaluated for virulence in the CBA mouse model of ascending UTI by co-challenges with each other and with the wild-type strain. Results from these experiments demonstrated conclusively that the MR/P fimbria was a critical bladder colonization factor of uropathogenic P. mirabilis and also suggested that the ability to switch off the expression of MR/P fimbria might be important for kidney colonization.     

Targeted plasmid integration into the human genome by an engineered zinc-finger recombinase

The development of new methods for gene addition to mammalian genomes is necessary to overcome the limitations of conventional genetic engineering strategies. Although a variety of DNA-modifying enzymes have been used to directly catalyze the integration of plasmid DNA into mammalian genomes, there is still an unmet need for enzymes that target a single specific chromosomal site. We recently engineered zinc-finger recombinase (ZFR) fusion proteins that integrate plasmid DNA into a synthetic target site in the human genome with exceptional specificity. In this study, we present a two-step method for utilizing these enzymes in any cell type at randomly-distributed target site locations. The piggyBac transposase was used to insert recombinase target sites throughout the genomes of human and mouse cell lines. The ZFR efficiently and specifically integrated a transfected plasmid into these genomic target sites and into multiple transposons within a single cell. Plasmid integration was dependent on recombinase activity and the presence of recombinase target sites. This work demonstrates the potential for broad applicability of the ZFR technology in genome engineering, synthetic biology and gene therapy.     

The molecular basis for the specificity of fimE in the phase variation of type 1 fimbriae of Escherichia coli K-12

The expression of type 1 fimbriae in Escherichia coli is phase variable, with cells switching between fimbriate (ON) and afimbriate (OFF) phases. The phase variation is dependent on the orientation of a 314 bp DNA element (the switch) that undergoes DNA inversion. DNA inversion requires either fimB or fimE, site-specific recombinases that differ in both specificity and activity. Whereas fimB promotes recombination with little orientational bias, fimE promotes recombination in the ON-to-OFF direction exclusively. In wild-type cells, fimE activity predominates and, hence, most bacteria are afimbriate. Here, it is shown that fimE specificity is caused by two different, but complementary, mechanisms. First, FimE shows a strong preference for the switch in the ON orientation as a substrate for recombination. Differences in the nucleotide sequence of the recombinase binding sites is a key factor in determining FimE specificity, although one or more additional cis-active sites that flank the fim switch also appear to be involved. Secondly, the orientation of the switch controls fimE in cis, most probably to control recombinase expression.     

Mycoplasma pulmonis Vsa proteins and polysaccharide modulate adherence to pulmonary epithelial cells

The Mycoplasma pulmonis Vsa proteins are a family of size- and phase-variable lipoproteins that shield the mycoplasmas from complement and modulate attachment to abiotic surfaces. Mycoplasmas producing a long Vsa protein hemadsorb poorly and yet are proficient at colonizing rats and mice. The effect of the length of the Vsa protein on the attachment of mycoplasmas to epithelial cells has not been previously explored. We find that independent of Vsa isotype, mycoplasmas producing a long Vsa protein with many tandem repeats adhere poorly to murine MLE-12 cells compared with mycoplasmas producing a short Vsa. We also find that mutants lacking the EPS-I polysaccharide of M.?pulmonis exhibited decreased adherence to MLE-12 cells, even though it has been shown previously that such mutants have an enhanced ability to form a biofilm.     

DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages

Sequencing of the 7 kb immC region from four P1-related phages identified a novel DNA recombinase that exhibits many Cre-like characteristics, including recombination in mammalian cells, but which has a distinctly different DNA specificity. DNA sequence comparison to the P1 immC region showed that all phages had related DNA terminase, C1 repressor and DNA recombinase genes. Although these genes from phages P7, ϕw39 and p15B were highly similar to those from P1, those of phage D6 showed significant divergence. Moreover, the D6 sequence showed evidence of DNA deletion and substitution in this region relative to the other phages. Characterization of the D6 site-specific DNA recombinase (Dre) showed that it was a tyrosine recombinase closely related to the P1 Cre recombinase, but that it had a distinct DNA specificity for a 32 bp DNA site (rox). Cre and Dre are heterospecific: Cre did not catalyze recombination at rox sites and Dre did not catalyze recombination at lox sites. Like Cre, Dre catalyzed both integrative and excisive recombination and required no other phage-encoded proteins for recombination. Dre-mediated recombination in mammalian cells showed that, like Cre, no host bacterial proteins are required for efficient Dre-mediated site-specific DNA recombination.     

Integration host factor stimulates both FimB- and FimE-mediated site-specific DNA inversion that controls phase variation of type 1 fimbriae expression in Escherichia coli

The site-specific DNA inversion that controls phase variation of type 1 fimbriation in E. coli is catalysed by two recombinases, FimB and FimE. Efficient inversion by either recombinase also requires the leucine-responsive regulatory protein (Lrp). In addition, FimB recombination is stimulated by the integration host factor (IHF). The effect of IHF on FimE inversion has not previously been reported. Here it is shown that IHF stimulates FimE recombination; in strain MG1655, mutants containing lesions in either the α ( ihfA ) or β ( ihfB ) subunits of IHF show a marked decrease in both FimB- (100-fold) and FimE (15 000-fold)-promoted switching. IHF is shown to bind with high affinity to sites both adjacent to (site I) and within (site II) the fim invertible element. Furthermore, mutations in site I or site II that lower the affinity of IHF binding in vitro were found to lower the frequency of FimE and/or FimB recombination in vivo . Although site I and site II mutations in combination have an effect on FimB-promoted switching comparable to that of IHF knockout mutations (100-fold), the cis site mutations have a much less marked effect (100-fold) on FimE-promoted switching.