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1.  A novel transposon construct expressing PhoA with potential for studying protein expression and translocation in Mycoplasma gallisepticum  
BMC Microbiology  2012;12:138.
Background
Mycoplasma gallisepticum is a major poultry pathogen and causes severe economic loss to the poultry industry. In mycoplasmas lipoproteins are abundant on the membrane surface and play a critical role in interactions with the host, but tools for exploring their molecular biology are limited.
Results
In this study we examined whether the alkaline phosphatase gene (phoA ) from Escherichia coli could be used as a reporter in mycoplasmas. The promoter region from the gene for elongation factor Tu (ltuf) and the signal and acylation sequences from the vlhA 1.1 gene, both from Mycoplasma gallisepticum , together with the coding region of phoA , were assembled in the transposon-containing plasmid pISM2062.2 (pTAP) to enable expression of alkaline phosphatase (AP) as a recombinant lipoprotein. The transposon was used to transform M. gallisepticum strain S6. As a control, a plasmid containing a similar construct, but lacking the signal and acylation sequences, was also produced (pTP) and also introduced into M. gallisepticum . Using a colorimetric substrate for detection of alkaline phosphatase activity, it was possible to detect transformed M. gallisepticum . The level of transcription of phoA in organisms transformed with pTP was lower than in those transformed with pTAP, and alkaline phosphatase was not detected by immunoblotting or enzymatic assays in pTP transformants, eventhough alkaline phosphatase expression could be readily detected by both assays in pTAP transformants. Alkaline phosphatase was shown to be located in the hydrophobic fraction of transformed mycoplasmas following Triton X-114 partitioning and in the membrane fraction after differential fractionation. Trypsin proteolysis confirmed its surface exposure. The inclusion of the VlhA lipoprotein signal sequence in pTAP enabled translocation of PhoA and acylation of the amino terminal cysteine moiety, as confirmed by the effect of treatment with globomycin and radiolabelling studies with [14 C]palmitate. PhoA could be identified by mass-spectrometry after separation by two-dimensional electrophoresis.
Conclusion
This is the first study to express PhoA as a lipoprotein in mycoplasmas. The pTAP plasmid will facilitate investigations of lipoproteins and protein translocation across the cell membrane in mycoplasmas, and the ease of detection of these transformants makes this vector system suitable for the simultaneous screening and detection of cloned genes expressed as membrane proteins in mycoplasmas.
doi:10.1186/1471-2180-12-138
PMCID: PMC3438114  PMID: 22770122
Mycoplasma gallisepticum ; Lipoprotein; Membrane protein; Reporter gene; phoA ; Alkaline phosphatase
2.  Experimental Evolution of a Plant Pathogen into a Legume Symbiont 
PLoS Biology  2010;8(1):e1000280.
Following acquisition of a rhizobial symbiotic plasmid, adaptive mutations in the virulence pathway allowed pathogenic Ralstonia solanacearum to evolve into a legume symbiont under plant selection.
Rhizobia are phylogenetically disparate α- and β-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen in symbiosis with legumes. Ample evidence indicates that horizontal transfer of symbiotic plasmids/islands has played a crucial role in rhizobia evolution. However, adaptive mechanisms that allow the recipient genomes to express symbiotic traits are unknown. Here, we report on the experimental evolution of a pathogenic Ralstonia solanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating and infecting symbionts. Two types of adaptive mutations in the hrpG-controlled virulence pathway of R. solanacearum were identified that are crucial for the transition from pathogenicity towards mutualism. Inactivation of the hrcV structural gene of the type III secretion system allowed nodulation and early infection to take place, whereas inactivation of the master virulence regulator hrpG allowed intracellular infection of nodule cells. Our findings predict that natural selection of adaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobia evolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading to symbiosis.
Author Summary
Most leguminous plants can form a symbiosis with members of a group of soil bacteria known as rhizobia. On the roots of their hosts, some rhizobia elicit the formation of specialized organs, called nodules, that they colonize intracellularly and within which they fix nitrogen to the benefit of the plant. Rhizobia do not form a homogenous taxon but are phylogenetically dispersed bacteria. How such diversity has emerged is a fascinating, but only partly documented, question. Although horizontal transfer of symbiotic plasmids or groups of genes has played a major role in the spreading of symbiosis, such gene transfer alone is usually unproductive because genetic or ecological barriers restrict evolution of symbiosis. Here, we experimentally evolved the usually phytopathogenic bacterium Ralstonia solanacearum, which was carrying a rhizobial symbiotic plasmid into legume-nodulating and -infecting symbionts. From resequencing the bacterial genomes, we showed that inactivation of a single regulatory gene allowed the transition from pathogenesis to legume symbiosis. Our findings indicate that following the initial transfer of symbiotic genes, subsequent genome adaptation under selection in the plant has been crucial for the evolution and diversification of rhizobia.
doi:10.1371/journal.pbio.1000280
PMCID: PMC2796954  PMID: 20084095
3.  Occurrence, Plasticity, and Evolution of the vpma Gene Family, a Genetic System Devoted to High-Frequency Surface Variation in Mycoplasma agalactiae▿ † 
Journal of Bacteriology  2009;191(13):4111-4121.
Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits a very versatile surface architecture by switching multiple, related lipoproteins (Vpmas) on and off. In the type strain, PG2, Vpma phase variation is generated by a cluster of six vpma genes that undergo frequent DNA rearrangements via site-specific recombination. To further comprehend the degree of diversity that can be generated at the M. agalactiae surface, the vpma gene repertoire of a field strain, 5632, was analyzed and shown to contain an extended repertoire of 23 vpma genes distributed between two loci located 250 kbp apart. Loci I and II include 16 and 7 vpma genes, respectively, with all vpma genes of locus II being duplicated at locus I. Several Vpmas displayed a chimeric structure suggestive of homologous recombination, and a global proteomic analysis further indicated that at least 13 of the 16 Vpmas can be expressed by the 5632 strain. Because a single promoter is present in each vpma locus, concomitant Vpma expression can occur in a strain with duplicated loci. Consequently, the number of possible surface combinations is much higher for strain 5632 than for the type strain. Finally, our data suggested that insertion sequences are likely to be involved in 5632 vpma locus duplication at a remote chromosomal position. The role of such mobile genetic elements in chromosomal shuffling of genes encoding major surface components may have important evolutionary and epidemiological consequences for pathogens, such as mycoplasmas, that have a reduced genome and no cell wall.
doi:10.1128/JB.00251-09
PMCID: PMC2698505  PMID: 19376859
4.  Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation 
Molecular Microbiology  2008;67(6):1196-1210.
Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits antigenic diversity by switching the expression of multiple surface lipoproteins called Vpmas (Variable proteins of M. agalactiae). Although phase variation has been shown to play important roles in many host–pathogen interactions, the biological significance and the mechanism of Vpma oscillations remain largely unclear. Here, we demonstrate that all six Vpma proteins are expressed in the type strain PG2 and all undergo phase variation at an unusually high frequency. Furthermore, targeted gene disruption of the xer1 gene encoding a putative site-specific recombinase adjacent to the vpma locus was accomplished via homologous recombination using a replicon-based vector. Inactivation of xer1 abolished further Vpma switching and the ‘phase-locked’ mutants (PLMs) continued to steadily express only a single Vpma product. Complementation of the wild-type xer1 gene in PLMs restored Vpma phase variation thereby proving that Xer1 is essential for vpma inversions. The study is not only instrumental in enhancing our ability to understand the role of Vpmas in M. agalactiae infections but also provides useful molecular approaches to study potential disease factors in other ‘difficult-to-manipulate’ mycoplasmas.
doi:10.1111/j.1365-2958.2007.06103.x
PMCID: PMC2268961  PMID: 18248580
5.  Surface Diversity in Mycoplasma agalactiae Is Driven by Site-Specific DNA Inversions within the vpma Multigene Locus 
Journal of Bacteriology  2002;184(21):5987-5998.
The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas. Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis. In this study, six vpma genes were characterized in the M. agalactiae type strain PG2. All vpma genes clustered within an 8-kb region and shared highly conserved 5′ untranslated regions, lipoprotein signal sequences, and short N-terminal sequences. Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5′ untranslated region of all vpma genes. This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus. An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the λ integrase family of site-specific xer recombinases. It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M. agalactiae.
doi:10.1128/JB.184.21.5987-5998.2002
PMCID: PMC135373  PMID: 12374833
6.  Expression of the pMGA Genes of Mycoplasma gallisepticum Is Controlled by Variation in the GAA Trinucleotide Repeat Lengths within the 5′ Noncoding Regions 
Infection and Immunity  1998;66(12):5833-5841.
We analyzed the segment of DNA which contains the expressed pMGA gene from one strain of Mycoplasma gallisepticum in normal (strain S6) cells and in cells in which pMGA1.1 gene expression had ceased as a consequence of in vitro culture in the presence of pMGA1.1-specific antibodies. Sequence analysis of isolates lacking pMGA1.1 expression revealed that this gene, which is typically expressed, exhibited sequence changes within a region 5′ to its promoter. Specifically, pMGA1.1+ cells contained a (GAA)12 motif upstream of the promoter, whereas in pMGA1.1− cells the corresponding region contained a (GAA)10 motif; when such cells were grown in medium no longer containing pMGA-specific antibodies, pMGA1.1 was reexpressed and the 5′ (GAA)12 motif was restored. Two other genes, pMGA1.9 and pMGA1.2, were also shown to acquire a (GAA)12 motif in clones which expressed these genes. The results imply the evolution by the pMGA genes of M. gallisepticum of a novel transcriptional requirement which facilitates rapid and reversible switches in the pMGA expression pattern.
PMCID: PMC108738  PMID: 9826362
7.  Expression of Two Members of the pMGA Gene Family of Mycoplasma gallisepticum Oscillates and Is Influenced by pMGA-Specific Antibodies 
Infection and Immunity  1998;66(6):2845-2853.
Certain monoclonal antibodies and polyclonal antisera directed to pMGA, the major protein of Mycoplasma gallisepticum, were tested for the ability to influence the surface phenotype of the cell population which resulted from their inclusion in growth medium. The polyclonal antiserum and one monoclonal antibody (MAb 66) resulted in an alteration of surface phenotype; specifically, populations of cells grown either on plates or in broth cultures which contained these reagents ceased the expression of pMGA and instead expressed an antigenically unrelated new polypeptide (p82). Upon the removal of antibody, the progeny of these cells regained pMGA expression and produced antigenically sectored colonies. The basis of this switch between pMGA+ and pMGA− states was shown to be transcriptional. The p82 polypeptide, the expression of which resulted from growth of cells in antibodies, was another member of the pMGA gene family and was located just downstream from the pMGA gene normally expressed by the M. gallisepticum cells used. Collectively the results of this work suggest that this organism has evolved an unusual means of altering the antigenic composition of its surface in response to antibodies or to other environmental cues.
PMCID: PMC108280  PMID: 9596758

Results 1-7 (7)