Type B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage.

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM ß-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins.

Complementation in trans of Porphyromonas gingivalis Lipopolysaccharide Biosynthetic Mutants Demonstrates Lipopolysaccharide Exchange.

Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host.IMPORTANCEPorphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.

Type IX Secretion System Cargo Proteins Are Glycosylated at the C Terminus with a Novel Linking Sugar of the Wbp/Vim Pathway.

Porphyromonas gingivalis and Tannerella forsythia use the type IX secretion system to secrete cargo proteins to the cell surface where they are anchored via glycolipids. In P. gingivalis, the glycolipid is anionic lipopolysaccharide (A-LPS), of partially known structure. Modified cargo proteins were deglycosylated using trifluoromethanesulfonic acid and digested with trypsin or proteinase K. The residual modifications were then extensively analyzed by tandem mass spectrometry. The C terminus of each cargo protein was amide-bonded to a linking sugar whose structure was deduced to be 2-N-seryl, 3-N-acetylglucuronamide in P. gingivalis and 2-N-glycyl, 3-N-acetylmannuronic acid in T. forsythia The structures indicated the involvement of the Wbp pathway to produce 2,3-di-N-acetylglucuronic acid and a WbpS amidotransferase to produce the uronamide form of this sugar in P. gingivalis The wbpS gene was identified as PGN_1234 as its deletion resulted in the inability to produce the uronamide. In addition, the P. gingivalisvimA mutant which lacks A-LPS was successfully complemented by the T. forsythiavimA gene; however, the linking sugar was altered to include glycine rather than serine. After removal of the acetyl group at C-2 by the putative deacetylase, VimE, VimA presumably transfers the amino acid to complete the biosynthesis. The data explain all the enzyme activities required for the biosynthesis of the linking sugar accounting for six A-LPS-specific genes. The linking sugar is therefore the key compound that enables the attachment of cargo proteins in P. gingivalis and T. forsythia We propose to designate this novel linking sugar biosynthetic pathway the Wbp/Vim pathway.IMPORTANCEPorphyromonas gingivalis and Tannerella forsythia, two pathogens associated with severe gum disease, use the type IX secretion system (T9SS) to secrete and attach toxic arrays of virulence factor proteins to their cell surfaces. The proteins are tethered to the outer membrane via glycolipid anchors that have remained unidentified for more than 2 decades. In this study, the first sugar molecules (linking sugars) in these anchors are identified and found to be novel compounds. The novel biosynthetic pathway of these linking sugars is also elucidated. A diverse range of bacteria that do not have the T9SS were found to have the genes for this pathway, suggesting that they may synthesize similar linking sugars for utilization in different systems. Since the cell surface attachment of virulence factors is essential for virulence, these findings reveal new targets for the development of novel therapies.

Quantitative proteomic analysis of the type IX secretion system mutants in Porphyromonas gingivalis.

Porphyromonas gingivalis is an anaerobic, gram-negative human oral pathogen highly associated with chronic periodontitis. P. gingivalis utilizes the type IX secretion system (T9SS) to transport many of its virulence factors including the gingipains to the cell surface. The T9SS is comprised of at least 16 proteins and the involvement of these 16 proteins in the T9SS has been verified by creating gene deletion mutants in P. gingivalis. These T9SS mutants are regularly utilized to understand how these proteins function together to allow the secretion of the T9SS substrates. We performed label-free quantitative proteomic analysis on the T9SS protein mutants in P. gingivalis to understand the relative abundance of each T9SS component in different mutants. The T9SS components were reduced in abundance in the porK, porL, porM, porN, sov and porT mutants, whereas they were increased in the porE, porU, porV, porZ and porQ mutants. Sov and PorW appear to be the lowest in abundance and PorV the highest amongst all the T9SS components in P. gingivalis wild-type strain. These results are consistent with the proposed role of Sov as the translocation pore in the outer membrane and PorV as the shuttle protein that transports the T9SS substrates between sub-complexes. Together, the label-free quantitative proteomics analyses showed that different T9SS mutants have vastly different abundances of the T9SS components. This knowledge will greatly assist in interpreting the phenotype of the T9SS mutants as well as selecting the right mutant for exploring the role of an individual component.

Localization of Outer Membrane Proteins in Treponema denticola by Quantitative Proteome Analyses of Outer Membrane Vesicles and Cellular Fractions.

The identification and localization of outer membrane proteins (Omps) and lipoproteins in pathogenic treponemes such as T. denticola (periodontitis) and T. pallidum (syphilis) has been challenging. In this study, label-free quantitative proteomics using MaxQuant was applied to naturally produced outer membrane vesicles (OMVs) and cellular fractions to identify 1448 T. denticola proteins. Of these, 90 proteins were localized to the outer membrane (OM) comprising 59 lipoproteins, 25 ß-barrel proteins, and six other putative OM-associated proteins. Twenty-eight lipoproteins were localized to the inner membrane (IM), and 43 proteins were assigned to the periplasm. The signal cleavage regions of the OM and IM lipoprotein sequences were different and may reveal the signals for their differential localization. Proteins significantly enriched in OMVs included dentilisin, proteins containing leucine-rich repeats, and several lipoproteins containing FGE-sulfatase domains. Blue native PAGE analysis enabled the native size of the dentilisin complex and Msp to be determined and revealed that the abundant ß-barrel Omps TDE2508 and TDE1717 formed large complexes. In addition to the large number of integral Omps and potentially surface-located lipoproteins identified in T. denticola, many such proteins were also newly identified in T. pallidum through homology, generating new targets for vaccine development in both species.

PorV is an Outer Membrane Shuttle Protein for the Type IX Secretion System.

Porphyromonas gingivalis is a keystone pathogen associated with chronic periodontitis. Major virulence factors named gingipains (cysteine proteinases, RgpA, RgpB and Kgp) are secreted via the Type IX Secretion System (T9SS). These, together with approximately 30 other proteins, are secreted to the cell surface and anchored to the outer membrane by covalent modification to anionic lipopolysaccharide (A-LPS) via the novel Gram negative sortase, PorU. PorU is localised on the cell surface and cleaves the C-terminal domain signal (CTD) of T9SS substrates and conjugates their new C-termini to A-LPS. A 440 kDa-attachment complex was identified in the wild-type (WT) comprising of PorU:PorV:PorQ:PorZ. In mutant strains, sub-complexes comprising PorU:PorV or PorQ:PorZ were also identified at smaller native sizes suggesting that PorU and PorZ are anchored to the cell surface via interaction with the PorV and PorQ outer membrane proteins, respectively. Analysis of porU mutants and a CTD cleavage mutant revealed accumulation of immature T9SS substrates in a PorV-bound form. Quantitative label-free proteomics of WT whole cell lysates estimated that the proportion of secretion channels:attachment complexes:free PorV:T9SS substrates was 1:6:110:2000 supporting a role for PorV as a shuttle protein delivering secreted proteins to the attachment complex for CTD signal cleavage and A-LPS modification.

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers.

The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum. Proteins secreted by the T9SS have an N-terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C-terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C-terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components.

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism.

The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria.

The Porphyromonas gingivalis ferric uptake regulator orthologue binds hemin and regulates hemin-responsive biofilm development.

Porphyromonas gingivalis is a Gram-negative pathogen associated with the biofilm-mediated disease chronic periodontitis. P. gingivalis biofilm formation is dependent on environmental heme for which P. gingivalis has an obligate requirement as it is unable to synthesize protoporphyrin IX de novo, hence P. gingivalis transports iron and heme liberated from the human host. Homeostasis of a variety of transition metal ions is often mediated in Gram-negative bacteria at the transcriptional level by members of the Ferric Uptake Regulator (Fur) superfamily. P. gingivalis has a single predicted Fur superfamily orthologue which we have designated Har (heme associated regulator). Recombinant Har formed dimers in the presence of Zn2+ and bound one hemin molecule per monomer with high affinity (Kd of 0.23 µM). The binding of hemin resulted in conformational changes of Zn(II)Har and residue 97Cys was involved in hemin binding as part of a predicted -97C-98P-99L- hemin binding motif. The expression of 35 genes was down-regulated and 9 up-regulated in a Har mutant (ECR455) relative to wild-type. Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation. A truncated Zn(II)Har bound the promoter region of dnaA (PGN_0001), one of the up-regulated genes in the ECR455 mutant. This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability. ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability. P. gingivalis possesses a hemin-binding Fur orthologue that regulates hemin-dependent biofilm formation.

Blue native-PAGE analysis of membrane protein complexes in Porphyromonas gingivalis.

Membrane complexes of Porphyromonas gingivalis were analyzed using two dimensional-blue native-PAGE. The molecular mass of the gingipain complexes, RgpA and Kgp, ranged from 450 kDa to greater than 1200 kDa, and did not change in single rgpA and kgp mutants indicating that the proteolytically processed polyproteins were independently capable of forming complexes. The outer membrane protein, LptO, which is essential for gingipain secretion, was found in up to seven different complex sizes. PG0026, also important for secretion, was observed to interact with the largest LptO complex [VII] at 480 kDa, supporting a cooperative role in secretion. Two pro-form RgpB intermediates formed a complex before cleavage of their C-terminal secretion signal domains (CTDs) such that complex formation may occur during secretion and processing. This may also be the case for other CTD-proteins as not only modified, mature RgpB, but also CPG70 was found to exist as multi-subunit complexes. RagA and RagB were observed in three different complex sizes. Elimination of the abundant gingipains enabled the identification of many inner and outer membrane protein complexes: TonB:ExbB:ExbD, Omp85, P51:PG2168, PorK:PorN, PG0056, PG0241, PG1430 and five proposed respiratory chain complexes (Mmd, Nqr, Rnf, Frd/Sdh and Atp). BIOLOGICAL SIGNIFICANCE: Porphyromonas gingivalis is a major oral pathogen associated with chronic periodontitis in humans. Secreted gingipains are considered major virulence factors of this pathogen and are secreted by a newly described type IX secretion system. This work has used 2D-BN-PAGE and MS to demonstrate that mature gingipains can independently form complexes and that substrate intermediates and mature secreted proteins of the type IX secretion system form multi-subunit complexes. Based on this work we propose that the substrates of this secretion system are secreted as large multi-subunit protein complexes. Two known important components of the secretion machinery, PG0026 and the integral outer membrane protein, LptO, were found to interact which would anchor PG0026 to the outer membrane and perhaps aid in the function of PG0026 to cleave the CTD from secreted substrates. The work has also identified more than 100 membrane proteins forming multi-subunit complexes.

Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors.

Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces outer membrane vesicles (OMVs) that carry a cargo of virulence factors. In this study, the proteome of OMVs was determined by LC-MS/MS analyses of SDS-PAGE fractions, and a total of 151 OMV proteins were identified, with all but one likely to have originated from either the outer membrane or periplasm. Of these, 30 exhibited a C-terminal secretion signal known as the CTD that localizes them to the cell/vesicle surface, 79 and 27 were localized to the vesicle membrane and lumen respectively while 15 were of uncertain location. All of the CTD proteins along with other virulence factors were found to be considerably enriched in the OMVs, while proteins exhibiting the OmpA peptidoglycan-binding motif and TonB-dependent receptors were preferentially retained on the outer membrane of the cell. Cryo-transmission electron microscopy analysis revealed that an electron dense surface layer known to comprise CTD proteins accounted for a large proportion of the OMVs' volume providing an explanation for the enrichment of CTD proteins. Together the results show that P. gingivalis is able to specifically concentrate and release a large number of its virulence factors into the environment in the form of OMVs.

A novel transposon construct expressing PhoA with potential for studying protein expression and translocation in Mycoplasma gallisepticum.

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 [14C]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.

PG0026 is the C-terminal signal peptidase of a novel secretion system of Porphyromonas gingivalis.

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) of ∼70-80 amino acid residues that is essential for their secretion and attachment to the cell surface. The CTD itself has not been detected in mature substrates, suggesting that it may be removed by a novel signal peptidase. More than 10 proteins have been shown to be essential for the proper functioning of the secretion system, and one of these, PG0026, is a predicted cysteine proteinase that also contains a CTD, suggesting that it may be a secreted component of the secretion system and a candidate for being the CTD signal peptidase. A PG0026 deletion mutant was constructed along with a PG0026C690A targeted mutant encoding an altered catalytic Cys residue. Analysis of clarified culture fluid fractions by SDS-PAGE and mass spectrometry revealed that the CTD was released intact into the surrounding medium in the wild type strain, but not in the PG0026 mutant strains. Western blot experiments revealed that the maturation of a model substrate was stalled at the CTD-removal step specifically in the PG0026 mutants, and whole cell ELISA experiments demonstrated partial secretion of substrates to the cell surface. The CTD was also shown to be accessible at the cell surface in the PG0026 mutants, suggesting that the CTD was secreted but could not be cleaved. The data indicate that PG0026 is responsible for the cleavage of the CTD signal after substrates are secreted across the OM.

The outer membrane protein LptO is essential for the O-deacylation of LPS and the co-ordinated secretion and attachment of A-LPS and CTD proteins in Porphyromonas gingivalis.

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) essential for secretion and attachment to the cell surface. Inactivation of lptO (PG0027) or porT produced mutants that lacked surface protease activity and an electron-dense surface layer. Both mutants showed co-accumulation of A-LPS and unmodified CTD proteins in the periplasm. Lipid profiling by mass spectrometry showed the presence of both tetra- and penta-acylated forms of mono-phosphorylated lipid A in the wild-type and porT mutant, while only the penta-acylated forms of mono-phosphorylated lipid A were found in the lptO mutant, indicating a specific role of LptO in the O-deacylation of mono-phosphorylated lipid A. Increased levels of non-phosphorylated lipid A and the presence of novel phospholipids in the lptO mutant were also observed that may compensate for the missing mono-phosphorylated tetra-acylated lipid A in the outer membrane (OM). Molecular modelling predicted LptO to adopt a ß-barrel structure characteristic of an OM protein, supported by the enrichment of LptO in OM vesicles. The results suggest that LPS deacylation by LptO is linked to the co-ordinated secretion of A-LPS and CTD proteins by a novel secretion and attachment system to form a structured surface layer.

Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation.

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.

Surface diversity in Mycoplasma agalactiae is driven by site-specific DNA inversions within the vpma multigene locus.

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 lambda 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.

A gene family in Mycoplasma imitans closely related to the pMGA family of Mycoplasma gallisepticum.

The avian pathogen Mycoplasma gallisepticum possesses a large gene family encoding lipoproteins which function as haemagglutinins. Representative species of the pneumoniae phylogenetic group of mycoplasmas were examined for the presence of genes homologous to members of this multigene family. Antisera against the pMGA1.1 lipoprotein recognized a 35 kDa protein in Mycoplasma imitans, but did not recognize proteins of Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma pirum, Mycoplasma penetrans of Mycoplasma iowae in Western blots. A fragment of the pMGA 1.2 gene and oligonucleotide probes complementary to highly conserved coding and non-coding regions of pMGA genes bound to fragments of genomic DNA of M. imitans, but not to the genomes of M. genitalium, M. pneumoniae, M. pirum or M. penetrans, and only one probe bound to a fragment of the M. iowae genome. One homologue of the pMGA genes was amplified from the M. imitans genome by PCR and used as a probe to clone a 3.1 kbp DNA fragment from a library of HindIII-digested M. imitans genomic DNA. The contiguous DNA sequence of the PCR and HindIII clones was predicted to encode one complete and one partial ORF which shared some peptide sequence identity with the pMGA genes, including the signal peptidase II cleavage site and the proline-rich amino-terminal region. Like the pMGA genes, the M. imitans genes were found to be members of a large gene family, with an association with GAA trinucleotide repeats, a feature which distinguishes these two families from the homologous vlhA gene family in Mycoplasma synoviae. The identification of these gene families in three phylogenetically distinct avian mycoplasma species, but not in human mycoplasmas, suggests their horizontal transfer between species infecting the same host.

Size and genomic location of the pMGA multigene family of Mycoplasma gallisepticum.

The pMGA multigene family encodes variant copies of the cell surface haemagglutinin of Mycoplasma gallisepticum. Quantitative Southern blotting, using an oligonucleotide probe complementary to a region conserved in the leader sequence of all known pMGA genes, was used to estimate the number of members of the family in the genome of seven strains of M. gallisepticum. The number of copies estimated to be present in the genome varied from 32 in strain F to 70 in strain R, indicating that the pMGA gene family may be second in size only to the tRNA family among prokaryotes. If all members of the pMGA family are of similar length to those which have been characterized, a minimum of 79 kb (7.7%) of the genome of strain S6, 82 kb (8.2%) of PG31 and 168 kb (16%) of the genome of strain R is dedicated to encoding variants of the same haemagglutinin. The GAA repeat motif identified in the intergenic region between all characterized pMGA genes appeared to be a feature common to most, if not all, pMGA genes, and furthermore probably exclusive to them. The genomic locations of members of the pMGA family were determined by PFGE and Southern blot hybridization of M. gallisepticum strain S6. The hybridizing regions were localized to four separate regions on the chromosome. The pMGA genes are likely to be predominantly arranged as tandem repeats within these regions, similar to the restricted regions for which the genomic sequence has been determined.