PhoA gene fusions in Legionella pneumophila generated in vivo using a new transposon, MudphoA

To enable effective use of phoA gene fusions in Legionella pneumophila, we constructed MudphoA, a derivative of the mini-Mu phage Mu dII4041, which is capable of generating gene fusions to the Escherichia coli alkaline phosphatase gene (EC 3.1.3.1). Although an existing fusion-generating transposon, TnphoA, has been a useful tool for studying secreted proteins in other bacteria, this transposon and other Tn5 derivatives transpose inefficiently in Legionella pneumophila, necessitating the construction of a more effective vector for use in this pathogen. Using MudphoA we generated fusions to an E. coli gene encoding a periplasmic protein and to an L. pneumophila gene encoding an outer membrane protein; both sets of fusions resulted in alkaline phosphatase activity. We have begun to use MudphoA to mutate secreted proteins of L. pneumophila specifically, since this subset of bacterial proteins is most likely to be involved in host-bacterial interactions. This modified transposon may be useful for studies of other bacteria that support transposition of Mu, but not Tn5, derivatives.     

The use of Tn phoA in Erwinia amylovora to generate random fusions of alkaline phosphatase to extracytoplasmic proteins

Abstract: Tn phoA has been introduced with high efficiency into the chromosome of the phytopathogenic bacterium Erwinia amylovora . Transposition occurred with apparent randomness and single insertions were predominant. Fusion proteins were detected in cell envelope fractions from PhoA⁺ mutants and a range of alkaline phosphatase activities was onserved. The results provide the first evidence that sequences encoding signal peptides are present in E. amylovora and that Tn phoA may be a valuable tool for the study of the translocation, regulation and function of E. amylovora extracytoplasmic proteins. In particular, Tn phoA mutagenesis should be applicable to the identification of cell envelope proteins involved in the virulence of this organism.     

Construction of transposon Tn3phoA: its application in defining the membrane topology of the Agrobacterium tumefaciens DNA transfer proteins

Protein fusion with the Escherichia coli alkaline phosphatase is used extensively for the analysis of the topology of membrane proteins. To study the topology of the Agrobacterium T-DNA transfer proteins, we constructed a transposon, Tn 3phoA . The transposon mobilizes into plasmids at a high frequency, is stable after transposition, can produce phoA translational fusions and can be used for the analysis of protein topology directly in the bacterium of interest. For studies on the DNA transfer proteins, an Agrobacterium strain deficient in phoA under our experimental conditions was constructed by chemical mutagenesis. A plasmid containing virB and virD4 was used as a target for mutagenesis. Twenty-eight unique phoA -positive clones that mapped to eight virB genes were isolated. Multiple insertions throughout VirB1, VirB5, VirB7, VirB9 and VirB10 indicated that these proteins primarily face the periplasm. Insertions in VirB2, VirB6 and VirB8 allowed the identification of their periplasmic domains. No insertions were found in VirB3, VirB4 and VirB11. These proteins either lack or have a short periplasmic domain. No insertions mapped to VirD4 either. To study VirD4 topology, targeted phoA fusions and random lacZ fusions were constructed. Analysis of the fusion proteins indicated that VirD4 contains a single periplasmic domain near the N-terminus, and most of the protein lies in the cytoplasm. A hypothetical model for the T-DNA transport pore is presented.     

The inhibitory effect of Mycoplasma fermentans on tumour necrosis factor (TNF)-alpha-induced apoptosis resides in the membrane lipoproteins

Mycoplasma have been shown to be involved in the alteration of several eukaryotic cell functions, such as cytokine production, gene expression and more. We have previously reported that infection of human myelomonocytic U937 cell line with live Mycoplasma fermentans (M. fermentans) inhibited tumour necrosis factor (TNF-alpha)-induced apoptosis. Mycoplasmal membrane lipoproteins are considered to be the most potent initiators of inflammatory reactions in mycoplasmal infections. The aim of this study was to clarify whether the inhibitory effect on TNFalpha-induced apoptosis is exerted by M. fermentans lipoproteins (LPMf). A significant reduction in TNFalpha-induced apoptosis was demonstrated by stimulation of U937 cells with M. fermentans total proteins, LPMf or MALP-2 (M. fermentans synthetic lipopeptide), but not with M. fermentans hydrophilic protein preparation (AqMf). To investigate the mechanism of M. fermentans antiapoptotic effect, the reduction of mitochondrial transmembrane potential (delta psi m) was measured. M. fermentans total proteins LPMf and MALP-2, but not AqMf, inhibited the reduction of delta psi m. In addition, M. fermentans total proteins LPMf and MALP-2, but not AqMf, downregulated the formation of active caspase-8. NF-kappaB was transactivated in cells treated with M. fermentans lipoproteins, and was essential for host cell survival, but not for the inhibition of TNFalpha-induced apoptosis by LPMf. Our results suggest that the inhibitory effect exerted by M. fermentans on TNFalpha-induced apoptosis in U937 cells is due to the membrane lipoproteins of these bacteria.     

Identification of mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.

The activity of bacterial alkaline phosphatase (PhoA) is dependent on it being exported across the plasma membrane. A plasmid vector (pJEM11) allowing fusions between phoA and genes encoding exported proteins was constructed to study protein export in mycobacteria. Introduction of the Mycobacterium fortuitum beta-lactamase gene (blaF*) into this vector led to the production in M. smegmatis of protein fusions with PhoA activity. A genomic library from M. tuberculosis was constructed in pJEM11 and screened in M. smegmatis for clones with PhoA activity. Sequences of the M. tuberculosis inserts directing the production of protein fusions in these PhoA-positive clones were determined. They include part of the already-known exported 19-kDa lipoprotein, a sequence with similarities to the exported 28-kDa antigen from M. leprae, a sequence encoding a protein sharing conserved amino acid motifs with stearoyl-acyl-carrier-protein desaturases, and unknown sequences. This approach thus appears to identify sequences directing protein export, and we expect that more extensive screening of such libraries will lead to a better understanding of protein export in M. tuberculosis.     

Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector

Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence-encoding region. Library construction with Sau3A-digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum-AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non-cleavable hydrophobic membrane-spanning sequences. Triton X-114 detergent phase partitioning of individual T. pallidum-AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]-palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N-terminal signal sequence containing a consensus leader peptidase II recognition site. The DNA sequence of Tp75 also indicates that this is a previously unreported T. pallidum lipoprotein. T. pallidum-AP fusion proteins which partitioned into the hydrophobic detergent phase but did not incorporate palmitate were also identified. DNA and amino acid analysis of one such clone, Tp70, showed no cleavable signal but had a significant hydrophobic region of approximately 20 residues, consistent with a membrane-spanning domain. Immunoblot analysis of T. pallidum-AP fusions detected with a monoclonal antibody specific for AP identified several fusion proteins which migrated as doublets separated in apparent electrophoretic mobility by no more than 3 kDa. [35S]-methionine pulse-chase incorporation showed that the doublet AP fusions represented precursor and processed forms of the same protein. DNA and amino acid sequence analysis of clones expressing processed fusion proteins demonstrated hydrophobic N-terminal signal sequences containing consensus leader peptidase I recognition sites.     

The Mycoplasma fermentans prophage phiMFV1: genome organization, mobility and variable expression of an encoded surface protein

The approximately 16 kb genome of the Mycoplasma fermentans phiMFV1 prophage is described, and its mobility, replication and effect on the mycoplasma surface phenotype are demonstrated. In various M. fermentans strains, phiMFV1 was either absent or integrated at diverse (and sometimes multiple) chromosomal sites, each marked by a conserved TTTTTA target sequence that is duplicated upon integration. Precise excision, replication of an extrachromosomal form and loss of phiMFV1 from the mycoplasmal genome were documented in a series of clonal derivatives of M. fermentans propagated in culture. Of 18 open reading frames (ORFs) encoded by phiMFV1, most can be ascribed functions related to phage biology, whereas one encodes a unique coiled-coil membrane surface protein, Mem, that was confirmed to be expressed in propagating populations of M. fermentans. With the exception of Mem and other minor ORFs, the striking similarity between the deduced proteomes of phiMFV1 and the recently described phiMAV1 of arthritogenic strains of Mycoplasma arthritidis, along with the prominent gene synteny between these elements, provides the taxonomic basis for a new family of prophage. Their coding features are consistent with long-term residence in mycoplasma genomes and the divergence of species within a phylogenetic clade of mycoplasmas. The unique Mem protein expressed from phiMFV1 and the unique hypothetical surface lipoproteins encoded by phiMAV1 and phiMFV1 also suggest that prophage-associated genes may provide specific, selectable phenotypic traits during co-evolution of mycoplasma species with their respective mammalian hosts. Retention of these labile prophage elements in organisms with such drastically reduced genome sizes implies a significant role in adaptation and survival.     

Sequence requirements of Escherichia coli attTn7, a specific site of transposon Tn7 insertion.

Transposon Tn7 transposes at high frequency to a specific site, attTn7, in the Escherichia coli chromosome. We devised a quantitative assay for Tn7 transposition in which Tn7-end derivatives containing the cis-acting transposition sequences of Tn7 transpose from a bacteriophage lambda vector upon infection into cells containing the Tn7-encoded transposition proteins. We used this assay to identify a 68-base-pair DNA segment containing the sequences essential for attTn7 target activity. This segment is positioned asymmetrically with respect to the specific point of Tn7 insertion in attTn7 and lacks obvious homology to the sequences at the ends of Tn7 which participate directly in transposition. We also show that some sequences essential for attTn7 target activity are contained within the protein-coding sequence of a bacterial gene.     

Identification of genes encoding exported Mycobacterium tuberculosis proteins using a Tn552'phoA in vitro transposition system

Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552'phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552'phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosis databases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.     

Haloferax volcanii twin-arginine translocation substates include secreted soluble, C-terminally anchored and lipoproteins

Recent in silico and in vivo studies have suggested that the majority of proteins destined for secretion in the haloarchaea are trafficked through the twin-arginine translocation (Tat) pathway. The presence of lipobox motifs in most haloarchaeal Tat signal sequences is intriguing as: (i) bioinformatic searches of archaeal genomes have not identified lipoprotein biogenesis enzymes and (ii) there are no known Tat substrates containing both a twin-arginine and a bona fide lipobox. We have examined six computationally designated Tat substrates in the haloarchaeon Haloferax volcanii to verify previous computational predictions and to initiate studies of lipoprotein biogenesis via the Tat pathway. Our results confirmed that the six candidate proteins were not only Tat substrates, but also belonged to diverse classes of secretory proteins. Analysis of predicted lipoprotein Tat substrates revealed that they are anchored to the archaeal membrane in a cysteine-dependent manner. Interestingly, despite the absence of an archaeal lipoprotein signal peptidase II (SPase II) homologue, the SPase II inhibitor globomycin impeded cell growth and specifically prevented maturation of lipoproteins. Together, this work not only represents the first experimental demonstration of a lipoprotein Tat substrate, but also indicates the presence of an unidentified lipoprotein biogenesis pathway in archaea.     

Characterization of DNA topoisomerase activity in two strains of Mycoplasma fermentans and in Mycoplasma pirum.

DNA topoisomerases (topos) are essential enzymes that participate in many cellular processes involving DNA. The presence of the DNA-gyrase genes in various mycoplasmas has been reported elsewhere. However, the characterization of DNA topo activity in mycoplasmas has not been previously undertaken. In this study, we characterized the topo activity in extracts of Mycoplasma fermentans K7 and incognitus and in Mycoplasma pirum, as well as in partially purified extract of M. fermentans K7. The topo activity in these microorganisms had the following properties. (i) The relaxation of supercoiled DNA was ATP dependent. (ii) ATP independent relaxation activity was not detected. (iii) Supercoiling of relaxed topoisomers was not observed. (iv) The relaxation activity was inhibited by DNA gyrase and topo IV antagonists (novobiocin and oxolinic acid) and by eukaryotic topo II (m-AMSA [4'-(9-acridylamino)methanesulfon-m-anisidide]) and topo I antagonists (camptothecin). Other eukaryotic topo II antagonists (teniposide and etoposide) did not affect the topo relaxation activity. (v) Two polypeptides of 66 and 180 kDa were found to be associated with the mycoplasma topo activity. These results suggest that the properties of the topo enzyme in these mycoplasma species resemble those of the bacterial topo IV and the eukaryotic and the bacteriophage T4 topo II. The findings that mycoplasma topo is inhibited by both eukaryotic topo II and topo I antagonists and that m-AMSA and camptothecin inhibited the growth of M. fermentans K7 in culture support our conclusion that these mycoplasma species have topo with unique properties.     

Use of TnphoA to detect genes for exported proteins in Escherichia coli: identification of the plasmid-encoded gene for a periplasmic acid phosphatase.

The structural gene (appA) for the periplasmic acid phosphatase (optimum pH 2.5) of Escherichia coli was cloned into a plasmid by using a combination of in vivo and in vitro techniques. The position and orientation of the appA gene within the cloned DNA fragment were identified by using fusions to the alkaline phosphatase gene (phoA) generated by Tn5 IS50L::phoA (TnphoA) insertions. For TnphoA-generated hybrid proteins to have high enzymatic activity, it appears that the phoA gene must be fused to a target gene coding for a signal which promotes protein export. The approach used to identify the appA gene thus appears to provide a simple general means of selectively identifying genes encoding membrane and secreted proteins.     

Active lipoprotein precursors in the Gram-positive eubacterium Lactococcus lactis

Lipid-modified proteins play important roles at the interface between eubacterial cells and their environment. The importance of lipoprotein processing by signal peptidase II (SPase II) is underscored by the fact that this enzyme is essential for viability of the Gram-negative eubacterium Escherichia coli. In contrast, SPase II is not essential for growth and viability of the Gram-positive eubacterium Bacillus subtilis. This could be due to alternative amino-terminal lipoprotein processing, which was shown previously to occur in SPase II mutants of B. subtilis. Alternatively, uncleaved lipoprotein precursors might be functional. To explore further the importance of lipoprotein processing in Gram-positive eubacteria, an SPase II mutant strain of Lactococcus lactis was constructed. Although some of the 39 (predicted) lactococcal lipoproteins, such as PrtM and OppA, are essential for growth in milk, the growth of SPase II mutant L. lactis cells in this medium was not affected. Furthermore, the activity of the strictly PrtM-dependent extracellular protease PrtP, which is required for casein degradation, was not impaired in the absence of SPase II. Importantly, no alternative processing of pre-PrtM and pre-OppA was observed in cells lacking SPase II. Taken together, these findings show for the first time that authentic lipoprotein precursors retain biological activity.     

Development of a cloning system in Mycoplasma pulmonis

A system suitable for recombinant DNA manipulation in mycoplasmas was developed using the cloned antibiotic-resistance genes of Tn4001 and Tn916. An integrative plasmid containing one of the resistance markers was inserted into the genome of Mycoplasma pulmonis to form a recipient strain. This was accomplished by transformation and homologous recombination between chromosomal DNA sequences cloned onto the integrative plasmid. A second vector, the cloning vector, containing the same plasmid replicon and alternate resistance marker, carried cloned foreign DNA. When transformed into mycoplasmal recipients, homologous recombination between plasmid sequences resulted in integration of the cloning vector and foreign DNA. A Brucella abortus gene coding for a 31-kDa protein and the P1 structural gene and operon from Mycoplasma pneumoniae were introduced to examine the feasibility of developing mycoplasma as cloning hosts. Recombinant plasmids as large as 20 kb were inserted into M. pulmonis, and the integrated foreign DNA was stably maintained. The maximum size of clonable DNA was not determined, but plasmids larger than 22 kb have not been transformed into mycoplasmas using polyethylene glycol. Also the size of genome (800-1200 kb) may affect the stability of larger inserts of foreign DNA. This system is applicable to any mycoplasma capable of transformation, homologous recombination and expression of these resistance markers. Because of their lack of a cell wall, mycoplasmas may be useful cloning hosts for membrane or excreted protein genes from other sources.     

Characterization of an IS element in Mycoplasma orale that is highly homologous to M. fermentans ISLE

In a previous study, using a primer set designed from Mycoplasma fermentans, we amplified a PCR fragment from Mycoplasma orale similar to the 206-bp DNA fragment amplified from M. fermentans insertion-sequence-like element (ISLE). The presence of this similar ISLE fragment has the potential to cause confusion in the PCR diagnosis of M. fermentans and M. orale, which have significantly different clinical scenarios. An ISLE from three different M. orale strains was amplified by using a primer set designed from sequence within the left and right terminal stem and loop (S&L) structures flanking the ISLE of M. fermentans. Sequence analysis showed that the M. orale ISLE is 93% identical to that of M. fermentans at the nucleotide level and codes for two open reading frames also found in the M. fermentans ISLE. This is the first finding that two different mycoplasma species harbor highly homologous IS elements. This finding has great significance in clinical diagnosis and suggests a possibility of horizontal transfer of an IS element between two different mycoplasma species.     

Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen.     

Involvement of small GTPases in Mycoplasma fermentans membrane lipoproteins-mediated activation of macrophages.

Mycoplasma fermentans lipoproteins (LAMPf) are capable of activating macrophages and inducing the secretion of proinflammatory cytokines. We have recently reported that mitogen-activated protein kinase (MAPK) pathways and NF-kappaB and activated protein 1 (AP-1) play a crucial role in the activation induced by this bacterial compound. To further elucidate the mechanisms by which LAMPf mediate the activation of macrophages, we assessed the effects of inhibiting small G proteins Rac, Cdc42, and Rho. The Rho-specific inhibitor C3 enzyme completely abolished the secretion of tumor necrosis factor alpha by macrophages stimulated with LAMPf and also inhibited the activation of extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 kinase. In addition, we have shown that LAMPf stimulate Cdc42 and that inhibition of Cdc42 or Rac by dominant negative mutants abrogates LAMPf-mediated activation of JNK and transactivation of NF-kappaB and AP-1 in the murine macrophage cell line RAW 264.7. These results indicate that small G proteins Rho, Cdc42, and Rac are involved in the cascade of events leading to the macrophage activation by mycoplasma lipoproteins.     

Distribution of ecto 5'-nucleotidase on Mycoplasma species associated with arthritis

The enzyme ecto 5'-nucleotidase (5'N) was found to be active on 8/14 strains of Mycoplasma fermentans, K(m) (+/-S.D.) 3.8+/-2.8 microM 5'-AMP, and on the type strain of Mycoplasma pulmonis, K(m) 0.63 microM 5'-AMP. The six M. fermentans strains lacking 5'N activity were related by restriction fragment length polymorphism typing. At pH 8.5, the type strains of Mycoplasma arthritidis, Mycoplasma buccale and Ureaplasma urealyticum showed a relatively non-specific phosphatase activity against 5'-AMP but no activity was shown by the type strains of Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma orale, Mycoplasma penetrans, Mycoplasma pneumoniae and Mycoplasma salivarium at this pH. M. fermentans has been reported from rheumatoid joints, which show a raised 5'N activity on their synovial cells and in their fluid which may be associated directly or indirectly with the mycoplasma.