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1.  Genome-Scale Analysis of Mycoplasma agalactiae Loci Involved in Interaction with Host Cells 
PLoS ONE  2011;6(9):e25291.
Mycoplasma agalactiae is an important pathogen of small ruminants, in which it causes contagious agalactia. It belongs to a large group of “minimal bacteria” with a small genome and reduced metabolic capacities that are dependent on their host for nutrients. Mycoplasma survival thus relies on intimate contact with host cells, but little is known about the factors involved in these interactions or in the more general infectious process. To address this issue, an assay based on goat epithelial and fibroblastic cells was used to screen a M. agalactiae knockout mutant library. Mutants with reduced growth capacities in cell culture were selected and 62 genomic loci were identified as contributing to this phenotype. As expected for minimal bacteria, “transport and metabolism” was the functional category most commonly implicated in this phenotype, but 50% of the selected mutants were disrupted in coding sequences (CDSs) with unknown functions, with surface lipoproteins being most commonly represented in this category. Since mycoplasmas lack a cell wall, lipoproteins are likely to be important in interactions with the host. A few intergenic regions were also identified that may act as regulatory sequences under co-culture conditions. Interestingly, some mutants mapped to gene clusters that are highly conserved across mycoplasma species but located in different positions. One of these clusters was found in a transcriptionally active region of the M. agalactiae chromosome, downstream of a cryptic promoter. A possible scenario for the evolution of these loci is discussed. Finally, several CDSs identified here are conserved in other important pathogenic mycoplasmas, and some were involved in horizontal gene transfer with phylogenetically distant species. These results provide a basis for further deciphering functions mediating mycoplasma-host interactions.
doi:10.1371/journal.pone.0025291
PMCID: PMC3179502  PMID: 21966487
2.  Experimental Infections with Mycoplasma agalactiae Identify Key Factors Involved in Host-Colonization 
PLoS ONE  2014;9(4):e93970.
Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs.
doi:10.1371/journal.pone.0093970
PMCID: PMC3974822  PMID: 24699671
3.  Genes Found Essential in Other Mycoplasmas Are Dispensable in Mycoplasma bovis 
PLoS ONE  2014;9(6):e97100.
Mycoplasmas are regarded to be useful models for studying the minimum genetic complement required for independent survival of an organism. Mycoplasma bovis is a globally distributed pathogen causing pneumonia, mastitis, arthritis, otitis media and reproductive tract disease, and genome sequences of three strains, the type strain PG45 and two strains isolated in China, have been published. In this study, several Tn4001 based transposon constructs were generated and used to create a M. bovis PG45 insertional mutant library. Direct genome sequencing of 319 independent insertions detected disruptions in 129 genes in M. bovis, 48 of which had homologues in Mycoplasma mycoides subspecies mycoides SC and 99 of which had homologues in Mycoplasma agalactiae. Sixteen genes found to be essential in previous studies on other mycoplasma species were found to be dispensable. Five of these genes have previously been predicted to be part of the core set of 153 essential genes in mycoplasmas. Thus this study has extended the list of non-essential genes of mycoplasmas from that previously generated by studies in other species.
doi:10.1371/journal.pone.0097100
PMCID: PMC4045577  PMID: 24897538
4.  Being Pathogenic, Plastic, and Sexual while Living with a Nearly Minimal Bacterial Genome 
PLoS Genetics  2007;3(5):e75.
Mycoplasmas are commonly described as the simplest self-replicating organisms, whose evolution was mainly characterized by genome downsizing with a proposed evolutionary scenario similar to that of obligate intracellular bacteria such as insect endosymbionts. Thus far, analysis of mycoplasma genomes indicates a low level of horizontal gene transfer (HGT) implying that DNA acquisition is strongly limited in these minimal bacteria. In this study, the genome of the ruminant pathogen Mycoplasma agalactiae was sequenced. Comparative genomic data and phylogenetic tree reconstruction revealed that ∼18% of its small genome (877,438 bp) has undergone HGT with the phylogenetically distinct mycoides cluster, which is composed of significant ruminant pathogens. HGT involves genes often found as clusters, several of which encode lipoproteins that usually play an important role in mycoplasma–host interaction. A decayed form of a conjugative element also described in a member of the mycoides cluster was found in the M. agalactiae genome, suggesting that HGT may have occurred by mobilizing a related genetic element. The possibility of HGT events among other mycoplasmas was evaluated with the available sequenced genomes. Our data indicate marginal levels of HGT among Mycoplasma species except for those described above and, to a lesser extent, for those observed in between the two bird pathogens, M. gallisepticum and M. synoviae. This first description of large-scale HGT among mycoplasmas sharing the same ecological niche challenges the generally accepted evolutionary scenario in which gene loss is the main driving force of mycoplasma evolution. The latter clearly differs from that of other bacteria with small genomes, particularly obligate intracellular bacteria that are isolated within host cells. Consequently, mycoplasmas are not only able to subvert complex hosts but presumably have retained sexual competence, a trait that may prevent them from genome stasis and contribute to adaptation to new hosts.
Author Summary
Mycoplasmas are cell wall–lacking prokaryotes that evolved from ancestors common to Gram-positive bacteria by way of massive losses of genetic material. With their minimal genome, mycoplasmas are considered to be the simplest free-living organisms, yet several species are successful pathogens of man and animal. In this study, we challenged the commonly accepted view in which mycoplasma evolution is driven only by genome down-sizing. Indeed, we showed that a significant amount of genes underwent horizontal transfer among different mycoplasma species that share the same ruminant hosts. In these species, the occurrence of a genetic element that can promote DNA transfer via cell-to-cell contact suggests that some mycoplasmas may have retained or acquired sexual competence. Transferred genes were found to encode proteins that are likely to be associated with mycoplasma–host interactions. Sharing genetic resources via horizontal gene transfer may provide mycoplasmas with a means for adapting to new niches or to new hosts and for avoiding irreversible genome erosion.
doi:10.1371/journal.pgen.0030075
PMCID: PMC1868952  PMID: 17511520
5.  Being Pathogenic, Plastic, and Sexual while Living with a Nearly Minimal Bacterial Genome 
PLoS Genetics  2007;3(5):e75.
Mycoplasmas are commonly described as the simplest self-replicating organisms, whose evolution was mainly characterized by genome downsizing with a proposed evolutionary scenario similar to that of obligate intracellular bacteria such as insect endosymbionts. Thus far, analysis of mycoplasma genomes indicates a low level of horizontal gene transfer (HGT) implying that DNA acquisition is strongly limited in these minimal bacteria. In this study, the genome of the ruminant pathogen Mycoplasma agalactiae was sequenced. Comparative genomic data and phylogenetic tree reconstruction revealed that ∼18% of its small genome (877,438 bp) has undergone HGT with the phylogenetically distinct mycoides cluster, which is composed of significant ruminant pathogens. HGT involves genes often found as clusters, several of which encode lipoproteins that usually play an important role in mycoplasma–host interaction. A decayed form of a conjugative element also described in a member of the mycoides cluster was found in the M. agalactiae genome, suggesting that HGT may have occurred by mobilizing a related genetic element. The possibility of HGT events among other mycoplasmas was evaluated with the available sequenced genomes. Our data indicate marginal levels of HGT among Mycoplasma species except for those described above and, to a lesser extent, for those observed in between the two bird pathogens, M. gallisepticum and M. synoviae. This first description of large-scale HGT among mycoplasmas sharing the same ecological niche challenges the generally accepted evolutionary scenario in which gene loss is the main driving force of mycoplasma evolution. The latter clearly differs from that of other bacteria with small genomes, particularly obligate intracellular bacteria that are isolated within host cells. Consequently, mycoplasmas are not only able to subvert complex hosts but presumably have retained sexual competence, a trait that may prevent them from genome stasis and contribute to adaptation to new hosts.
Author Summary
Mycoplasmas are cell wall–lacking prokaryotes that evolved from ancestors common to Gram-positive bacteria by way of massive losses of genetic material. With their minimal genome, mycoplasmas are considered to be the simplest free-living organisms, yet several species are successful pathogens of man and animal. In this study, we challenged the commonly accepted view in which mycoplasma evolution is driven only by genome down-sizing. Indeed, we showed that a significant amount of genes underwent horizontal transfer among different mycoplasma species that share the same ruminant hosts. In these species, the occurrence of a genetic element that can promote DNA transfer via cell-to-cell contact suggests that some mycoplasmas may have retained or acquired sexual competence. Transferred genes were found to encode proteins that are likely to be associated with mycoplasma–host interactions. Sharing genetic resources via horizontal gene transfer may provide mycoplasmas with a means for adapting to new niches or to new hosts and for avoiding irreversible genome erosion.
doi:10.1371/journal.pgen.0030075
PMCID: PMC1868952  PMID: 17511520
6.  Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of Mycoplasma agalactiae, AvgC, with the Use of Synthetic Peptides  
Infection and Immunity  2002;70(1):171-176.
As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection.
doi:10.1128/IAI.70.1.171-176.2002
PMCID: PMC127643  PMID: 11748179
7.  Large-Scale Transposon Mutagenesis of Mycoplasma pulmonis 
Molecular microbiology  2008;69(1):67-76.
To obtain mutants for the study of the basic biology and pathogenic mechanisms of mycoplasmas, the insertion site of transposon Tn4001T was determined for 1,700 members of a library of Mycoplasma pulmonis mutants. After evaluating several criteria for gene disruption, we concluded that 321 of the 782 protein coding regions were inactivated. The dispensable and essential genes of M. pulmonis were compared to those reported for Mycoplasma genitalium and Bacillus subtilis. Perhaps the most surprising result of the current study was that unlike other bacteria, ribosomal proteins S18 and L28 were dispensable. Carbohydrate transport and the susceptibility of selected mutants to UV irradiation were examined to assess whether active transposition of Tn4001T within the genome would confound phenotypic analysis. In contrast to earlier reports suggesting that mycoplasmas were limited in their DNA repair machinery, mutations in recA, uvrA, uvrB and uvrC resulted in a DNA-repair deficient phenotype. A mutant with a defect in transport of N-acetylglucosamine was identified.
doi:10.1111/j.1365-2958.2008.06262.x
PMCID: PMC2453687  PMID: 18452587
8.  A Metabolic Enzyme as a Primary Virulence Factor of Mycoplasma mycoides subsp. mycoides Small Colony 
Journal of Bacteriology  2005;187(19):6824-6831.
During evolution, pathogenic bacteria have developed complex interactions with their hosts. This has frequently involved the acquisition of virulence factors on pathogenicity islands, plasmids, transposons, or prophages, allowing them to colonize, survive, and replicate within the host. In contrast, Mycoplasma species, the smallest self-replicating organisms, have regressively evolved from gram-positive bacteria by reduction of the genome to a minimal size, with the consequence that they have economized their genetic resources. Hence, pathogenic Mycoplasma species lack typical primary virulence factors such as toxins, cytolysins, and invasins. Consequently, little is known how pathogenic Mycoplasma species cause host cell damage, inflammation, and disease. Here we identify a novel primary virulence determinant in Mycoplasma mycoides subsp. mycoides Small Colony (SC), which causes host cell injury. This virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2 formed by l-α-glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected with M. mycoides subsp. mycoides SC in the presence of physiological amounts of glycerol, H2O2 is released inside the cells prior to cell death. This process can be inhibited with monospecific anti-GlpO antibodies.
doi:10.1128/JB.187.19.6824-6831.2005
PMCID: PMC1251598  PMID: 16166545
9.  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
10.  Emergence of Atypical Mycoplasma agalactiae Strains Harboring a New Prophage and Associated with an Alpine Wild Ungulate Mortality Episode 
Applied and Environmental Microbiology  2012;78(13):4659-4668.
The bacterium Mycoplasma agalactiae is responsible for contagious agalactia (CA) in small domestic ruminants, a syndrome listed by the World Organization for Animal Health and responsible for severe damage to the dairy industry. Recently, we frequently isolated this pathogen from lung lesions of ibexes during a mortality episode in the French Alps. This situation was unusual in terms of host specificity and tissue tropism, raising the question of M. agalactiae emergence in wildlife. To address this issue, the ibex isolates were characterized using a combination of approaches that included antigenic profiles, molecular typing, optical mapping, and whole-genome sequencing. Genome analyses showed the presence of a new, large prophage containing 35 coding sequences (CDS) that was detected in most but not all ibex strains and has a homolog in Mycoplasma conjunctivae, a species causing keratoconjunctivitis in wild ungulates. This and the presence in all strains of large integrated conjugative elements suggested highly dynamic genomes. Nevertheless, M. agalactiae strains circulating in the ibex population were shown to be highly related, most likely originating from a single parental clone that has also spread to another wild ungulate species of the same geographical area, the chamois. These strains clearly differ from strains described in Europe so far, including those found nearby, before CA eradication a few years ago. While M. agalactiae pathogenicity in ibexes remains unclear, our data showed the emergence of atypical strains in Alpine wild ungulates, raising the question of a role for the wild fauna as a potential reservoir of pathogenic mycoplasmas.
doi:10.1128/AEM.00332-12
PMCID: PMC3370481  PMID: 22522685
11.  The liposoluble proteome of Mycoplasma agalactiae: an insight into the minimal protein complement of a bacterial membrane 
BMC Microbiology  2010;10:225.
Background
Mycoplasmas are the simplest bacteria capable of autonomous replication. Their evolution proceeded from gram-positive bacteria, with the loss of many biosynthetic pathways and of the cell wall. In this work, the liposoluble protein complement of Mycoplasma agalactiae, a minimal bacterial pathogen causing mastitis, polyarthritis, keratoconjunctivitis, and abortion in small ruminants, was subjected to systematic characterization in order to gain insights into its membrane proteome composition.
Results
The selective enrichment for M. agalactiae PG2T liposoluble proteins was accomplished by means of Triton X-114 fractionation. Liposoluble proteins were subjected to 2-D PAGE-MS, leading to the identification of 40 unique proteins and to the generation of a reference 2D map of the M. agalactiae liposoluble proteome. Liposoluble proteins from the type strain PG2 and two field isolates were then compared by means of 2D DIGE, revealing reproducible differences in protein expression among isolates. An in-depth analysis was then performed by GeLC-MS/MS in order to achieve a higher coverage of the liposoluble proteome. Using this approach, a total of 194 unique proteins were identified, corresponding to 26% of all M. agalactiae PG2T genes. A gene ontology analysis and classification for localization and function was also carried out on all protein identifications. Interestingly, the 11.5% of expressed membrane proteins derived from putative horizontal gene transfer events.
Conclusions
This study led to the in-depth systematic characterization of the M. agalactiae liposoluble protein component, providing useful insights into its membrane organization.
doi:10.1186/1471-2180-10-225
PMCID: PMC2941501  PMID: 20738845
12.  The Surface Protein Srr-1 of Streptococcus agalactiae Binds Human Keratin 4 and Promotes Adherence to Epithelial HEp-2 Cells▿  
Infection and Immunity  2007;75(11):5405-5414.
Streptococcus agalactiae is frequently the cause of bacterial sepsis and meningitis in neonates. In addition, it is a commensal bacterium that colonizes the mammalian gastrointestinal tract. During its commensal and pathogenic lifestyles, S. agalactiae colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present study, the serine-rich repeat protein Srr-1 from S. agalactiae was functionally investigated. Immunofluorescence microscopy showed that Srr-1 was localized on the surface of streptococcal cells. The Srr-1 protein was shown to interact with a 62-kDa protein in human saliva, which was identified by matrix-assisted laser desorption ionization-time-of-flight analysis as human keratin 4 (K4). Immunoblot and enzyme-linked immunosorbent assay experiments allowed us to narrow down the K4 binding domain in Srr-1 to a region of 157 amino acids (aa). Furthermore, the Srr-1 binding domain of K4 was identified in the C-terminal 255 aa of human K4. Deletion of the srr-1 gene in the genome of S. agalactiae revealed that this gene plays a role in bacterial binding to human K4 and that it is involved in adherence to epithelial HEp-2 cells. Binding to immobilized K4 and adherence to HEp-2 cells were restored by introducing the srr-1 gene on a shuttle plasmid into the srr-1 mutant. Furthermore, incubation of HEp-2 cells with the K4 binding domain of Srr-1 blocked S. agalactiae adherence to epithelial cells in a dose-dependent fashion. This is the first report describing the interaction of a bacterial protein with human K4.
doi:10.1128/IAI.00717-07
PMCID: PMC2168289  PMID: 17709412
13.  Variable Lipoprotein Genes of Mycoplasma agalactiae Are Activated In Vivo by Promoter Addition via Site-Specific DNA Inversions  
Infection and Immunity  2003;71(7):3821-3830.
Mycoplasma agalactiae, the etiological agent of contagious agalactia of small ruminants, has a family of related genes (avg genes) which encode surface lipoprotein antigens that undergo phase variation. A series of 13 M. agalactiae clonal isolates, obtained from one chronically infected animal over a period of 7 months, were found to undergo major rearrangement events within the avg genomic locus. We show that these rearrangements regulate the phase-variable expression of individual avg genes. Northern blot analysis and reverse transcription-PCR showed that only one avg gene is transcribed, while the other avg genes are transcriptionally silent. Sequence analysis and primer extension experiments with two M. agalactiae clonal isolates showed that a specific 182-bp avg 5′ upstream region (avg-B2) that is present as a single chromosomal copy serves as an active promoter and exhibits a high level of homology with the vsp promoter of the bovine pathogen Mycoplasma bovis. PCR analysis showed that each avg gene is associated with the avg-B2 promoter in a subpopulation of cells that is present in each subclone. Multiple sequence-specific sites for DNA recombination (vis-like), which are presumably recognized by site-specific recombinase, were identified within the conserved avg 5′ upstream regions of all avg genes and were found to be identical to the recombination sites of the M. bovis vsp locus. In addition, a gene encoding a member of the integrase family of tyrosine site-specific recombinases was identified adjacent to the variable avg locus. The molecular genetic basis for avg phase-variable expression appears to be mediated by site-specific DNA inversions occurring in vivo that allow activation of a silent avg gene by promoter addition. A model for the control of avg genes is proposed.
doi:10.1128/IAI.71.7.3821-3830.2003
PMCID: PMC162021  PMID: 12819065
14.  Distribution and diversity of mycoplasma plasmids: lessons from cryptic genetic elements 
BMC Microbiology  2012;12:257.
Background
The evolution of mycoplasmas from a common ancestor with Firmicutes has been characterized not only by genome down-sizing but also by horizontal gene transfer between mycoplasma species sharing a common host. The mechanisms of these gene transfers remain unclear because our knowledge of the mycoplasma mobile genetic elements is limited. In particular, only a few plasmids have been described within the Mycoplasma genus.
Results
We have shown that several species of ruminant mycoplasmas carry plasmids that are members of a large family of elements and replicate via a rolling-circle mechanism. All plasmids were isolated from species that either belonged or were closely related to the Mycoplasma mycoides cluster; none was from the Mycoplasma bovis-Mycoplasma agalactiae group. Twenty one plasmids were completely sequenced, named and compared with each other and with the five mycoplasma plasmids previously reported. All plasmids share similar size and genetic organization, and present a mosaic structure. A peculiar case is that of the plasmid pMyBK1 from M. yeatsii; it is larger in size and is predicted to be mobilizable. Its origin of replication and replication protein were identified. In addition, pMyBK1 derivatives were shown to replicate in various species of the M. mycoides cluster, and therefore hold considerable promise for developing gene vectors. The phylogenetic analysis of these plasmids confirms the uniqueness of pMyBK1 and indicates that the other mycoplasma plasmids cluster together, apart from the related replicons found in phytoplasmas and in species of the clade Firmicutes.
Conclusions
Our results unraveled a totally new picture of mycoplasma plasmids. Although they probably play a limited role in the gene exchanges that participate in mycoplasma evolution, they are abundant in some species. Evidence for the occurrence of frequent genetic recombination strongly suggests they are transmitted between species sharing a common host or niche.
doi:10.1186/1471-2180-12-257
PMCID: PMC3541243  PMID: 23145790
Mycoplasma,Plasmid,Replication,Rep protein,Gene transfer,Evolution,Expression vector,Mycoplasma mycoides,Mycoplasma capricolum,Mycoplasma yeatsii
15.  Genome of Mycoplasma arthritidis▿ †  
Infection and Immunity  2008;76(9):4000-4008.
The genomes of several species of mycoplasma have been sequenced. Most of these species rely on the glycolytic pathway for energy production, with the one exception of Ureaplasma, a species that breaks down urea as its principle source of acquiring energy. Several species, including as Mycoplasma arthritidis, are nonglycolytic and can use arginine as their source of energy. Described here are the genome sequence and a transposon library of M. arthritidis. The genome of 820,453 bp is typical in size for a mycoplasma and contains two large families of genes that are predicted to code for phase-variable proteins. The transposon library was constructed using a minitransposon that inserts stably into the mycoplasma genome. Of the 635 predicted coding regions, 218 were disrupted in a library of 1,100 members. Dispensable genes included the gene coding for the MAM superantigen and genes coding for ribosomal proteins S15, S18, and L15.
doi:10.1128/IAI.00516-08
PMCID: PMC2519406  PMID: 18573899
16.  Transposon Mutagenesis Identifies Genes Associated with Mycoplasma pneumoniae Gliding Motility 
Journal of Bacteriology  2006;188(17):6335-6345.
The wall-less prokaryote Mycoplasma pneumoniae, a common cause of chronic respiratory tract infections in humans, is considered to be among the smallest and simplest known cells capable of self-replication, yet it has a complex architecture with a novel cytoskeleton and a differentiated terminal organelle that function in adherence, cell division, and gliding motility. Recent findings have begun to elucidate the hierarchy of protein interactions required for terminal organelle assembly, but the engineering of its gliding machinery is largely unknown. In the current study, we assessed gliding in cytadherence mutants lacking terminal organelle proteins B, C, P1, and HMW1. Furthermore, we screened over 3,500 M. pneumoniae transposon mutants individually to identify genes associated with gliding but dispensable for cytadherence. Forty-seven transformants having motility defects were characterized further, with transposon insertions mapping to 32 different open reading frames widely distributed throughout the M. pneumoniae genome; 30 of these were dispensable for cytadherence. We confirmed the clonality of selected transformants by Southern blot hybridization and PCR analysis and characterized satellite growth and gliding by microcinematography. For some mutants, satellite growth was absent or developed more slowly than that of the wild type. Others produced lawn-like growth largely devoid of typical microcolonies, while still others had a dull, asymmetrical leading edge or a filamentous appearance of colony spreading. All mutants exhibited substantially reduced gliding velocities and/or frequencies. These findings significantly expand our understanding of the complexity of M. pneumoniae gliding and the identity of possible elements of the gliding machinery, providing a foundation for a detailed analysis of the engineering and regulation of motility in this unusual prokaryote.
doi:10.1128/JB.00698-06
PMCID: PMC1595379  PMID: 16923901
17.  Identification and Functional Mapping of the Mycoplasma fermentans P29 Adhesin  
Infection and Immunity  2002;70(9):4925-4935.
Initial adherence interactions between mycoplasmas and mammalian cells are important for host colonization and may contribute to subsequent pathogenic processes. Despite significant progress toward understanding the role of specialized, complex tip structures in the adherence of some mycoplasmas, particularly those that infect humans, less is known about adhesins through which other mycoplasmas of this host bind to diverse cell types, even though simpler surface components are likely to be involved. We show by flow cytometric analysis that a soluble recombinant fusion protein (FP29), representing the abundant P29 surface lipoprotein of Mycoplasma fermentans, binds human HeLa cells and inhibits M. fermentans binding to these cells, in both a quantitative and a saturable manner, whereas analogous fusion proteins representing other mycoplasma surface proteins did not. Constructs representing nested N- or C-terminal truncations of FP29 allowed initial mapping of this specific adherence function to a central region of the P29 sequence containing a 36-amino-acid disulfide loop. A derivative of FP29 containing a mutation converting one participating Cys to Ser, precluding intrachain disulfide bond formation, retained full activity. Together these results suggest that the direct interaction of M. fermentans with a ligand on the HeLa cell surface involves a limited segment of the P29 surface lipoprotein and requires neither the disulfide bond nor the contribution of adjacent portions of the protein. Earlier results indicating phase-variable display of monoclonal antibody surface epitopes on P29, now recognized to be outside this ligand binding region, raise the possibility that variation of mycoplasma surface architecture might alter the presentation of the binding region and the adherence phenotype. Preliminary results further indicated that FP29 could inhibit binding to HeLa cells by Mycoplasma hominis, a distinct human mycoplasma species displaying the phase-variable adhesin Vaa, but not that by Mycoplasma capricolum, an organism infecting caprine species. This result raises the additional, testable possibility that a common host cell ligand for two human mycoplasma species may be recognized through structurally dissimilar adhesins that undergo phase variation by two distinct mechanisms, governing protein expression (Vaa) or surface masking (P29).
doi:10.1128/IAI.70.9.4925-4935.2002
PMCID: PMC128281  PMID: 12183538
18.  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
19.  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
20.  Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase (lpd), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants  
Infection and Immunity  2006;74(2):931-939.
To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.
doi:10.1128/IAI.74.2.931-939.2006
PMCID: PMC1360363  PMID: 16428737
21.  Mycoplasmas: sophisticated, reemerging, and burdened by their notoriety. 
Emerging Infectious Diseases  1997;3(1):21-32.
Mycoplasmas are most unusual self-replicating bacteria, possessing very small genomes, lacking cell wall components, requiring cholesterol for membrane function and growth, using UGA codon for tryptophan, passing through "bacterial-retaining" filters, and displaying genetic economy that requires a strict dependence on the host for nutrients and refuge. In addition, many of the mycoplasmas pathogenic for humans and animals possess extraordinary specialized tip organelles that mediate their intimate interaction with eucaryotic cells. This host-adapted survival is achieved through surface parasitism of target cells, acquisition of essential biosynthetic precursors, and in some cases, subsequent entry and survival intracellularly. Misconceptions concerning the role of mycoplasmas in disease pathogenesis can be directly attributed to their biological subtleties and to fundamental deficits in understanding their virulence capabilities. In this review, we highlight the biology and pathogenesis of these procaryotes and provide new evidence that may lead to increased appreciation of their role as human pathogens.
PMCID: PMC2627593  PMID: 9126441
22.  rgf Encodes a Novel Two-Component Signal Transduction System of Streptococcus agalactiae  
Infection and Immunity  2002;70(5):2434-2440.
The adhesion of gram-positive bacteria to extracellular matrix (ECM) proteins is regarded as an important determinant of pathogenicity. A comparison of the adhesion of Streptococcus agalactiae strain O90R to different ECM proteins showed that the most pronounced binding could be observed for immobilized fibrinogen. To investigate the genetic determinants of S. agalactiae fibrinogen binding, a pGhost9:ISS1 mutant library was screened for mutants displaying reduced agglutination of fibrinogen-coated latex beads. A putative two-component signal transduction system was identified and designated rgfBDAC. It comprises genes encoding a putative response regulator of 218 amino acids and a putative histidine kinase of 426 amino acids. Comparison of the deduced proteins with the GenBank database revealed a significant similarity to quorum-sensing systems of gram-positive pathogens. Transcription analysis of the rgf locus showed that the encoding genes are located on one transcript. To further characterize the influence of the putative histidine kinase encoded in the rgf locus on the adhesion of S. agalactiae to immobilized fibrinogen, a targeted mutant of rgfC was generated. In comparison to the wild-type strain this mutant demonstrated altered fibrinogen binding capacities depending on bacterial cell density. Transcription analysis of secreted and surface-localized S. agalactiae proteins in the wild type and the rgfC mutant strain revealed that mRNA levels of the C5a peptidase gene scpB were increased in the mutant strain while the transcription of the secreted CAMP factor gene cfb was unaffected by this mutation. Based on these results, we hypothesize that rgf regulates the expression of bacterial cell surface components.
doi:10.1128/IAI.70.5.2434-2440.2002
PMCID: PMC127907  PMID: 11953380
23.  Electrophoretic Analysis of Indian Isolates of Mycoplasma agalactiae and Mycoplasma bovis by SDS-PAGE and Immunoblotting 
Mycoplasma agalactiae and Mycoplasma bovis both are responsible for respiratory conditions in sheep and goats. M. agalactiae is a major pathogen of sheep and goats and accounts for almost 90% of outbreaks of contagious agalactia syndrome in goats and almost 100% in sheep. On the basis of clinical signs and cultural, morphological, and biochemical characterization it is almost impossible to differentiate between both the species. Moreover, due to presence of genomic and proteomic similarity most of the time routine diagnostic tests fail to differentiate between them. Hence the present study was conducted to find out the protein profile of isolates of both the species by SDS-PAGE and to find out the cross-reacting as well as differentiating immunogenic proteins by Immunoblotting, which can be of immunoprophylactic as well as diagnostic values. The study revealed 6-7 major immunogenic cross-reactive proteins with the presence of two important non-cross-reacting species specific polypeptides particularly 25.50 and 24.54 kDa in M. agalactiae and M. bovis, respectively, that might be of diagnostic values.
doi:10.1155/2014/892421
PMCID: PMC3998006  PMID: 24808973
24.  Mycoplasma polysaccharide protects against complement 
Microbiology  2012;158(Pt 7):1867-1873.
Although they lack a cell wall, mycoplasmas do possess a glycocalyx. The interactions between the glycocalyx, mycoplasmal surface proteins and host complement were explored using the murine pathogen Mycoplasma pulmonis as a model. It was previously shown that the length of the tandem repeat region of the surface lipoprotein Vsa is associated with susceptibility to complement-mediated killing. Cells producing a long Vsa containing about 40 repeats are resistant to complement, whereas strains that produce a short Vsa of five or fewer repeats are susceptible. We show here that the length of the Vsa protein modulates the affinity of the M. pulmonis EPS-I polysaccharide for the mycoplasma cell surface, with more EPS-I being associated with mycoplasmas producing a short Vsa protein. An examination of mutants that lack EPS-I revealed that planktonic mycoplasmas were highly susceptible to complement killing even when the Vsa protein was long, demonstrating that both EPS-I and Vsa length contribute to resistance. In contrast, the mycoplasmas were resistant to complement even in the absence of EPS-I when the cells were encased in a biofilm.
doi:10.1099/mic.0.058222-0
PMCID: PMC3542145  PMID: 22504437
25.  Homologue of Macrophage-Activating Lipoprotein in Mycoplasma gallisepticum Is Not Essential for Growth and Pathogenicity in Tracheal Organ Cultures 
Journal of Bacteriology  2003;185(8):2538-2547.
While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M. pneumoniae and M. genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M. agalactiae and M. fermentans. The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M. fermentans and lipoprotein P48 of M. agalactiae. The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M. gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47− mutant and wild-type M. gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.
doi:10.1128/JB.185.8.2538-2547.2003
PMCID: PMC152605  PMID: 12670978

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