The complete genome of Mycoplasma gallisepticum strain R low has been sequenced. The genome is composed of 996 422 bp with an overall G+C content of 31 mol%. It contains 742 putative coding DNA sequences (CDSs), representing a 91 % coding density. Function has been assigned to 469 of the CDSs, while 150 encode conserved hypothetical proteins and 123 remain as unique hypothetical proteins. The genome contains two copies of the rRNA genes and 33 tRNA genes. The origin of replication has been localized based on sequence analysis in the region of the dnaA gene. The vlhA family (previously termed pMGA) contains 43 genes distributed among five loci containing 8, 2, 9, 12 and 12 genes. This family of genes constitutes 10?4 % (103 kb) of the total genome. Two CDSs were identified immediately downstream of gapA and crmA encoding proteins that share homology to cytadhesins GapA and CrmA. Based on motif analysis it is predicted that 80 genes encode lipoproteins and 149 proteins contain multiple transmembrane domains. The authors have identified 75 proteins putatively involved in transport of biomolecules, 12 transposases, and a number of potential virulence factors. The completion of this sequence has spawned multiple projects directed at defining the biological basis of M. gallisepticum. INTRODUCTIONPhylogenetic analyses indicate that mycoplasmas (class Mollicutes) have undergone a degenerative evolution from related, low G+C content, Gram-positive eubacteria (Rogers et al., 1985;Woese et al., 1980). The reduction of the mycoplasma genome has resulted in the loss of the cell wall and has limited the biosynthetic capabilities of these organisms. As a consequence of this loss of biosynthetic machinery, mycoplasmas are obligate parasites and rely on the uptake of many essential molecules from their environment.Mycoplasmas have long been considered model systems for defining the minimal set of genes required for a living cell (Morowitz, 1984). For this reason, it was not surprising when Mycoplasma genitalium (580 kb) was selected as one of the first targets for complete genome sequencing (Fraser et al., 1995). Since this initial report, the genomes of four additional mycoplasmas have been sequenced, Mycoplasma pneumoniae (816 kb; Dandekar et al., 2000;Himmelreich et al., 1996), Ureaplasma urealyticum (752 kb; Glass et al., 2000), Mycoplasma pulmonis (964 kb; Chambaud et al., 2001) and Mycoplasma penetrans (1358 kb; Sasaki et al., 2000). Theoretical and experimental approaches have estimated the minimum number of essential mycoplasma genes to be between 265 and 350 (Hutchison et al., 1999;Mushegian & Koonin, 1996).Abbreviations: CDS, coding DNA sequence; COGs, conserved orthologous groups.The GenBank accession number for the sequence reported in this paper is AE015450. Mycoplasma gallisepticum is an avian pathogen involved in chronic respiratory disease in chickens resulting in considerable economic losses in poultry production. Infection with this bacterium is spread by aerosol exposure and egg transmission. Outbreaks spread...

Comparison of the phenotypic expression of Mycoplasma gallisepticum strain R low (passage 15) to that of strain R high (passage 164) revealed that three proteins, i.e., the cytadhesin molecule GapA, a 116-kDa protein (p116), and a 45-kDa protein (p45), are missing in strain R high. Sequence analysis confirmed that the insertion of an adenine 105 bp downstream of the gapA translational start codon resulted in premature termination of translation in R high. A second adenine insertion had also occurred at position 907. Restoration of expression of wild-type gapA in R high (clone designated GT5) allowed us to evaluate the extent to which the diminished cytadherence capacity could be attributed to GapA alone. The results indicated that GT5 attached to the same limited extent as the parental R high, from which it was derived. The cytadherence capability of the parental R high was not restored solely by gapA complementation alone, indicating that either p116 or p45 or both may play a role in the overall cytadherence process. The gene encoding p116 was found to be immediately downstream of gapA in the same operon and was designated crmA. This gene exhibited striking homology to genes encoding molecules with cytadhesin-related functions in both Mycoplasma pneumoniae and Mycoplasma genitalium. Transcriptional analysis revealed that crmA is not transcribed in R high. We are currently constructing a shuttle vector containing both the wild-type gapA and crmA for transformation into R high to assess the role of CrmA in the cytadherence process.Consistent with the interactions of typical noninvasive pathogenic bacteria and their host cells, Mycoplasma gallisepticum must first establish a specific and firm attachment to its target cell through a process known as cytadhesion in order to avoid rapid clearance by innate host defense mechanisms. This is a prerequisite for the initiation of the processes that result in host cell alterations and pathogenesis. Recent studies on the molecular mechanisms of M. gallisepticum cytadherence have revealed a complex multifactorial cytadhesion process involving the coordinate action of the primary cytadhesin molecule, GapA, while implicating additional proteins as potential cytadherence-related molecules in a manner reminiscent of the complex cytadherence mechanism of the human respiratory pathogen Mycoplasma pneumoniae. M. pneumoniae cytadherence has been demonstrated to involve the coordinate action of the primary cytadhesin molecule, P1 (11,12,21), in concert with an array of high-molecular-weight accessory proteins (7,8). Prolonged colonization and survival of M. gallisepticum in the host requires maximal utilization of cytadhesin molecules in association with the cytadherence-related molecules. This ultimately confers a selective advantage for that strain of pathogenic M. gallisepticum over those with diminished cytadherence capacity. The aim of this study was to characterize the molecular basis for the diminished cytadherence capacity as well as to investigate the biochemical and molecul...

Abstract. During the period from January 2002 to March 2007, infections by melanized fungi were identified with greater frequency in aquarium-maintained leafy seadragons (Phycodurus eques) and weedy seadragons (Phyllopteryx taeniolatus), pivotal species to the educational and environmental concerns of the aquarium industry and conservation groups. The objective of this study was to characterize the pathology and identify fungi associated with phaeohyphomycotic lesions in these species. Samples from 14 weedy and 6 leafy seadragons were received from 2 institutions and included fresh, frozen, and formalin-fixed tissues from necropsy and biopsy specimens. Fresh and frozen tissues were cultured for fungi on Sabouraud dextrose agar only or both Sabouraud dextrose agar and inhibitory mold agar with gentamicin and chloramphenicol at 30uC. Isolates were processed for morphologic identification and molecular sequence analysis of the internal transcribed spacer region and D1/D2 domains of the large subunit ribosomal RNA gene. Lesions were extensive and consisted of parenchymal and vascular necrosis with fungal invasion of gill (11/20), kidney (14/20), and other coelomic viscera with or without cutaneous ulceration (13/20). Exophiala sp. isolates were obtained from 4 weedy and 3 leafy seadragons and were identified to species level in 6 of 7 instances, namely Exophiala angulospora (1) and a novel species of Exophiala (5), based on nucleotide sequence comparisons and phylogenetic analyses. Disseminated phaeohyphomycosis represents an important pathologic condition of both weedy and leafy seadragons for which 2 species of Exophiala, 1 a novel species, have been isolated.

Mycoplasma gallisepticum is a significant respiratory and reproductive pathogen of domestic poultry. While the complete genomic sequence of the virulent, low-passage M. gallisepticum strain R (R low ) has been reported, genomic determinants responsible for differences in virulence and host range remain to be completely identified. Here, we utilize genome sequencing and microarray-based comparative genomic data to identify these genomic determinants of virulence and to elucidate genomic variability among strains of M. gallisepticum. Analysis of the high-passage, attenuated derivative of R low , R high , indicated that relatively few total genomic changes (64 loci) occurred, yet they are potentially responsible for the observed attenuation of this strain. In addition to previously characterized mutations in cytadherence-related proteins, changes included those in coding sequences of genes involved in sugar metabolism. Analyses of the genome of the M. gallisepticum vaccine strain F revealed numerous differences relative to strain R, including a highly divergent complement of vlhA surface lipoprotein genes, and at least 16 genes absent or significantly fragmented relative to strain R. Notably, an R low isogenic mutant in one of these genes (MGA_1107) caused significantly fewer severe tracheal lesions in the natural host compared to virulent M. gallisepticum R low . Comparative genomic hybridizations indicated few genetic loci commonly affected in F and vaccine strains ts-11 and 6/85, which would correlate with proteins affecting strain R virulence. Together, these data provide novel insights into inter-and intrastrain M. gallisepticum genomic variability and the genetic basis of M. gallisepticum virulence.

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.Mycoplasma gallisepticum is the primary etiologic agent of the chronic respiratory disease complex in chickens and infectious sinusitis in turkeys. Primary inflammatory responses in the respiratory tract associated with infection are sinusitis, tracheitis, bronchitis, and airsacculitis. Colonization of the respiratory tract leads to ciliostasis and deciliation of the tracheal epithelium, allowing secondary infection by other bacterial and viral pathogens, such as Newcastle disease virus, infectious bronchitis virus, and Escherichia coli (24). Common signs of M. gallisepticum infection include nasal discharge, tracheal rales, weight loss, and decreased egg production. Mycoplasma gallisepticum is highly contagious in commercial chicken and turkey flocks, spreading horizontally in populations through aerosol, dust, and feathers and vertically transmitted through eggs (8,10,24,40). Its economic impact on the poultry industry is significant, leading to millions of dollars of loss each year. Efforts to improve disease prevention and control programs require increased research to identify novel virulenceassociated determinants. These determinants may provide the basis for new and promising vaccines or targets for other antimicrobial ...