Increasing concentrations of competitor lipid (diluted from a 10?mM stock in absolute ethanol) were added to the MilA/1,8-ANS complex, the solution mixed for 30 s, incubated again for 5?min, and fluorescence then measured. (P226) protein, encoded by (the full-length product of which has a predicted molecular weight of 303?kDa), had lipase activity. The predicted sequence of MilA contains glycosaminoglycan binding motifs, as well as multiple copies of a domain of unknown function (DUF445) that is also found in apolipoproteins. We mutagenized the gene to facilitate expression of a series of regions spanning the gene in contributes to the bovine respiratory disease complex and poses a significant threat to livestock production worldwide (1, 2), resulting in its inclusion in the EU-funded DISCONTOOLS project in their disease database (https://www.discontools.eu/database.html) (3). Clinical disease caused by includes mastitis, arthritis, genital disorders, keratoconjunctivitis, and otitis media (4, 5). The survival, replication, and virulence of mycoplasmas depend to a large extent on their ability to colonize and, as a consequence of their limited biosynthetic capabilities, to capture and import host-derived nutrients. The acquisition of nutrients by this important group of pathogens depends on a number of poorly characterized membrane-associated proteins. In spite of the considerable increase in genome sequence data over the past decade, many mycoplasma proteins remain hypothetical. Many predicted mycoplasma gene products, other than those involved in housekeeping functions, have little or no detectable sequence similarity to those characterized in other bacteria (6), limiting our capacity to extrapolate gene function from studies in other bacteria. Improved understanding of the molecular pathogenesis of has been identified as one of the crucial gaps in understanding about this pathogen (12). These functional MJN110 studies are likely to require concern of the possibility that many of them will play a number of roles during contamination, as the evolutionary forces that have driven the reduction in the genome of the mycoplasmas are likely to have limited functional redundancy and selected for acquisition of multifunctionality in proteins. Some progress has been made in predicting gene function in mycoplasmas by examining metabolomic differences between wild-type organisms and mutants in which specific genes have been disrupted by insertion of a transposon (13,C15). Functional prediction methods can complement primary sequence annotations. While computational three-dimensional protein structural modeling has limitations, it can suggest biochemical functions and/or biological functions of a protein about which relatively little is known (16). As many of the proteins of have no known function and many have homology only with other mycoplasma proteins of unknown function, structural modeling and comparisons using the Protein Data Lender (PDB) structural database may provide hints about their functions, providing a starting point for functional studies on purified proteins. The gene (MBOVPG45_0710) of encodes a very large membrane protein that was shown in previous studies to be detected by antibodies from experimentally infected calves (17). The NR4A2 amino terminal end of the protein was shown to have lipase activity, but the functions of the carboxyl 75% of MilA have not been investigated. While it is likely that this role of the remainder of the protein may be linked to the lipase activity that was detected previously (17), given the prevalence of proteins with moonlighting functions in mycoplasmas it is appropriate to consider the possibility of other activities as well. Previous studies aimed at determining the dispensability of genes in using transposon mutagenesis did not detect any mutants in which was disrupted MJN110 (18, 19), suggesting that this gene may be essential in (18). The aim of this study was to further investigate the function of MilA, initially by using bioinformatic approaches to identify potential functional domains and predict likely activities. These predicted activities were explored by expressing different regions of the full-length protein in and assessing their biochemical function and its homologues. The coding sequence of the gene is located between nucleotides 814575 and 822587 in the genome of strain PG45 (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”CP002188″,”term_id”:”312950072″,”term_text”:”CP002188″CP002188). The region upstream of contains a gene that has not been assigned a function (MBOVPG45_0709, a hypothetical protein), while the region downstream has a putative cluster. The physicochemical and other features of MilA were predicted using ProtParam (ExPASy). MilA had a predicted molecular weight of 303?kDa and an isoelectric point of 8.71, MJN110 while its closest MJN110 relative, the product of MAG_6100 of (168?kDa) and (174?kDa), and isoelectric points greater than 8.0, with the exception of those in (4.66), (5.07), (5.12), (5.93) and (5.97). Open in a separate windows FIG 1 Physical map of PG45 gene lies downstream of and and is followed by a pseudogene.
Increasing concentrations of competitor lipid (diluted from a 10?mM stock in absolute ethanol) were added to the MilA/1,8-ANS complex, the solution mixed for 30 s, incubated again for 5?min, and fluorescence then measured