Eubacterial transducers are transmembrane, methyl-accepting proteins central to chemotaxis systems and share common structural features. We identified a large family of transducer proteins in the Archaeon Halobacterium salinarium using a site-specific multiple antigenic peptide antibody raised against 23 amino acids, representing the highest homology region of eubacterial transducers. This immunological observation was confirmed by isolating 13 methyl-accepting taxis genes using a 27-mer oligonucleotide probe, corresponding to conserved regions between the eubacterial and first halobacterial phototaxis transducer gene htrI. On the basis of the comparison of the predicted structural domains of these transducers, we propose that at least three distinct subfamilies of transducers exist in the Archaeon H. salinarium: (i) (SRI and SRII), mediate "color vision" (4). Both are retinalcontaining proteins distinct from the retinal-based, lightdriven ion pumps bacteriorhodopsin and halorhodopsin (5-7). SRI is the photoreceptor for green-red and UV light stimuli, and SRII is the photoreceptor for blue light (7-10). Even in the absence of the dedicated sensory rhodopsins, proton pumping by bacteriorhodopsin generates phototaxis responses, probably because of effects on membrane potential or proton motive force (11,12).We and others (13-15) identified a family of methylaccepting taxis proteins with (apparent) molecular masses between 90 and 135 kDa as components of both the photosensory and chemosensory systems in H. salinarium. These proteins are altered in levels of methylation by positive and negative chemostimuli (13) To identify and isolate other transducers involved in halobacterial signal transduction, we used immunological, genetic, and biochemical approaches. This paper describes for the first time the identification and isolation of all the methylaccepting taxis genes, and the localization of their products in the Archaeon H. salinarium.
Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.
We have previously reported the purification of rats testis galactosyl receptor, an equivalent to the Ca(2+)-dependent (C-type) minor variant of rat hepatic lectin-2/3 (RHL-2/3). We now report the purification of galactosyl receptor from rat sperm and its immunolocalization in the intact rat testis and sperm by polyclonal antibodies prepared using multiple antigen peptides (MAP) as immunogens. Two MAP antigens (designated 27-mer and 28-mer), corresponding to amino acid sequences of the carbohydrate-recognition domain (galactose) and adjacent Ca(2+)-binding sites of RHL-2/3, were used for immunization. Anti-RHL-2/3, anti-p27, and anti-p28 sera crossreacted with rat hepatocyte RHL-2/3 and its rat testis and sperm equivalent, galactosyl receptor, purified by chromatofocusing followed by galactose-Hydropore-EP affinity chromatography. Neither anti-p27 nor anti-p28 sera cross-reacted with the major hepatocyte variant, RHL-1. A RHL-1-equivalent was not detected in rat testis and sperm. Immunofluorescence studies demonstrated that anti-p27 and anti-p28 sera recognize galactosyl receptor sites at the Sertoli cell-spermatogenic cell interface and on the dorsal surface of the sperm head, overlying the acrosome. The characteristic crescent-shaped immunoreactive pattern in sperm was lost after induction of the acrosome reaction. Further studies should determine whether antisera to MAP antigens 27-mer and 28-mer, corresponding to specific protein motifs, can serve as immunological probes for examining cell-cell interaction events during spermatogenesis and at fertilization.
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