Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.

The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the different Brucella species, Brucella melitensis is considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence in B. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the two B. melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up-or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.Brucellosis is a major infectious disease afflicting humans and a wide range of domesticated animals worldwide. The disease is caused by several Brucella species, which are aerobic, nonmotile, gram-negative, facultative intracellular coccobacilli. The genus Brucella belongs to the ␣-2 subgroup of the class Proteobacteria. It is subdivided, on the basis of its pathogenicity and host preference, into six nomen species: Brucella abortus, B. canis, B. melitensis, B. neotomae, B. ovis, and B. suis (12). In addition, a new strain affecting marine mammals was recently isolated and tentatively named B. maris (32). On the basis of DNA hybridization data, it was suggested that all of these organisms be placed into a single species, B. melitensis (39). Among the various nomen species, B. abortus, B. canis, B. suis, B. maris, and B. melitensis have been reported to infect humans (21,35,13). B. melitensis is a pathogen of goats and sheep and is considered the most virulent species for humans. Human infection can result from either occupational contact or ingestion of contaminated food.Vaccination and eradication of infected hosts have been key factors in the control of brucellosis. Rev 1, an attenuated strain of virulent B. melitensis, was developed in 1957 (19). It is considered the most effective vaccine for the control of brucellosis in small ruminants and was used in comprehensive vaccination programs in many countries, including Saudi Arabia, Kuwait, Mongolia, Spain, and Turkey (8).Our laboratory has been involved in a comprehensive analysis of the B. melitensis 16M proteome, and initial results have been published recently (40). Previous proteomics studies using B. melitensis cells grown under different conditions have been reported (36, 37), and initial work on the B. abortus proteome has been described (26, 29). A comparative study was conducted with B. abortus vaccine strains S19 and RB51 and...

Sulfite reductase (SiR) from Escherichia coli has a alpha 8 beta 4 subunit structure, where alpha 8 is a flavoprotein (SiR-FP) containing both FAD and FMN as prosthetic groups. It also exhibits a NADPH:flavin oxidoreductase activity with exogenous riboflavin, FMN, and FAD serving as substrates. The flavin reductase activity may function during activation of ribonucleotide reductase or during ferrisiderophore reduction. A plasmid containing cysJ gene, coding for the alpha subunit, overexpresses flavin reductase activity by 100-fold, showing that alpha is the site of free flavin reduction. The overproducer allows a fast and simple preparation of large amounts of the flavoprotein. Kinetic studies of its flavin reductase activity demonstrates a ping-pong bisubstrate-biproduct reaction mechanism. NADP+ inhibition studies show that both substrates, NADPH and free flavins, bind to the same site. While the FAD cofactor mediates the electron transfer between NADPH and free flavins, the FMN cofactor is not essential since a FMN-depleted SiR-FP retains a large proportion of activity. In contradiction with previous reports, SiR-FP is found to contain 1.6-1.7 flavin per alpha subunit. This result, together with the sequence homology between SiR-FP and NADPH-cytochrome P-450 reductase, suggests a new model for the structure of the protein with one FMN and one FAD prosthetic group per alpha subunit.

Bacillus anthracis, the causative agent of anthrax, secretes numerous proteins into the extracellular environment during infection. A comparative proteomic approach was employed to elucidate the differences among the extracellular proteomes (secretomes) of three isogenic strains of B. anthracis that differed solely in their plasmid contents. The strains utilized were the wild-type virulent B. anthracis RA3 (pXO1 ؉ pXO2 ؉ ) and its two nonpathogenic derivative strains: the toxigenic, nonencapsulated RA3R (pXO1 ؉ pXO2 ؊ ) and the totally cured, nontoxigenic, nonencapsulated RA3:00 (pXO1 ؊ pXO2 ؊ ). Comparative proteomics using two-dimensional gel electrophoresis followed by computer-assisted gel image analysis was performed to reveal unique, up-regulated, or down-regulated secretome proteins among the strains. In total, 57 protein spots, representing 26 different proteins encoded on the chromosome or pXO1, were identified by peptide mass fingerprinting. S-layer-derived proteins, such as Sap and EA1, were most frequently observed. Many sporulation-associated enzymes were found to be overexpressed in strains containing pXO1؉ . This study also provides evidence that pXO2 is necessary for the maximal expression of the pXO1-encoded toxins lethal factor (LF), edema factor (EF), and protective antigen (PA). Several newly identified putative virulence factors were observed; these include enolase, a high-affinity zinc uptake transporter, the peroxide stress-related alkyl hydroperoxide reductase, isocitrate lyase, and the cell surface protein A.Bacillus anthracis, the etiologic agent of anthrax, is a grampositive, rod-shaped, nonmotile facultative anaerobic and sporeforming bacterium (35). Anthrax is a disease of wildlife, livestock, and humans that begins when spores of B. anthracis enter a host, are engulfed by macrophages, germinate, and then cause septicemia. This pathogen's virulence, along with the spore's resistance to adverse environmental conditions and facility to be weaponized, has made it a significant agent of bioterrorism (1).In addition to a 5.2-Mb circular chromosome, fully virulent strains of B. anthracis harbor two plasmids: pXO1 and pXO2 (35). The pXO1 plasmid (181.6 kb) encodes the toxins lethal factor (LF) and edema factor (EF) and the protective antigen (PA) responsible for the translocation of EF and LF into the host's cytosol (35). The cascade of events leading to toxin entry into host cells has been described (7). The genes encoding LF, EF, and PA are designated lef, cya, and pagA, respectively (8,50,63). Proteins required for a proteinous capsule biosynthesis (CapBCA) and depolymerization (Dep) are encoded on pXO2 (94.8 kb) (29,60). Strains lacking either or both plasmids are attenuated in most animal hosts (22). The transcriptions of lef, cya, pagA, and capB have all been shown to be coordinately induced by the presence of bicarbonate-CO 2 , while temperature has been shown to be important for toxin, but not for CapB, production (55). High CO 2 tension is believed to simulate conditions encountere...