Mariner mutagenesis of Brucella melitensis reveals genes with previously uncharacterized roles in virulence and survival

Abstract

Background: Random gene inactivation used to identify cellular functions associated with virulence and survival of Brucella spp has relied heavily upon the use of the transposon Tn5 that integrates at G/C base pairs. Transposons of the mariner family do not require species-specific host factors for efficient transposition, integrate nonspecifically at T/A base pairs, and, at a minimum, provide an alternative approach for gene discovery. In this study, plasmid vector pSC189, containing both the hyperactive transposase C9 and transposon terminal inverted repeats flanking a kanamycin resistance gene, were used to deliver Himar1 transposable element into the B. melitensis genome. Conjugation was performed efficiently and rapidly in less than one generation in order to minimize the formation of siblings while assuring the highest level of genome coverage.

Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

“…2B). These data are in line with the significant attenuation of a B. melitensis mucR mutant described previously (23) and suggest that the link between MucR and virulence is a conserved relationship in Brucella strains.…”

“…1). The slow growth of the B. abortus mucR mutant is intriguing given that a B. melitensis mucR mutant does not have the same overt growth defect (data not shown) (23). Recent experiments do, however, indicate that a B. melitensis 16 M-derived mucR mutant has a more subtle growth defect than its B. abortus counterpart.…”

Brucella abortus Depends on Pyruvate Phosphate Dikinase and Malic Enzyme but Not on Fbp and GlpX Fructose-1,6-Bisphosphatases for Full Virulence in Laboratory Models

“…Genes (and their putative functions) identified in this manner include, in B. suis 1330, a gguA homologue (gluP, presumably involved in sugar uptake), some erythritol catabolism genes, gnd (6-phosphogluconate dehydrogenase, required for using glucose through the pentose phosphate pathway), rbsK (ribose kinase), pyc (anaplerotic pyruvate carboxylase), pgi (phosphoglucose isomerase), and genes related to the biosynthesis of amino acids (14)(15)(16); in B. melitensis 16M, dbsA and ugpA (ribose and glycerol-3-phosphate transport-ers, respectively), glpD (glycerol-3-phosphate dehydrogenase), and some erythritol catabolism genes (17,18); and in B. abortus 2308, gluP (previously shown to encode an active glucose and galactose transporter in this species [19]), gnd (6-phosphogluconate dehydrogenase), gltD (glutamate synthase), and gcvB (glycine dehydrogenase) (20). Also in B. abortus 2308, dxs (an isoprenoid biosynthesis transketolase) and mocC (rhizopine or inositol catabolism) were identified as expressed in macrophages using a fluorescent reporter (21).…”

Comparison of Genomes of Brucella melitensis M28 and the B. melitensis M5-90 Derivative Vaccine Strain Highlights the Translation Elongation Factor Tu Gene tuf2 as an Attenuation-Related Gene

“…A systematic review of the literature by Crasta et al (17) tabulated 236 potential virulence factors and an additional 53 genes previously implicated in Brucella virulence (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). Of these 289 potential virulence factors, only BM28_A0655 (encoding a soluble lytic murein transglycosylase), which is involved in amino sugar metabolism and N-glycan biosynthesis, showed consistent differences between M5-90 and the three virulent strains (28). Other prominent virulence factors in Brucella spp., including urease (ure), cytochrome oxidase (cydDCAB), nitric oxide reductase (norD), superoxide dismutase (sod), the type IV secretion system (T4SS), Brucella virulence factor A (bvfA), and a two-component regulatory system (bvrS-bvrR), are all intact in the M5-90 genome (7).…”

“…We believe that systematic modification of these other functions, in conjunction with tuf2 mutants of M28, and host-response studies will together provide a comprehensive account of the attenuation of the M5-90 vaccine strain. A systematic review of the literature by Crasta et al (17) tabulated 236 potential virulence factors and an additional 53 genes previously implicated in Brucella virulence (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). Of these 289 potential virulence factors, only BM28_A0655 (encoding a soluble lytic murein transglycosylase), which is involved in amino sugar metabolism and N-glycan biosynthesis, showed consistent differences between M5-90 and the three virulent strains (28).…”