A type IV secretion system similar to the VirB system of the phytopathogen Agrobacterium tumefaciens is essential for the intracellular survival and multiplication of the mammalian pathogen Brucella. Reverse transcriptase-PCR showed that the 12 genes encoding the Brucella suis VirB system form an operon. Semiquantitative measurements of virB mRNA levels by slot blotting showed that transcription of the virB operon, but not the flanking genes, is regulated by environmental factors in vitro. Flow cytometry used to measure green fluorescent protein expression from the virB promoter confirmed the data from slot blots. Fluorescence-activated cell sorter analysis and fluorescence microscopy showed that the virB promoter is induced in macrophages within 3 h after infection. Induction only occurred once the bacteria were inside the cells, and phagosome acidification was shown to be the major signal inducing intracellular expression. Because phagosome acidification is essential for the intracellular multiplication of Brucella, we suggest that it is the signal that triggers the secretion of unknown effector molecules. These effector molecules play a role in the remodeling of the phagosome to create the unique intracellular compartment in which Brucella replicates.

Bacteria of the genusTransposon mutagenesis is the most frequently used approach in the identification of genes involved in the virulence of bacterial pathogens (28). Classical transposon mutagenesis strategies are limited by the fact that each mutant must be tested individually to identify an attenuated phenotype. For example, Fields et al. tested 10,000 Salmonella enterica serovar Typhimurium Tn10 mutants individually to obtain less than 100 attenuated mutants (21). The use of modified transposons which generate active fusions with envelope proteins often essential for bacterial virulence, such as TnphoA or TnblaM, has been adopted in an attempt to increase the chances of identifying virulence factors (24,42). Recently, a modification of the classical transposon mutagenesis technique, called signature-tagged transposon mutagenesis (STM), has been described in which the transposon in each mutant carries a unique 80-bp DNA tag which has a variable central portion and constant flanking regions (29). The unique tag allows the detection of a given mutant within a complex pool of mutants by hybridization with a probe obtained by PCR with primers based on the constant regions. Animals are infected with a pool of mutants and, at an appropriate time after infection, the surviving bacteria are recovered. The tags in the recovered bacteria are amplified and labeled by PCR and then used to probe an array of all the tags present in the inoculum. Mutants with attenuated virulence are identified as those whose tags are absent (not amplified) from the bacteria recovered from the infected animal. This system was originally used to identify genes involved in the virulence of serovar Typhimurium, including the Salmonella pathogenicity island 2 (29, 52). Brucella is a small gram-negative, facultative intracellular pathogen that infects animals and humans, inducing abortion in pregnant susceptible hosts and producing chronic infections with recurrent bacteremia (31). The physiopathology of brucellosis remains poorly understood. Brucella spp. infect and multiply within both professional and nonprofessional phagocytes. As with many intracellular pathogens, Brucella seems to use phagocytes as vehicles to disseminate within the organism, allowing it to colonize organs of the reticuloendothelial system and the trophoblasts in the placenta (49). Brucella invades cells, blocks phagosome-lysosome fusion, and replicates within a novel intracellular membrane-bound compartment (14, 45). Very little is known about the genetic basis of Brucella virulence; stress response proteins (20,33,35) and smooth lipopolysaccharide (LPS) (25) have been shown to be required for virulence in in vitro and animal models. Recently, a two-component system, BvrAS (57), and a type IV secretion system, VirB (42), have been identified as essential virulence factors. We have used STM to identify new Brucella virulence factors. STM technology has so far been applied essentially to animal virulence models (8,28). In this study, we report a further application of STM, ...

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PacI and SpeI restriction maps were obtained for the two chromosomes of each of the six species of the genus Brucella: B. melitensis, B. abortus, B. suis, B. canis, B. ovis, and B. neotomae. Three complementary techniques were used: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping method. For each type strain, a unique I-SceI site was introduced in each of the two replicons, and the location of SpeI sites was determined by linearization at the unique site, partial digestion, and end labeling of the fragments. The restriction and genetic maps of the six species were highly conserved. However, numerous small insertions or deletions, ranging from 1 to 34 kb, were observed by comparison with the map of the reference strain of the genus, B. melitensis 16M. A 21-kb SpeI fragment specific to B. ovis was found in the small chromosome of this species. A 640-kb inversion was demonstrated in the B. abortus small chromosome. All of these data allowed the construction of a phylogenetic tree, which reflects the traditional phenetic classification of the genus.Brucella is a small gram-negative bacterium pathogenic for animals and occasionally for humans. The genus is divided into six species on the basis of cultural, metabolic, and antigenic characteristics: B. melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae (6). However, it was shown by DNA-DNA hybridization that the genus is more probably monospecific (42). In a previous work (1), we found that each of the socalled species exhibited a specific macrorestriction pattern by digestion of genomic DNA with XbaI, with only minor variations between biovars within a species. Recently, a physical map was proposed for the genus type strain, B. melitensis 16M, by a cross-hybridization method (30). We showed that the Brucella genome was constituted by two circular chromosomes with sizes of about 2.05 and 1.15 Mb.In this study, we established the PacI and SpeI restriction maps of the two chromosomes of the other reference strains in the genus Brucella: B. abortus 544, B. suis 1330, B. canis 62/290, B. ovis 63/290, and B. neotomae 5K33. Three different but complementary techniques were employed: hybridization with the two replicons as probes, cross-hybridization of restriction fragments, and a new mapping strategy using I-SceI. I-SceI is an endonuclease encoded by a group I intron of the Saccharomyces cerevisiae mitochondrial 21S rRNA gene (31). Recently, by using artificially inserted I-SceI sites, a physical map of chromosome XI of S. cerevisiae (40) and a physical map of YAC inserts (5) were made. Since bacterial genomes lack natural I-SceI sites, we showed that the introduction of a unique restriction site recognized by I-SceI could be a useful tool for the study of the genome organization (20). In this study, we used an indirect end-labeling method to localize the SpeI sites, derived from a previously described procedure involving partial digestion of linearized DNA, followed by Southern blotting and h...

Analysis of the entire Agrobacterium tumefaciens C58 genome by pulsed-field gel electrophoresis (PFGE) reveals four replicons: two large molecules of 3,000 and 2,100 kb, the 450-kb cryptic plasmid, and the 200-kb Ti plasmid. Digestion by PacI or SwaI generated 12 or 14 fragments, respectively. The two megabase-sized replicons, used as probes, hybridize with different restriction fragments, showing that these replicons are two independent genetic entities. A 16S rRNA probe and genes encoding functions essential to the metabolism of the organism were found to hybridize with both replicons, suggesting their chromosomal nature. In PFGE, megabase-sized circular DNA does not enter the gel. The 2.1-Mb chromosome always generated an intense band, while the 3-Mb band was barely visible. After linearization of the DNA by X-irradiation, the intensity of the 3-Mb band increased while that of the 2.1-Mb remained constant. This suggests that the 3-Mb chromosome is circular and that the 2.1-Mb chromosome is linear. To confirm this hypothesis, genomic DNA, trapped in an agarose plug, was first submitted to PFGE to remove any linear DNA present. The plug was then recovered, and the remaining DNA was digested with either PacI or SwaI and then separated by PFGE. The fragments corresponding to the small chromosome were found to be absent, while those corresponding to the circular replicon remained, further proof of the linear nature of the 2.1-Mb chromosome.