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Genetic organization and iron-responsive regulation of the Brucella abortus 2,3-dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle.

ABSTRACT: Brucella abortus reportedly produces the monocatechol siderophore 2,3-dihydroxybenzoic acid (2,3-DHBA) in response to iron limitation. Nucleotide sequence analysis of the cloned DHBA biosynthesis locus from virulent B. abortus 2308 and genetic complementation of defined Escherichia coli mutants were used to identify the B. abortus genes (designated dhbC, -B, and -A) responsible for synthesis of this siderophore. Reverse transcriptase PCR analysis of total RNA with dhb-specific primers demonstrated that dhbC, -B, and -A are transcribed as components of an operon, together with dhbE, a functional homolog of the Escherichia coli entE gene. Homologs of the E. coli entD and Vibrio cholerae vibH genes were also detected in the flanking regions immediately adjacent to the B. abortus dhbCEBA operon, suggesting that B. abortus has the genetic capacity to produce a more complex 2,3-DHBA-based siderophore. Slot blot hybridization experiments and primer extension analysis showed that transcription of the B. abortus dhbCEBA operon originates from two iron-regulated promoters located upstream of dhbC. Consistent with their iron-dependent regulation, both of the dhbCEBA promoter sequences contain typical consensus Fur-binding motifs. Although previously published studies have shown that 2,3-DHBA production is not required for the establishment and maintenance of chronic spleen infection by B. abortus in mice, experimental infection of pregnant cattle with the B. abortus dhbC mutant BHB1 clearly showed that production of this siderophore is essential for wild-type virulence in the natural ruminant host.

SUBMITTER: Bellaire BH

PROVIDER: S-EPMC152065 | biostudies-literature | 2003 Apr

REPOSITORIES: biostudies-literature

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Genetic organization and iron-responsive regulation of the Brucella abortus 2,3-dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle.

Bellaire Bryan H BH Elzer Philip H PH Hagius Sue S Walker Joel J Baldwin Cynthia L CL Roop R Martin RM

Infection and immunity 20030401 4

Brucella abortus reportedly produces the monocatechol siderophore 2,3-dihydroxybenzoic acid (2,3-DHBA) in response to iron limitation. Nucleotide sequence analysis of the cloned DHBA biosynthesis locus from virulent B. abortus 2308 and genetic complementation of defined Escherichia coli mutants were used to identify the B. abortus genes (designated dhbC, -B, and -A) responsible for synthesis of this siderophore. Reverse transcriptase PCR analysis of total RNA with dhb-specific primers demonstrat ...[more]

PMID: 12654793

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Project description:BackgroundAzerbaijan currently ranks thirteenth in global incidence of human brucellosis, with an estimated annual incidence through 2000 at over 50 cases per million. Brucella melitensis has been isolated from patients and is thought to have been acquired through contact with small ruminants or as a foodborne infection. To reduce the burden of human brucellosis, the Azerbaijani government began in 2002, a nationwide vaccination control campaign in small ruminants. There is serological evidence of bovine brucellosis (presumably due to Brucella abortus) in Azerbaijan, but no prevalence estimates were available when this study started in March 2017. The aim of this study was to isolate and identify Brucella spp. from cow milk in the Ganja region, where brucellosis takes a heavy toll on humans and livestock.ResultsBlood and milk samples were collected from cows (n = 1075) in early lactation (up to 90-days) in farms that had a history of previous positive serological results and abortions. Twenty-two out of 57 milk samples collected from seropositive cows, showed growth on Farrell's media, when incubated with 5% CO2. Eight additional milk samples showed growth in the absence of CO2. The classical biotyping classified them as Brucella abortus (22) and Brucella melitensis (8). RT-PCR confirmed that strains belonged to the genus Brucella. MLVA profiles were obtained for DNA extracted from two B. abortus and six B. melitensis strains. While the B. abortus genetic profile was described in the MLVA database, matching the profile of B. abortus strains isolated in East Europe, Central Asia and China, we found a new genotype for the B. melitensis strains isolated in Azerbaijan, clustering with strains belonging to the American clade, rarely identified in the region.ConclusionDespite the implementation of the vaccination program in small ruminants, our results suggest that spill-over events of B. melitensis from small ruminants to cattle have occurred. However, cattle are likely to be primarily infected with B. abortus, which warranted the implementation of a bovine brucellosis program. Such a program started in fall 2017. In the Ganja region, cattle should be considered as a potential source of B. abortus and B. melitensis for humans.

Project description:The YbeY endoribonuclease is one of the best-conserved proteins across the kingdoms of life. In the present study, we demonstrated that YbeY in Brucella abortus is linked to a variety of important activities, including proper cellular morphology, mRNA transcript levels, and virulence. Deletion of ybeY in B. abortus led to a small-colony phenotype when the bacteria were grown on agar medium, as well as to significant aberrations in the morphology of the bacterial cell as evidenced by electron microscopy. Additionally, compared to the parental strain, the ΔybeY strain was significantly attenuated in both macrophage and mouse models of infection. The ΔybeY strain also showed increased sensitivities to several in vitro-applied stressors, including bile acid, hydrogen peroxide, SDS, and paraquat. Transcriptomic analysis revealed that a multitude of mRNA transcripts are dysregulated in the ΔybeY strain, and many of the identified mRNAs encode proteins involved in metabolism, nutrient transport, transcriptional regulation, and flagellum synthesis. We subsequently constructed gene deletion strains of the most highly dysregulated systems, and several of the YbeY-linked gene deletion strains exhibited defects in the ability of the bacteria to survive and replicate in primary murine macrophages. Taken together, these data establish a clear role for YbeY in the biology and virulence of Brucella; moreover, this work further illuminates the highly varied roles of this widely conserved endoribonuclease in bacteria.IMPORTANCEBrucella spp. are highly efficient bacterial pathogens of animals and humans, causing significant morbidity and economic loss worldwide, and relapse of disease often occurs following antibiotic treatment of human brucellosis. As such, novel therapeutic strategies to combat Brucella infections are needed. Ribonucleases in the brucellae are understudied, and these enzymes represent elements that may be potential targets for future treatment approaches. The present work demonstrates the importance of the YbeY endoribonuclease for cellular morphology, efficient control of mRNA levels, and virulence in B. abortus Overall, the results of this study advance our understanding of the critical roles of YbeY in the pathogenesis of the intracellular brucellae and expand our understanding of this highly conserved RNase.

Dataset Information

Genetic organization and iron-responsive regulation of the Brucella abortus 2,3-dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle.

Publications

Genetic organization and iron-responsive regulation of the Brucella abortus 2,3-dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle.

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