Cloning and characterization of the glucokinase gene of Brucella abortus 19 and identification of three other genes.
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ABSTRACT:
SUBMITTER: Essenberg RC
PROVIDER: S-EPMC177474 | biostudies-other | 1995 Nov
REPOSITORIES: biostudies-other
Publications
Cloning and characterization of the glucokinase gene of Brucella abortus 19 and identification of three other genes.
Journal of bacteriology 19951101 21
A clone from Brucella abortus 19 complemented an Escherichia coli strain deficient in phosphorylation of glucose. Open reading frames similar to E. coli mepA, glk, and genes encoding ATP-coupled exporters were found in the sequence. A fourth affected growth on minimal media of the ptsI glk strain with various carbon sources. ...[more]
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Project description:Elucidating the function of proteins <50 amino acids in length is no small task. Nevertheless, small proteins can play vital roles in the lifestyle of bacteria and influence the virulence of pathogens; thus, the investigation of the small proteome is warranted. Recently, our group identified the Brucella abortus protein VtlR as a transcriptional activator of four genes, one of which is the well-studied small regulatory RNA AbcR2, while the other three genes encode hypothetical small proteins, two of which are highly conserved among the order Rhizobiales This study provides evidence that all three genes encode authentic small proteins and that all three are highly expressed under oxidative stress, low-pH, and stationary-phase growth conditions. Fractionation of the cells revealed that the proteins are localized to the membranes of B. abortus We demonstrate that the small proteins under the transcriptional control of VtlR are not accountable for attenuation observed with the B. abortusvtlR deletion strain. However, there is an association between VtlR-regulated genes and growth inhibition in the presence of the sugar l-fucose. Subsequent transcriptomic analyses revealed that B. abortus initiates the transcription of a locus encoding a putative sugar transport and utilization system when the bacteria are cultured in the presence of l-fucose. Altogether, our observations characterize the role of the VtlR-controlled small proteins BAB1_0914, BAB2_0512, and BAB2_0574 in the biology of B. abortus, particularly in the capacity of the bacteria to utilize l-fucose.IMPORTANCE Despite being one of the most common zoonoses worldwide, there is currently no human vaccine to combat brucellosis. Therefore, a better understanding of the pathogenesis and biology of Brucella spp., the causative agent of brucellosis, is essential for the discovery of novel therapeutics against these highly infectious bacteria. In this study, we further characterize the virulence-associated transcriptional regulator VtlR in Brucella abortus Our findings not only shed light on our current understanding of a virulence related genetic system in Brucella spp. but also increase our knowledge of small proteins in the field of bacteriology.
| S-EPMC6112010 | biostudies-literature
Project description:The animal pathogen Brucella abortus contains a gene cgt, which complemented Sinorhizobium meliloti nodule development (ndvA) and Agrobacterium tumefaciens chromosomal virulence (chvA) mutants. Complemented strains recovered the presence of anionic cyclic beta-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic beta-1,2-glucan in the periplasm. Nucleotide sequencing revealed that B. abortus cgt contains a 1,797-bp open reading frame coding for a predicted membrane protein of 599 amino acids (65.9 kDa) that is 58.5 and 59.9% identical to S. meliloti NdvA and A. tumefaciens ChvA, respectively. Additionally, B. abortus cgt, like S. meliloti ndvA and A. tumefaciens chvA possesses ATP-binding motifs and the ABC signature domain features of a typical ABC transporter. Characterization of Cgt was carried out by the construction of null mutants in B. abortus 2308 and S19 backgrounds. Both mutants do not transport cyclic beta-1,2-glucan to the periplasm, as shown by the absence of anionic cyclic glucan, and they display reduced virulence in mice and defective intracellular multiplication in HeLa cells. These results suggest that cyclic beta-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor.
| S-EPMC375185 | biostudies-literature
Project description:The genetic basis for chronic persistence of Brucella abortus in lymphoid organs of mice, cows, and humans is currently unknown. We identified B. abortus genes involved in chronic infection, by assessing the ability of 178 signature-tagged mutants to establish and maintain persistent infection in mice. Each mutant was screened for its ability to colonize the spleens of mice at 2 and 8 weeks after inoculation. Comparison of the results from both time points identified two groups of mutants attenuated for chronic infection in mice. The first group was not recovered at either 2 or 8 weeks postinfection and was therefore defective in establishing infection. Mutants in this group carried transposon insertions in genes involved in lipopolysaccharide biosynthesis (wbkA), in aromatic amino acid biosynthesis, and in type IV secretion (virB1 and virB10). The second group, which was recovered at wild-type levels 2 weeks postinfection but not 8 weeks postinfection was able to establish infection but was unable to maintain chronic infection. One mutant in this group carried a transposon insertion in a gene with homology to gcvB of Mycobacterium tuberculosis, encoding glycine dehydrogenase, an enzyme whose activity is increased during the state of nonreplicating persistence. These results suggest that some mechanisms for long-term persistence may be shared among chronic intracellular pathogens. Furthermore, identification of two groups of genes, those required for initiating infection and those required only for long-term persistence, suggests that B. abortus uses distinct sets of virulence determinants to establish and maintain chronic infection in mice.
| S-EPMC101704 | biostudies-literature
Project description:Smooth lipopolysaccharide (LPS) of Brucella abortus has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. While the protective properties of LPS against complement are well accepted, there is still some controversy about the capacity of rough mutants to replicate intracellularly. The B. abortus phosphoglucomutase gene (pgm) was cloned, sequenced, and disrupted. The gene has a high index of identity to Agrobacterium tumefaciens pgm but is not part of the glycogen operon. A B. abortus null mutant lacks LPS O antigen but has an LPS core with an electrophoretic profile undistinguishable from that of the wild-type core, suggesting that glucose, galactose, or a derivative of these sugars may be part of the linkage between the core and the O antigen. This mutant is unable to survive in mice but replicates in HeLa cells, indicating that the complete LPS is not essential either for invasion or for intracellular multiplication. This behavior suggests that the LPS may play a role in extracellular survival in the animal, probably protecting the cell against complement-mediated lysis, but is not involved in intracellular survival.
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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.
| S-EPMC8845251 | biostudies-literature
Project description:The aim of this work is to unveil the gene regulatory networks - crucial for the different stages of intracellular survival - in the bovine and zoonotic pathogen Brucella abortus.These data are part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
2015-03-17 | E-ERAD-333 | biostudies-arrayexpress
Project description:Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus.
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Project description:Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp. Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B. melitensis 16M and Brucella abortus 2308. Analysis of 45 SSH clones revealed several deletions on chromosomes of B. abortus and B. melitensis that encoded proteins of various metabolic pathways. A 640-kb inversion on chromosome II of B. abortus has been reported previously (S. Michaux Charachon, G. Bourg, E. Jumas Bilak, P. Guigue Talet, A. Allardet Servent, D. O'Callaghan, and M. Ramuz, J. Bacteriol. 179:3244-3249, 1997) and is further described in this study. One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293. The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp. As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region. The splitting of the PDEA gene most likely inactivated the function of this enzyme. A recombination mechanism responsible for this inversion is proposed.
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Project description:BackgroundGlucokinase (GK) is a metabolic enzyme encoded by the GCK gene and expressed in glucose-sensitive tissues, principally pancreatic islets cell and hepatocytes. The GK protein acts in pancreatic islets as a "glucose sensor" that couples fluctuations in the blood glucose concentration to changes in cellular function and insulin secretion. GCK and GK have proposed importance in the development and progression of diabetes mellitus and are potential therapeutic targets for diabetes treatment. The study was undertaken to determine the nucleotide sequence of feline pancreatic GK cDNA, predict the amino acid sequence and structure of the feline GK protein, and perform comparative bioinformatic analysis of feline cDNA and protein. Routine PCR techniques were used with cDNA from feline pancreas. Clones were assembled to obtain the full length cDNA. Protein prediction and modeling were performed using bioinformatic tools.ResultsFull-length feline pancreatic GK cDNA contains a 1398 nucleotide coding sequence with high identity to other pancreatic GK cDNAs. The deduced 465 amino acid feline protein has 15 amino acid substitutions not found in other mammalian GK proteins but maintains high structural homology with human GK. Feline pancreatic GK is highly conserved at nucleotide and protein levels. Residues crucial for substrate binding and catalysis are completely conserved in the feline protein.ConclusionMolecular analysis predicts that feline pancreatic GK functions similarly to other mammalian GK proteins.
| S-EPMC4066705 | biostudies-literature
Project description:Brucella spp. is a species of facultative intracellular Gram-negative bacteria that induces abortion and causes sterility in domesticated mammals and chronic undulant fever in humans. Important determinants of Brucella's virulence and potential for chronic infection include the ability to circumvent the host cell's internal surveillance system and the capability to proliferate within dedicated and non-dedicated phagocytes. Hence, identifying genes necessary for intracellular survival may hold the key to understanding Brucella infection. In the present study, microarray analysis reveals that 7.82% (244/3334) of all Brucella abortus genes were up-regulated and 5.4% (180/3334) were down-regulated in RAW264.7 cells, compared to free-living cells in TSB. qRT-PCR verification further confirmed a >5-fold up-regulation for fourteen genes. Functional analysis classified araC, ddp, and eryD as to partake in information storage and processing, alp, flgF and virB9 to be involved in cellular processes, hpcd and aldh to play a role in metabolism, mfs and nikC to be involved in both cellular processes and metabolism, and four hypothetical genes (bruAb1_1814, bruAb1_0475, bruAb1_1926, and bruAb1_0292) had unknown functions. Furthermore, we constructed a B. abortus 2308 mutant ?ddp where the ddp gene is deleted in order to evaluate the role of ddp in intracellular survival. Infection assay indicated significantly higher adherence and invasion abilities of the ?ddp mutant, however it does not survive well in RAW264.7 cells. Brucella may survive in hostile intracellular environment by modulating gene expression.
| S-EPMC3737221 | biostudies-literature