scholarly journals Molecular Cloning and Characterization of cgt, the Brucella abortus Cyclic β-1,2-Glucan Transporter Gene, and Its Role in Virulence

2004 ◽  
Vol 72 (4) ◽  
pp. 2263-2271 ◽  
Author(s):  
Mara S. Roset ◽  
Andrés E. Ciocchini ◽  
Rodolfo A. Ugalde ◽  
Nora Iñón de Iannino
Keyword(s):  
Brucella Abortus ◽  
Sinorhizobium Meliloti ◽  
Nodule Development ◽  
Nucleotide Sequencing ◽  
Nodule Occupancy ◽  
Transporter Gene ◽  
Binding Motifs ◽  
Reading Frame ◽  
Intracellular Multiplication

ABSTRACT 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 β-1,2-glucan, motility, tumor induction in A. tumefaciens, and nodule occupancy in S. meliloti, all traits strictly associated with the presence of cyclic β-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 β-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 β-1,2-glucan must be transported into the periplasmatic space to exert its action as a virulence factor.

scholarly journals Molecular Cloning and Characterization of cgs, theBrucella abortus Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB andAgrobacterium tumefaciens chvB Mutants

1998 ◽  
Vol 180 (17) ◽  
pp. 4392-4400 ◽  
Author(s):  
Nora Iñón de Iannino ◽  
Gabriel Briones ◽  
Marcelo Tolmasky ◽  
Rodolfo A. Ugalde
Keyword(s):  
Amino Acids ◽  
Membrane Protein ◽  
Rhizobium Meliloti ◽  
Nodule Development ◽  
Nucleotide Sequencing ◽  
Insertion Mutant ◽  
Reading Frame ◽  
Glucan Synthesis ◽  
Glucan Synthetase

ABSTRACT The animal pathogen Brucella abortus contains a gene,cgs, that complemented a Rhizobium melilotinodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved inRhizobium, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor inBrucella infection.

Gene Cloning, Nucleotide Sequencing, and Purification and Characterization of the Low-Specificityl-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

1998 ◽  
Vol 64 (2) ◽  
pp. 549-554 ◽  
Author(s):  
Ji-Quan Liu ◽  
Saeko Ito ◽  
Tohru Dairi ◽  
Nobuya Itoh ◽  
Michihiko Kataoka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Significant Loss ◽  
Site Directed Mutagenesis ◽  
Nucleotide Sequencing ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Reading Frame ◽  
Threonine Aldolase

ABSTRACT A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia colicells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictlyl specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, includingl-β-3,4-dihydroxyphenylserine,l-β-3,4-methylenedioxyphenylserine, andl-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificityl-TA from Saccharomyces cerevisiae,l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of thel-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.

scholarly journals Cloning and Characterization of a Rhizobium leguminosarum Gene Encoding a Bacteriocin with Similarities to RTX Toxins

1999 ◽  
Vol 65 (7) ◽  
pp. 2833-2840 ◽  
Author(s):  
Ivan J. Oresnik ◽  
Sunny Twelker ◽  
Michael F. Hynes
Keyword(s):  
Amino Acid ◽  
Sinorhizobium Meliloti ◽  
Insertional Mutagenesis ◽  
Rhizobium Leguminosarum ◽  
Nodule Occupancy ◽  
Bacteriocin Activity ◽  
Gene Encoding ◽  
Reading Frame ◽  
Rtx Toxins ◽  
Culture Supernatants

ABSTRACT A 3-kb region containing the determinant for bacteriocin activity from Rhizobium leguminosarum 248 was isolated and characterized by Tn5 insertional mutagenesis and DNA sequencing. Southern hybridizations showed that this bacteriocin was encoded on the plasmid pRL1JI and that homologous loci were not found in other unrelated R. leguminosarum strains. Tn5 insertional mutagenesis showed that mutations in the C-terminal half of the bacteriocin open reading frame apparently did not abolish bacteriocin activity. Analysis of the deduced amino acid sequence revealed that, similarly to RTX proteins (such as hemolysin and leukotoxin), this protein contains a characteristic nonapeptide repeated up to 18 times within the protein. In addition, a novel 19- to 25-amino-acid motif that occurred every 130 amino acids was detected. Bacteriocin bioactivity was correlated with the presence of a protein of approximately 100 kDa in the culture supernatants, and the bacteriocin bioactivity demonstrated a calcium dependence in bothR. leguminosarum and Sinorhizobium meliloti. A mutant of strain 248 unable to produce this bacteriocin was found to have a statistically significant reduction in competitiveness for nodule occupancy compared to two test strains in coinoculation assays. However, this strain was unable to compete any more successfully with a third test strain, 3841, than was wild-type 248.

scholarly journals Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development

2021 ◽  
Author(s):  
Paolo M Triozzi ◽  
Thomas B Irving ◽  
Henry W Schmidt ◽  
Zachary P Keyser ◽  
Sanhita Chakraborty ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Nodule Development ◽  
Symbiotic Association ◽  
Loss Of Function ◽  
Tissue Specific ◽  
Temporal Regulation ◽  
Positive Regulators ◽  
Sequential Activation

Abstract Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

scholarly journals A Highly Conserved Protein of Unknown Function Is Required by Sinorhizobium meliloti for Symbiosis and Environmental Stress Protection

2007 ◽  
Vol 190 (3) ◽  
pp. 1118-1123 ◽  
Author(s):  
Bryan W. Davies ◽  
Graham C. Walker
Keyword(s):  
Environmental Stress ◽  
Unknown Function ◽  
Sinorhizobium Meliloti ◽  
Environmental Stresses ◽  
Protein Family ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Stress Protection ◽  
Wide Range

ABSTRACT We report here the first characterization of the Sinorhizobium meliloti open reading frame SMc01113. The SMc01113 protein is a member of a highly conserved protein family, universal among bacteria. We demonstrate that the SMc01113 gene is absolutely required for S. meliloti symbiosis with alfalfa and also for the protection of the bacterium from a wide range of environmental stresses.

scholarly journals Succinoglycan Production Contributes to Acidic pH Tolerance in Sinorhizobium meliloti Rm1021

2017 ◽  
Vol 30 (12) ◽  
pp. 1009-1019 ◽  
Author(s):  
Justin P. Hawkins ◽  
Barney A. Geddes ◽  
Ivan J. Oresnik
Keyword(s):  
Molecular Weight ◽  
Sinorhizobium Meliloti ◽  
Degree Of Polymerization ◽  
Low Ph ◽  
Nodule Development ◽  
Low Molecular Weight ◽  
Nodule Occupancy ◽  
Acidic Ph ◽  
Ph Tolerance ◽  
Genes Encoding

In this work, the hypothesis that exopolysaccharide plays a role in the survival of Sinorhizobium meliloti at low pH levels is addressed. When S. meliloti was grown at pH 5.75, synthesis of succinoglycan increased, whereas synthesis of galactoglucan decreased. Succinoglycan that was isolated from cultures grown at low pH had a lower degree of polymerization relative to that which was isolated from cultures grown at neutral pH, suggesting that low–molecular weight (LMW) succinoglycan might play a role in adaptation to low pH. Mutants unable to produce succinoglycan or only able to produce high–molecular weight polysaccharide were found to be sensitive to low pH. However, strains unable to produce LMW polysaccharide were 10-fold more sensitive. In response to low pH, transcription of genes encoding proteins for succinoglycan, glycogen, and cyclic β(1-2) glucans biosynthesis increased, while those encoding proteins necessary for the biosynthesis of galactoglucan decreased. While changes in pH did not affect the production of glycogen or cyclic β(1-2) glucan, it was found that the inability to produce cyclic β(1-2) glucan did contribute to pH tolerance in the absence of succinoglycan. Finally, in addition to being sensitive to low pH, a strain carrying mutations in exoK and exsH, which encode the glycanases responsible for the cleavage of succinoglycan to LMW succinoglycan, exhibited a delay in nodulation and was uncompetitive for nodule occupancy. Taken together, the data suggest that the role for LMW succinoglycan in nodule development may be to enhance survival in the colonized curled root hair.

scholarly journals Molecular and genetic characterization of the interactions between the Drosophila stoned-B protein and DAP-160 (intersectin)

2005 ◽  
Vol 388 (1) ◽  
pp. 195-204 ◽  
Author(s):  
Leonard E. KELLY ◽  
A. Marie PHILLIPS
Keyword(s):  
Synaptic Vesicle ◽  
Stop Codon ◽  
Adaptor Protein ◽  
Binding Motifs ◽  
Reading Frame ◽  
Vesicle Recycling ◽  
Src Homology 3 ◽  
Synaptotagmin I ◽  
Src Homology

The stoned locus of Drosophila produces a dicistronic transcript and encodes two proteins, stoned-A (STNA) and stoned-B (STNB). Both proteins are located at synaptic terminals. The STNB protein contains a domain that has homology with the μ-subunit of the AP (adaptor protein) complex, as well as a number of NPF (Asp-Pro-Phe) motifs known to bind EH (Eps15 homology) domains. Mutations at the stoned locus interact synergistically with mutations at the shibire (dynamin) locus and alter synaptic vesicle endocytosis. The STNB protein has also been shown to interact with synaptic vesicles via synaptogamin-I. We initiated an investigation of the possible interaction of DAP-160 (dynamin-associated protein of 160 kDa), a Drosophila member of the intersectin family, with the STNB protein. We show here that both of the viable stoned alleles interacted with a genetic construct that reduces DAP-160 levels to 25% of normal. One of these stoned alleles contains a substitution resulting in a stop codon in the open reading frame encoding STNB. This allele also shows markedly reduced levels of both DAP-160 and dynamin. As anticipated, the NPF motifs in STNB are found to be high-affinity binding motifs for the EH domains of DAP-160. One of the SH3 (Src homology 3) domains of DAP-160 also interacts with STNB. Finally, we show that immunoprecipitation of STNB from fly head extracts co-precipitates with DAP-160, and we conclude that the interaction of the STNB protein with both synaptotagmin I and DAP-160 may regulate synaptic vesicle recycling by recruiting dynamin to a pre-fission complex.

scholarly journals Identification and Characterization of the Brucella abortus Phosphoglucomutase Gene: Role of Lipopolysaccharide in Virulence and Intracellular Multiplication

2000 ◽  
Vol 68 (10) ◽  
pp. 5716-5723 ◽  
Author(s):  
Juan E. Ugalde ◽  
Cecilia Czibener ◽  
Mario F. Feldman ◽  
Rodolfo A. Ugalde
Keyword(s):  
Brucella Abortus ◽  
Wild Type ◽  
Protective Properties ◽  
O Antigen ◽  
Important Virulence Factor ◽  
Intracellular Multiplication ◽  
Identification And Characterization ◽  
Precise Role

ABSTRACT 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.

scholarly journals A Lysozyme-Like Protein in Brucella abortus Is Involved in the Early Stages of Intracellular Replication

2013 ◽  
Vol 81 (3) ◽  
pp. 956-964 ◽  
Author(s):  
Mariela G. Del Giudice ◽  
Juan E. Ugalde ◽  
Cecilia Czibener
Keyword(s):  
Brucella Abortus ◽  
High Energy ◽  
Proper Function ◽  
Secretion Systems ◽  
Content Type ◽  
Early Stages ◽  
Energy Consuming ◽  
Intracellular Multiplication ◽  
Identification And Characterization

ABSTRACTSecretion of proteins in Gram-negative bacteria is a high-energy-consuming process that requires translocation across two membranes and a periplasmic space composed of a mesh-like layer, the peptidoglycan. To achieve this, bacteria have evolved complex secretion systems that cross these barriers, and in many cases there are specific peptidoglycanases that degrade the peptidoglycan to allow the proper assembly of the secretion machinery. We describe here the identification and characterization of a muramidase inBrucella abortusthat participates in the intracellular multiplication in professional and nonprofessional phagocytes. We demonstrated that this protein has peptidoglycanase activity, that a strain with a clean deletion of the gene displayed a defect in the early stages of the intracellular multiplication curve, and that this is dependent on the lytic activity. While neither the attachment nor the invasion of the strain was affected, we demonstrated that it had a defect in excluding the lysosomal marker LAMP-1 but not in acquiring the reticulum endoplasmic marker calnexin, indicating that the gene participates in the early stages of the intracellular trafficking but not in the establishment of the replicative niche. Analysis of the assembly status and functionality of the VirB secretion apparatus indicated that the mutant has affected the proper function of this central virulence factor.

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