scholarly journals The wzm gene located on the pRhico plasmid of Azospirillum brasilense Sp7 is involved in lipopolysaccharide synthesis

Microbiology ◽  
2009 ◽  
Vol 155 (3) ◽  
pp. 791-804 ◽  
Author(s):  
Anat Lerner ◽  
Yaacov Okon ◽  
Saul Burdman
Keyword(s):  
Azospirillum Brasilense ◽  
Structural Integrity ◽  
Sweet Corn ◽  
Cell Envelope ◽  
Osmotic Shock ◽  
Protein Composition ◽  
Monosaccharide Composition ◽  
Saline Stress ◽  
Wild Type ◽  
Azospirillum Brasilense Sp7

Several genes involved in the interaction between Azospirillum brasilense Sp7 and plants are located on the pRhico plasmid. Here we report the characterization of an Sp7 mutant strain with impairment of the pRhico-located gene wzm. This gene encodes an inner-membrane component of an ATP-binding cassette (ABC) transporter with similarity to transporters involved in surface polysaccharide export. Indeed, SDS-PAGE revealed that LPS synthesis is affected in the wzm mutant. No significant differences were observed between wild-type and mutant strains in exopolysaccharide (EPS) amount; however, several differences were observed between them in EPS monosaccharide composition, and only wild-type colonies stained positively with Congo red. Microscopy revealed that wzm mutant cells are longer and thinner, and exhibit several differences in their cell surface relative to the wild-type. The wzm mutant was more resistant to oxidative stress, starvation, desiccation, heat and osmotic shock than the wild-type. In contrast, the mutant was more susceptible than the wild-type to UV radiation and saline stress. The strains also differed in their susceptibility to different antibiotics. Differences between the strains were also observed in their outer-membrane protein composition. No differences were observed between strains in their ability to attach to sweet corn roots and seeds, and to promote growth under the tested conditions. As LPS plays an important role in cell envelope structural integrity, we propose that the pleiotropic phenotypic changes observed in the wzm mutant are due to its altered LPS relative to the wild-type.

scholarly journals Roles of Internal Cysteines in the Function, Localization, and Reactivity of the TraV Outer Membrane Lipoprotein Encoded by the F Plasmid

2002 ◽  
Vol 184 (11) ◽  
pp. 3126-3129 ◽  
Author(s):  
Robin L. Harris ◽  
Philip M. Silverman
Keyword(s):  
Outer Membrane ◽  
Cell Envelope ◽  
Osmotic Shock ◽  
Wild Type ◽  
F Plasmid ◽  
Outer Membrane Lipoprotein ◽  
Numerous Cell ◽  
Mixed Disulfides ◽  
Membrane Lipoprotein

ABSTRACT We have examined the functional role of two internal cysteine residues of the F-plasmid TraV outer membrane lipoprotein. Each was mutated to a serine separately and together to yield three mutant traV genes: traV C10S, traV C18S, and traV C10S/C18S. All three cysteine mutations complemented a traV mutant for DNA donor activity and for sensitivity to donor-specific bacteriophage; however, when measured by a transduction assay, the donor-specific DNA bacteriophage sensitivities of the traV C18S and, especially, traV C10S/C18S mutant strains were significantly less than those of the traV + and traV C10S strains. Thus, unlike the Agrobacterium tumefaciens T-plasmid-encoded VirB7 outer membrane lipoprotein, TraV does not require either internal cysteine to retain significant biological activity. By Western blot analysis, all three mutant TraV proteins were shown to accumulate in the outer membrane. However, by nonreducing gel electrophoresis, wild-type TraV and especially the TraVC18S mutant were shown to form mixed disulfides with numerous cell envelope proteins. This was not observed with the TraVC10S or TraVC10S/C18S proteins. Thus, it appears that TraV C10 is unusually reactive and that this reactivity is reduced by C18, perhaps by intramolecular oxidation. Finally, whereas the TraVC10S and TraVC18S proteins fractionated primarily with the outer membrane, as did the wild-type protein, the TraVC10S/C18S protein was found in osmotic shock fluid and inner membrane fractions as well as outer membrane fractions. Hence, at least one cysteine is required for the efficient localization of TraV to the outer membrane.

scholarly journals Involvement of glnB, glnZ, and glnD Genes in the Regulation of Poly-3-Hydroxybutyrate Biosynthesis by Ammonia in Azospirillum brasilense Sp7

2002 ◽  
Vol 68 (2) ◽  
pp. 985-988 ◽  
Author(s):  
Jun Sun ◽  
Anne Van Dommelen ◽  
Jan Van Impe ◽  
Jos Vanderleyden
Keyword(s):  
Growth Phase ◽  
Azospirillum Brasilense ◽  
Regulatory Genes ◽  
Wild Type ◽  
Active Growth ◽  
Phb Production ◽  
Azospirillum Brasilense Sp7

ABSTRACT The role of three key nitrogen regulatory genes, glnB (encoding the PII protein), glnZ (encoding the Pz protein), and glnD (encoding the GlnD protein), in regulation of poly-3-hydroxybutyrate (PHB) biosynthesis by ammonia in Azospirillum brasilense Sp7 was investigated. It was observed that glnB glnZ and glnD mutants produce substantially higher amounts of PHB than the wild type produces during the active growth phase. glnB and glnZ mutants have PHB production phenotypes similar to that of the wild type. Our results indicate that the PII-Pz system is apparently involved in nitrogen-dependent regulation of PHB biosynthesis in A. brasilense Sp7.

scholarly journals The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 4058-4068 ◽  
Author(s):  
Anat Lerner ◽  
Susana Castro-Sowinski ◽  
Angel Valverde ◽  
Hadas Lerner ◽  
Rachel Dror ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Plant Root ◽  
Extracellular Polysaccharide ◽  
Extracellular Polysaccharides ◽  
Wild Type ◽  
Polysaccharide Biosynthesis ◽  
Beneficial Effects ◽  
Mutant Strains ◽  
Azospirillum Brasilense Sp7 ◽  
Surface Polysaccharides

Azospirillum brasilense is a plant root-colonizing bacterium that exerts beneficial effects on the growth of many agricultural crops. Extracellular polysaccharides of the bacterium play an important role in its interactions with plant roots. The pRhico plasmid of A. brasilense Sp7, also named p90, carries several genes involved in synthesis and export of cell surface polysaccharides. We generated two Sp7 mutants impaired in two pRhico-located genes, noeJ and noeL, encoding mannose-6-phosphate isomerase and GDP-mannose 4,6-dehydratase, respectively. Our results demonstrate that in A. brasilense Sp7, noeJ and noeL are involved in lipopolysaccharide and exopolysaccharide synthesis. noeJ and noeL mutant strains were significantly altered in their outer membrane and cytoplasmic/periplasmic protein profiles relative to the wild-type strain. Moreover, both noeJ and noeL mutations significantly affected the bacterial responses to several stresses and antimicrobial compounds. Disruption of noeL, but not noeJ, affected the ability of the A. brasilense Sp7 to form biofilms. The pleiotropic alterations observed in the mutants could be due, at least partially, to their altered lipopolysaccharides and exopolysaccharides relative to the wild-type.

scholarly journals The ntrB and ntrC Genes Are Involved in the Regulation of Poly-3-Hydroxybutyrate Biosynthesis by Ammonia in Azospirillum brasilense Sp7

2000 ◽  
Vol 66 (1) ◽  
pp. 113-117 ◽  
Author(s):  
Jun Sun ◽  
Xuan Peng ◽  
Jan Van Impe ◽  
Jos Vanderleyden
Keyword(s):  
Growth Phase ◽  
Azospirillum Brasilense ◽  
Genetic Evidence ◽  
Wild Type ◽  
High Concentration ◽  
Active Growth ◽  
Phb Production ◽  
Azospirillum Brasilense Sp7 ◽  
In The Wild

ABSTRACT Azospirillum brasilense Sp7 and its ntrA(rpoN), ntrBC, and ntrC mutants have been evaluated for their capabilities of poly-3-hydroxybutyrate (PHB) accumulation in media with high and low ammonia concentrations. It was observed that the ntrBC and ntrC mutants can produce PHB in both low- and high-C/N-ratio media, while no significant PHB production was observed for the wild type or thentrA mutant in low-C/N-ratio media. Further investigation by fermentation analysis indicated that the ntrBC andntrC mutants were able to grow and accumulate PHB simultaneously in the presence of a high concentration of ammonia in the medium, while little PHB was produced in the wild type andntrA (rpoN) mutant during active growth phase. These results provide the first genetic evidence that thentrB and ntrC genes are involved in the regulation of PHB synthesis by ammonia in A. brasilenseSp7.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

scholarly journals Stress-Based High-Throughput Screening Assays to Identify Inhibitors of Cell Envelope Biogenesis

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 808
Author(s):  
Maurice Steenhuis ◽  
Corinne M. ten Hagen-Jongman ◽  
Peter van Ulsen ◽  
Joen Luirink
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
Stress Responses ◽  
High Throughput Screening ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Compound Screening ◽  
Outer Membrane Biogenesis

The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.

Molybdenum incorporation in denitrifying Azospirillum brasilense Sp7

1992 ◽  
Vol 38 (10) ◽  
pp. 1042-1047 ◽  
Author(s):  
Christian Chauret ◽  
Wilfredo L. Barraquio ◽  
Roger Knowles
Keyword(s):  
Nitrate Reductase ◽  
Key Words ◽  
Nitrate Reduction ◽  
Gel Electrophoresis ◽  
Azospirillum Brasilense ◽  
Paracoccus Denitrificans ◽  
Free Medium ◽  
Azospirillum Brasilense Sp7

Nondenaturating disc gel electrophoresis revealed that 99Mo was incorporated into the nitrate reductase of Azospirillum brasilense grown in the absence but not in the presence of tungstate. Under denitrifying conditions, A. brasilense grown in tungsten-free medium steadily accumulated 99Mo for 12 h. In contrast, Paracoccus denitrificans grown under the same conditions ceased uptake after 1 h. However, both bacteria were incapable of accumulating significant amounts of 99Mo in media containing 10 mM tungstate, even though nitrate was reduced by A. brasilense. Aerobically grown A. brasilense cells transported 99Mo more efficiently than anaerobically grown cells. Key words: Azospirillum brasilense, tungsten, molybdenum incorporation, nitrate reduction.

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