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Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate.

ABSTRACT: Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion of an Omega-Km interposon within the PHB synthase structural gene (phaC). The identification and sequence of the R. etli phaC gene are also reported here. Physiological studies showed that the PHB-negative mutant strain was unable to synthesize PHB and excreted more lactate, acetate, pyruvate, beta-hydroxybutyrate, fumarate, and malate than the wild-type strain. The NAD+/NADH ratio in the mutant strain was lower than that in the parent strain. The oxidative capacity of the PHB-negative mutant was reduced. Accordingly, the ability to grow in minimal medium supplemented with glucose or pyruvate was severely diminished in the mutant strain. We propose that in free life PHB synthesis sequesters reductive power, allowing the tricarboxylic acid cycle to proceed under conditions in which oxygen is a limiting factor. In symbiosis with Phaseolus vulgaris, the PHB-negative mutant induced nodules that prolonged the capacity to fix nitrogen.

SUBMITTER: Cevallos MA

PROVIDER: S-EPMC177850 | biostudies-other | 1996 Mar

REPOSITORIES: biostudies-other

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Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate.

Cevallos M A MA Encarnación S S Leija A A Mora Y Y Mora J J

Journal of bacteriology 19960301 6

Rhizobium etli accumulates poly-beta-hydroxybutyrate (PHB) in symbiosis and in free life. PHB is a reserve material that serves as a carbon and/or electron sink when optimal growth conditions are not met. It has been suggested that in symbiosis PHB can prolong nitrogen fixation until the last stages of seed development, but experiments to test this proposition have not been done until now. To address these questions in a direct way, we constructed an R. etli PHB-negative mutant by the insertion ...[more]

PMID: 8626293

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Project description:Recently, Rhizobium etli, in addition to Agrobacterium spp., has emerged as a prokaryotic species whose genome encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we show that the signal phenolic molecule acetosyringone (AS) induces R. etli vir gene expression both in an R. etli background and in an Agrobacterium tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter transfer DNA (T-DNA). Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium but not from nonhost plants. The early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a than of T-DNA transfer of pTiC58 of A. tumefaciensIMPORTANCE The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by the genome of a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into the plant cell genome. In this study, we explored the transcriptional regulation and induction of virulence genes in R. etli and show similarities to and differences from those of their A. tumefaciens counterparts, contributing to an understanding and a comparison of these two systems. Whereas most vir genes in R. etli follow an induction pattern similar to that of A. tumefaciens vir genes, a few significant differences may at least in part explain the variations in T-DNA transfer efficiency.

Project description:Gellan gum is a microbial exopolysaccharide, produced after aerobic fermentation using the Gram-negative bacterium strain Sphingomonas elodea ATCC 31461. Due to its unique structure and excellent physical characteristics, gellan gum has a broad range of applications in food, pharmaceutical, and other industries where it is used for stabilizing, emulsifying, thickening, and suspending. During the fermentative production of gellan, strain ATCC 31461 also accumulates large amounts of the metabolic by-products yellow carotenoid pigments and poly-?-hydroxybutyrate (PHB), which is decreasing the gellan production and increasing processing costs. A pigment PHB-free mutant was obtained by knocking out the phytoene desaturase gene (crtI) in the carotenoid biosynthetic pathway and the phaC gene, encoding a PHB synthase for the polymerization of PHB. Unfortunately, the double gene knockout mutant produced only 0.56 g liter-1 gellan. Furthermore, blocking PHB and carotenoid synthesis resulted in the accumulation of pyruvate, which reduced gellan production. To elevate gellan production, combined UV irradiation and ethyl methanesulfonate (EMS) mutagenesis treatment were used. A mutant strain with the same level of pyruvate as that of the wild-type strain and higher gellan production was isolated (1.35 g liter-1, 132.8% higher than the double gene knockout mutant and 14.4% higher than the wild-type strain ATCC 31461). In addition, a new gellan gum recovery method based on the new mutant strain was investigated, in which only 30% isopropanol was required, which is twice for the wild-type strains, and the performance of the final product was improved. Thus, the mutant strain could be an ideal strain for the commercial production of gellan.IMPORTANCE A carotenoid- and PHB-free double gene knockout strain mutant was constructed to simplify the purification steps normally involved in gellan production. However, the production of gellan gum was unexpectedly reduced. A mutant with 14.4% higher gellan production than that of the wild-type strain was obtained and isolated after employing UV and EMS combined mutagenesis. Based on this high-yield and low-impurity-producing mutant, a new recovery method requiring less organic solvent and fewer operating steps was developed. This method will effectively reduce the production costs and improve the economic benefits of large-scale gellan production.

Dataset Information

Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate.

Publications

Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate.

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