Dataset Information

Bacteriophytochrome controls carotenoid-independent response to photodynamic stress in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7.

ABSTRACT: Ever since the discovery of the role of bacteriophytochrome (BphP) in inducing carotenoid synthesis in Deinococcus radiodurans in response to light the role of BphPs in other non-photosynthetic bacteria is not clear yet. Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours a pair of BphPs out of which AbBphP1 is a homolog of AtBphP1 of Agrobacterium tumefaciens. By overexpression, purification, biochemical and spectral characterization we have shown that AbBphP1 is a photochromic bacteriophytochrome. Phenotypic study of the ?AbBphP1 mutant showed that it is required for the survival of A. brasilense on minimal medium under red light. The mutant also showed reduced chemotaxis towards dicarboxylates and increased sensitivity to the photooxidative stress. Unlike D. radiodurans, AbBphP1 was not involved in controlling carotenoid synthesis. Proteome analysis of the ?AbBphP1 indicated that AbBphP1 is involved in inducing a cellular response that enables A. brasilense in regenerating proteins that might be damaged due to photodynamic stress.

SUBMITTER: Kumar S

PROVIDER: S-EPMC3503143 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Bacteriophytochrome controls carotenoid-independent response to photodynamic stress in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7.

Kumar Santosh S Kateriya Suneel S Singh Vijay Shankar VS Tanwar Meenakshi M Agarwal Shweta S Singh Hina H Khurana Jitendra Paul JP Amla Devinder Vijay DV Tripathi Anil Kumar AK

Scientific reports 20121119

Ever since the discovery of the role of bacteriophytochrome (BphP) in inducing carotenoid synthesis in Deinococcus radiodurans in response to light the role of BphPs in other non-photosynthetic bacteria is not clear yet. Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours a pair of BphPs out of which AbBphP1 is a homolog of AtBphP1 of Agrobacterium tumefaciens. By overexpression, purification, biochemical and spectral characterization we have shown that AbBphP1 is a photochrom ...[more]

PMID: 23173079

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Project description:Carotenoids constitute an important component of the defense system against photooxidative stress in bacteria. In Azospirillum brasilense Sp7, a nonphotosynthetic rhizobacterium, carotenoid synthesis is controlled by a pair of extracytoplasmic function sigma factors (RpoEs) and their cognate zinc-binding anti-sigma factors (ChrRs). Its genome harbors two copies of the gene encoding geranylgeranyl pyrophosphate synthase (CrtE), the first critical step in the carotenoid biosynthetic pathway in bacteria. Inactivation of each of two crtE paralogs found in A. brasilense caused reduction in carotenoid content, suggesting their involvement in carotenoid synthesis. However, the effect of crtE1 deletion was more pronounced than that of crtE2 deletion. Out of the five paralogs of rpoH in A. brasilense, overexpression of rpoH1 and rpoH2 enhanced carotenoid synthesis. Promoters of crtE2 and rpoH2 were found to be dependent on RpoH2 and RpoE1, respectively. Using a two-plasmid system in Escherichia coli, we have shown that the crtE2 gene of A. brasilense Sp7 is regulated by two cascades of sigma factors: one consisting of RpoE1and RpoH2 and the other consisting of RpoE2 and RpoH1. In addition, expression of crtE1 was upregulated indirectly by RpoE1 and RpoE2. This study shows, for the first time in any carotenoid-producing bacterium, that the regulation of carotenoid biosynthetic pathway involves a network of multiple cascades of alternative sigma factors.ImportanceCarotenoids play a very important role in coping with photooxidative stress in prokaryotes and eukaryotes. Although extracytoplasmic function (ECF) sigma factors are known to directly regulate the expression of carotenoid biosynthetic genes in bacteria, regulation of carotenoid biosynthesis by one or multiple cascades of sigma factors had not been reported. This study provides the first evidence of the involvement of multiple cascades of sigma factors in the regulation of carotenoid synthesis in any bacterium by showing the regulation of a gene encoding geranylgeranyl pyrophosphate synthase (crtE2) by RpoE1?RpoH2?CrtE2 and RpoE2?RpoH1?CrtE2 cascades in A. brasilense It also provides an insight into existence of an additional cascade or cascades regulating expression of another paralog of crtE.

Project description:Chemotaxis is the movement of cells in response to gradients of diverse chemical cues. Motile bacteria utilize a conserved chemotaxis signal transduction system to bias their motility and navigate through a gradient. A central regulator of chemotaxis is the histidine kinase CheA. This cytoplasmic protein interacts with membrane-bound receptors, which assemble into large polar arrays, to propagate the signal. In the alphaproteobacterium Azospirillum brasilense, Che1 controls transient increases in swimming speed during chemotaxis, but it also biases the cell length at division. However, the exact underlying molecular mechanisms for Che1-dependent control of multiple cellular behaviors are not known. Here, we identify specific domains of the CheA1 histidine kinase implicated in modulating each of these functions. We show that CheA1 is produced in two isoforms: a membrane-anchored isoform produced as a fusion with a conserved seven-transmembrane domain of unknown function (TMX) at the N terminus and a soluble isoform similar to prototypical CheA. Site-directed and deletion mutagenesis combined with behavioral assays confirm the role of CheA1 in chemotaxis and implicate the TMX domain in mediating changes in cell length. Fluorescence microscopy further reveals that the membrane-anchored isoform is distributed around the cell surface while the soluble isoform localizes at the cell poles. Together, the data provide a mechanism for the role of Che1 in controlling multiple unrelated cellular behaviors via acquisition of a new domain in CheA1 and production of distinct functional isoforms.IMPORTANCE Chemotaxis provides a significant competitive advantage to bacteria in the environment, and this function has been transferred laterally multiple times, with evidence of functional divergence in different genomic contexts. The molecular principles that underlie functional diversification of chemotaxis in various genomic contexts are unknown. Here, we provide a molecular mechanism by which a single CheA protein controls two unrelated functions: chemotaxis and cell length. Acquisition of this multifunctionality is seemingly a recent evolutionary event. The findings illustrate a mechanism by which chemotaxis function may be co-opted to regulate additional cellular functions.

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Project description:Escherichia coli and Saccharomyces cerevisiae have been used extensively for heterologous production of a variety of secondary metabolites. Neither has an endogenous high-flux isoprenoid pathway, required for the production of terpenoids. Azospirillum brasilense, a nonphotosynthetic GRAS (generally recognized as safe) bacterium, produces carotenoids in the presence of light. The carotenoid production increases multifold upon inactivating a gene encoding an anti-sigma factor (ChrR1). We used this A. brasilense mutant (Car-1) as a host for the heterologous production of two high-value phytochemicals, geraniol and amorphadiene. Cloned genes (crtE1 and crtE2) of A. brasilense encoding native geranylgeranyl pyrophosphate synthases (GGPPS), when overexpressed and purified, did not produce geranyl pyrophosphate (GPP) in vitro Therefore, we cloned codon-optimized copies of the Catharanthus roseus genes encoding GPP synthase (GPPS) and geraniol synthase (GES) to show the endogenous intermediates of the carotenoid biosynthetic pathway in the Car-1 strain were utilized for the heterologous production of geraniol in A. brasilense Similarly, cloning and expression of a codon-optimized copy of the amorphadiene synthase (ads) gene from Artemisia annua also led to the heterologous production of amorphadiene in Car-1. Geraniol or amorphadiene content was estimated using gas chromatography-mass spectrometry (GC-MS) and GC. These results demonstrate that Car-1 is a promising host for metabolic engineering, as the naturally available endogenous pool of the intermediates of the carotenoid biosynthetic pathway of A. brasilense can be effectively utilized for the heterologous production of high-value phytochemicals.IMPORTANCE To date, the major host organisms used for the heterologous production of terpenoids, i.e., E. coli and S. cerevisiae, do not have high-flux isoprenoid pathways and involve tedious metabolic engineering to increase the precursor pool. Since carotenoid-producing bacteria carry endogenous high-flux isoprenoid pathways, we used a carotenoid-producing mutant of A. brasilense as a host to show its suitability for the heterologous production of geraniol and amorphadiene as a proof-of-concept. The advantages of using A. brasilense as a model system include (i) dispensability of carotenoids and (ii) the possibility of overproducing carotenoids through a single mutation to exploit high carbon flux for terpenoid production.

Dataset Information

Bacteriophytochrome controls carotenoid-independent response to photodynamic stress in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7.

Publications

Bacteriophytochrome controls carotenoid-independent response to photodynamic stress in a non-photosynthetic rhizobacterium, Azospirillum brasilense Sp7.

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