The Flagellar Set Fla2 in Rhodobacter sphaeroides Is Controlled by the CckA Pathway and Is Repressed by Organic Acids and the Expression of Fla1

Vega-Baray, Benjamín; Domenzain, Clelia; Rivera, Anet; Alfaro-López, Rocío; Gómez-César, Elidet; Poggio, Sebastián; Dreyfus, Georges; Camarena, Laura

doi:10.1128/jb.02429-14

Cited by 20 publications

(36 citation statements)

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“…All R. sphaeroides cells were grown in Sistrom's minimal medium at 30°C with orbital shaking (18); when indicated, kanamycin (25 g/ml) was added to the culture medium. For the isolation of Fla2 filament-hook basal bodies (HBBs), AM1 cells were grown in Sistrom medium without succinate and in the presence of Casamino Acids (0.2%) with orbital shaking (250 rpm) at 30°C as described previously (14).…”

Section: Methodsmentioning

confidence: 99%

Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides

et al. 2015

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Rhodobacter sphaeroides is a free-living alphaproteobacterium that contains two clusters of functional flagellar genes in its genome: one acquired by horizontal gene transfer (fla1) and one that is endogenous (fla2). We have shown that the Fla2 system is normally quiescent and under certain conditions produces polar flagella, while the Fla1 system is always active and produces a single flagellum at a nonpolar position. In this work we purified and characterized the structure and analyzed the composition of the Fla2 flagellum. The number of polar filaments per cell is 4.6 on average. By comparison with the Fla1 flagellum, the prominent features of the ultra structure of the Fla2 HBB are the absence of an H ring, thick and long hooks, and a smoother zone at the hook-filament junction. The Fla2 helical filaments have a pitch of 2.64 m and a diameter of 1.4 m, which are smaller than those of the Fla1 filaments. Fla2 filaments undergo polymorphic transitions in vitro and showed two polymorphs: curly (righthanded) and coiled. However, in vivo in free-swimming cells, we observed only a bundle of filaments, which should probably be left-handed. Together, our results indicate that Fla2 cell produces multiple right-handed polar flagella, which are not conventional but exceptional. ؊ mutant grown phototrophically and in the absence of organic acids. The Fla1 system produces a single lateral or subpolar flagellum, and the Fla2 system produces multiple polar flagella. The two kinds of flagella are never expressed simultaneously, and both are used for swimming in liquid media. The two sets of genes are certainly ready for responding to specific environmental conditions. The characterization of the Fla2 system will help us to understand its role in the physiology of this microorganism. Motility provides microorganisms with a fundamental survival advantage. Flagella are one of the most complex and effective organelles of locomotion, capable of propelling bacteria through liquids (swimming) and through viscous environments or over surfaces (swarming), and are widely used among Bacteria and Archaea. In addition, these organelles play an important role in adhesion to substrates and biofilm formation, and they contribute to the virulence process in pathogenic bacteria (1, 2).The bacterial flagellum is a rotary motor powered by the electrochemical proton or sodium potential. The morphology of the flagellum is similar among different bacterial species. Nevertheless, there are differences in their substructures, which are not yet clearly understood. The improvement in powerful microscopy techniques has revealed variations in the architecture of the bacterial flagellar motors (3, 4). The bacterial flagellum is a supramolecular complex made of about 30 different proteins with copy numbers that range from a few to thousands. The structure has been divided into three parts: filament, hook, and basal body. The basal body spans the bacterial cell envelope and comprises a rod and a series of rings. In the cytoplasm the basal body for...

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Section: Methodsmentioning

confidence: 99%

Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides

et al. 2015

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“…In R. sphaeroides, the expression of the fla2 genes is dependent on the transcriptional activator CtrA (21). We demonstrated that cheOp1 expression is also dependent on CtrA.…”

Section: Discussionmentioning

confidence: 70%

“…The expression of three chemotaxis proteins encoded in cheOp1 (Fig. 1) was evaluated by Western blotting using total cell extracts obtained from AM1 (25) and its derivative, EA1 (ΔctrA::aadA) (21). The proteins CheY2 and CheY5 were detected in AM1 but not in the absence of ctrA ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…WS8N is the wild-type strain that constitutively expresses the Fla1 flagellum but is unable to express the fla2 genes given that the two-component system CckA/ChpT/CtrA is inactive. The AM1 strain is a WS8N derivative that is unable to express the fla1 genes, given that it carries a mutation in the flagellar master activator fleQ and also carries a point mutation in cckA that activates the CckA/ChpT/CtrA two-component system (21,25); therefore, this strain has a Fla1 Ϫ Fla2 ϩ phenotype. cheOp1 expression is dependent on the master regulator of the fla2 genes.…”

Section: Resultsmentioning

confidence: 99%

“…Subsequently, the ctrA ϩ allele in the JV1 strain was replaced with the ΔctrA::hyg allele by using the plasmid pJQ200_ΔctrA::hyg. This plasmid was obtained by inserting a BglII fragment carrying the hyg cassette into the BamHI site of the pTZ_ctrAupdown plasmid (21).…”

Section: Methodsmentioning

confidence: 99%

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The Master Regulators of the Fla1 and Fla2 Flagella of Rhodobacter sphaeroides Control the Expression of Their Cognate CheY Proteins

et al. 2017

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Rhodobacter sphaeroides is an alphaproteobacterium that has two complete sets of flagellar genes. The fla1 set was acquired by horizontal transfer from an ancestral gammaproteobacterium and is the only set of flagellar genes that is expressed during growth under standard laboratory conditions. The products of these genes assemble a single, subpolar flagellum. In the absence of the Fla1 flagellum, a gain-of-function mutation in the histidine kinase CckA turns on the expression of the fla2 flagellar genes through the response regulator CtrA. The rotation of the Fla1 and Fla2 flagella is controlled by different sets of chemotaxis proteins. Here, we show that the expression of the chemotaxis proteins that control Fla2, along with the expression of the fla2 genes, is coordinated by CtrA, whereas the expression of the chemotaxis genes that control Fla1 is mediated by the master regulators of the Fla1 system. The coordinated expression of the chemosensory proteins with their cognate flagellar genes highlights the relevance of integrating the expression of the horizontally acquired fla1 genes with a chemosensory system independently of the regulatory proteins responsible for the expression of fla2 and its cognate chemosensory system. IMPORTANCE Gene acquisition via horizontal transfer represents a challenge to the recipient organism to adjust its metabolic and genetic networks to incorporate the new material in a way that represents an adaptive advantage. In the case of Rhodobacter sphaeroides, a complete set of flagellar genes was acquired and successfully coordinated with the native flagellar system. Here we show that the expression of the chemosensory proteins that control flagellar rotation is dependent on the master regulators of their corresponding flagellar system, minimizing the use of transcription factors required to express the native and horizontally acquired genes along with their chemotaxis proteins.

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Purification of Fla2 Flagella of Rhodobacter sphaeroides

Mora

Camarena

Dreyfus

2017

Methods in Molecular Biology

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The photosynthetic bacterium R. sphaeroides expresses two flagellar systems that are encoded by two complete gene clusters that have distinct phylogenetic origins. The isolation and purification of the Filament-Hook Basal Body (F-HBB) or the Hook Basal Body (HBB) structure is a troublesome task given the complexity of this nano-machine that is composed of multiple loosely bound substructures that can be lost during the isolation and purification procedure. A successful procedure requires adjustments to the standard method established for Salmonella. In this chapter, we describe a detailed protocol to isolate and purify the Fla2 F-HBB and HBB from R. sphaeroides a photosynthetic bacterium that has a complex intracellular membrane system that frequently interferes with isolation of high-quality samples.

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The Flagellar Set Fla2 in Rhodobacter sphaeroides Is Controlled by the CckA Pathway and Is Repressed by Organic Acids and the Expression of Fla1

Cited by 20 publications

References 78 publications

Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides

Structural Characterization of the Fla2 Flagellum of Rhodobacter sphaeroides

The Master Regulators of the Fla1 and Fla2 Flagella of Rhodobacter sphaeroides Control the Expression of Their Cognate CheY Proteins

Purification of Fla2 Flagella of Rhodobacter sphaeroides

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