The Importance of the Interaction of CheD with CheC and the Chemoreceptors Compared to Its Enzymatic Activity during Chemotaxis in Bacillus subtilis

Yuan, Wei; Glekas, George D.; Allen, George M; Walukiewicz, Hanna E.; Rao, Christopher V.; Ordal, George

doi:10.1371/journal.pone.0050689

Cited by 15 publications

(13 citation statements)

References 28 publications

(40 reference statements)

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“…Fractions containing the purified proteins were identified with SDS-PAGE. The fractions containing the purified protein were pooled and refolded by dialyzing three times against TKMD-G (50 mM Tris-Cl, pH 8.0, 5 mM MgCl 2 , 50 mM KCl, 0.1 mM dithiothreitol, 10% glycerol, pH 8.0) at 4°C for 2 h, followed by an overnight charge at 4°C (39). Gel filtration was employed to further purify the refolded lyases using a 16-mm-diameter HiPrep Sephacryl S-200 HR (GE Healthcare) gel filtration column charged with TKMD-G. Fractions were collected using TKMD-G at 0.5 ml per min to elute the purified protein.…”

Section: Methodsmentioning

confidence: 99%

Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

Badur

Jagtap

Yalamanchili

et al. 2015

Appl Environ Microbiol

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bAlginate lyases are enzymes that degrade alginate through ␤-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (k cat ) for alginate of 0.60 ؎ 0.02 s ؊1 , 3.7 ؎ 0.3 s ؊1 , 4.5 ؎ 0.5 s ؊1 , and 7.1 ؎ 0.2 s ؊1 , respectively. The K m values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ؎ 7 M, 22 ؎ 5 M, 60 ؎ 2 M, and 123 ؎ 6 M, respectively. AlyA and AlyB were found principally to cleave the ␤-1,4 bonds between ␤-D-mannuronate and ␣-L-guluronate and subunits; AlyD and AlyE were found to principally cleave the ␣-1,4 bonds involving ␣-L-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers. Alginate is an abundant polysaccharide found within the cell wall of brown seaweeds (1) and comprises approximately 40% of the dry weight of the plant (2). Alginate is a copolymer consisting of the 1,4-linked epimers ␣-L-guluronate (G) and ␤-Dmannuronate (M). The individual monomeric subunits are organized in short stretches of polyguluronate (polyG), polymannuronate (polyM), or alternating sequences of mannuronate and guluronate (polyMG) (3). Alginate recently has been considered as a source for bioenergy, as it offers several potential advantages over terrestrial biomass. In particular, the farming of algae does not impinge on arable land; hence, it avoids the conflict between food and energy (4, 5). Algae also display higher growth rates than terrestrial plants (6). Finally, algae lack crystalline cellulose and lignin (7,8), bypassing a key obstacle to biofuel production.Alginate lyases are enzymes that degrade alginate through the ␤-elimination of the glycosidic bond into smaller oligomers. These enzymes can be classified based on the specific dyad G-G (EC 4.2.2.11) (9, 10), M-M (EC 4.2.2.3) (11), and M-G/G-M (12) bonds that they cleave. Additionally, alginate lyases can have either endocleaving or exocleaving specificity, with the majority of alginate lyases having an endocleaving preference (3). Along with the substrate specificity, alginate lyases also are characterized by their structure, termed polysaccharide lyase (PL) families. A total of seven PL families have been identified: PL5, PL6, PL7, PL14, PL15, PL17, and PL18 (13, 14). The most prevalent of these families is PL7 (Carbohydrate Active Enzymes database; http://www .cazy.org). The PL7 domain contains a ␤-jelly roll that consists of ␤-sheets in an antiparallel, adjacent barrel forming a cleft (12). This cleft contains three adjacent ␤-sheets that contain the catalytic residues (15). PL7 contains enzymes...

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Section: Methodsmentioning

confidence: 99%

Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

Badur

Jagtap

Yalamanchili

et al. 2015

Appl Environ Microbiol

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“…Subsequent studies revealed that CheD also participates with CheC and CheYp in a negative feedback loop, which controls the amount of CheD free to bind and activate the receptors in response to CheYp (Chao et al, 2006;Muff and Ordal, 2007). The two roles are distinct: of the two, the feedback loop is thought to be more important for chemotaxis Yuan et al, 2012). Indeed, replacing the target glutamines with glutamates does not remove the requirement of CheD for optimal chemotaxis.…”

Section: Chedmentioning

confidence: 99%

“…CheC is a weak CheYp phosphatase and CheD is a receptor deamidase (Kristich and Ordal, 2002). However, the enzymatic functions of these two proteins are secondary to their role as adaptation proteins (Muff and Ordal, 2007;Glekas et al, 2012;Yuan et al, 2012). In particular, these two proteins participate in a negative feedback loop that regulates CheA activity in response to CheYp levels (Muff and Ordal, 2007;Glekas et al, 2012;Yuan et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

“…CheC appears to control the ability of CheD to activate the receptors through a competitive mechanism: CheC binds CheD along the same interface that CheD uses to bind the receptors. Furthermore, CheYp enhances the binding affinity between CheC and CheD (Muff and Ordal, 2007;Glekas et al, 2012;Yuan et al, 2012). When CheYp concentrations are high, CheC is thought to be a better binding partner for CheD than the receptors (Rao et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

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Interactions among the three adaptation systems of Bacillus subtilis chemotaxis as revealed by an in vitro receptor‐kinase assay

Walukiewicz

Tohidifar

Ordal

et al. 2014

Molecular Microbiology

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SummaryThe Bacillus subtilis chemotaxis pathway employs three systems for sensory adaptation: the methylation system, the CheC/CheD/CheYp system, and the CheV system. Little is known in general about how these three adaptation systems contribute to chemotaxis in B. subtilis and whether they interact with one another. To further understand these three adaptation systems, we employed a quantitative in vitro receptor-kinase assay. Using this assay, we were able to determine how CheD and CheV affect receptor-kinase activity as a function of the receptor modification state. CheD was found to increase receptor-kinase activity, where the magnitude of the increase depends on the modification state of the receptor. The principal new findings concern CheV. Little was known about this protein before now. Our data suggest that this protein has two roles depending on the modification state of the receptor, one for sensory adaptation when the receptors are modified (methylated) and the other for signal amplification when they are unmodified (unmethylated). In addition, our data suggest that methylation of site 630 tunes the strength of the CheV adaptation system. Collectively, our results provide new insight regarding the integrated function of the three adaptation systems in B. subtilis.

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“…Moreover, B. burgdorferi has a putative gene encoding CheD, which is not found in E. coli (5). CheD is relatively well characterized in Bacillus subtilis and Thermotoga maritima (27)(28)(29)(30). In B. subtilis, CheD plays an important role in motility and chemotaxis.…”

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confidence: 99%

Borrelia burgdorferi CheD Promotes Various Functions in Chemotaxis and the Pathogenic Life Cycle of the Spirochete

2016

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b Borrelia burgdorferi possesses a sophisticated chemotaxis signaling system; however, the roles of the majority of the chemotaxis proteins in the infectious life cycle have not yet been demonstrated. Specifically, the role of CheD during host colonization has not been demonstrated in any bacterium. Here, we systematically characterized the B. burgdorferi CheD homolog using genetics and biochemical and mouse-tick-mouse infection cycle studies. Bacillus subtilis CheD plays an important role in chemotaxis by deamidation of methyl-accepting chemotaxis protein receptors (MCPs) and by increasing the receptor kinase activity or enhancing CheC phosphatase activity, thereby regulating the levels of the CheY response regulator. Our biochemical analysis indicates that B. burgdorferi CheD significantly enhances CheX phosphatase activity by specifically interacting with the phosphatase. Moreover, CheD specifically binds two of the six MCPs, indicating that CheD may also modulate the receptor proteins. Although the motility of the cheD mutant cells was indistinguishable from that of the wild-type cells, the mutant did exhibit reduced chemotaxis. Importantly, the mutant showed significantly reduced infectivity in C3H/HeN mice via needle inoculation. Mouse-tickmouse infection assays indicated that CheD is dispensable for acquisition or transmission of spirochetes; however, the viability of cheD mutants in ticks is marginally reduced compared to that of the wild-type or complemented cheD spirochetes. These data suggest that CheD plays an important role in the chemotaxis and pathogenesis of B. burgdorferi. We propose potential connections between CheD, CheX, and MCPs and discuss how these interactions play critical roles during the infectious life cycle of the spirochete.

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The Importance of the Interaction of CheD with CheC and the Chemoreceptors Compared to Its Enzymatic Activity during Chemotaxis in Bacillus subtilis

Cited by 15 publications

References 28 publications

Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

Interactions among the three adaptation systems of Bacillus subtilis chemotaxis as revealed by an in vitro receptor‐kinase assay

Borrelia burgdorferi CheD Promotes Various Functions in Chemotaxis and the Pathogenic Life Cycle of the Spirochete

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