Missense mutations in a transmembrane domain of the Komagataeibacter xylinus BcsA lead to changes in cellulose synthesis

Abstract Background Cellulose is synthesized by an array of bacterial species. Komagataeibacter xylinus is the best characterized as it produces copious amounts of the polymer extracellularly. Despite many advances in the past decade, the mechanisms underlying cellulose biosynthesis are not completely understood. Elucidation of these mechanisms is essential for efficient cellulose production in industrial applications. Results In an effort to gain a better understanding of cellulose biosynthesis and its regulation, cellulose crystallization was investigated in K. xylinus mutants resistant to an inhibitor of cellulose I formation, pellicin. Through the use of forward genetics and site-directed mutagenesis, A449T and A449V mutations in the K. xylinus BcsA protein were found to be important for conferring high levels of pellicin resistance. Phenotypic analysis of the bcsAA449T and bcsAA449V cultures revealed that the mutations affect cellulose synthesis rates and that cellulose crystallinity is affected in wet pellicles but not dry ones. Conclusions A449 is located in a predicted transmembrane domain of the BcsA protein suggesting that the structure of the transmembrane domain influences cellulose crystallization either by affecting the translocation of the nascent glucan chain or by allosterically altering protein-protein interactions.

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Missense mutations in a transmembrane domain of the Komagataeibacter xylinus BcsA lead to changes in cellulose synthesis

10.21203/rs.2.9716/v2 ◽

2019 ◽

Author(s):

Luis Salgado ◽

Silvia Blank ◽

Reza Alipour Moghadam Esfahani ◽

Janice L. Strap ◽

Dario Bonetta

Keyword(s):

Transmembrane Domain ◽

Bacterial Species ◽

Industrial Applications ◽

Forward Genetics ◽

Site Directed Mutagenesis ◽

Cellulose Synthesis ◽

Missense Mutations ◽

Cellulose Biosynthesis ◽

Phenotypic Analysis ◽

Komagataeibacter Xylinus

Abstract Background Cellulose is synthesized by an array of bacterial species. Komagataeibacter xylinus is the best characterized as it produces copious amounts of the polymer extracellularly. Despite many advances in the past decade, the mechanisms underlying cellulose biosynthesis are not completely understood. Elucidation of these mechanisms is essential for efficient cellulose production in industrial applications. Results In an effort to gain a better understanding of cellulose biosynthesis and its regulation, cellulose crystallization was investigated in K. xylinus mutants resistant to an inhibitor of cellulose I formation, pellicin. Through the use of forward genetics and site-directed mutagenesis, A449T and A449V mutations in the K. xylinus BcsA protein were found to be important for conferring high levels of pellicin resistance. Phenotypic analysis of the bcsAA449T and bcsAA449V cultures revealed that the mutations affect cellulose synthesis rates and that cellulose crystallinity is affected in wet pellicles but not dry ones. Conclusions A449 is located in a predicted transmembrane domain of the BcsA protein suggesting that the structure of the transmembrane domain influences cellulose crystallization either by affecting the translocation of the nascent glucan chain or by allosterically altering protein-protein interactions.

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Missense mutations in a transmembrane domain of the Komagataeibacter xylinus BcsA lead to changes in cellulose synthesis

10.21203/rs.2.9716/v1 ◽

2019 ◽

Author(s):

Luis Salgado ◽

Silvia Blank ◽

Reza Alipour Moghadam Esfahani ◽

Janice L. Strap ◽

Dario Bonetta

Keyword(s):

Transmembrane Domain ◽

Bacterial Species ◽

Industrial Applications ◽

Forward Genetics ◽

Site Directed Mutagenesis ◽

Cellulose Synthesis ◽

Missense Mutations ◽

Cellulose Biosynthesis ◽

Phenotypic Analysis ◽

Komagataeibacter Xylinus

Abstract Background Cellulose is synthesized by an array of bacterial species. Komagataeibacter xylinus is the best characterized as it produces copious amounts of the polymer extracellularly. Despite many advances in the past decade, the mechanisms underlying cellulose biosynthesis are not completely understood. Elucidation of these mechanisms is essential for efficient cellulose production in industrial applications. Results In an effort to gain a better understanding of cellulose biosynthesis and its regulation, cellulose crystallization was investigated in K. xylinus mutants resistant to an inhibitor of cellulose I formation, pellicin. Through the use of forward genetics and site-directed mutagenesis, A449T and A449V mutations in the K. xylinus BcsA protein were found to be important for conferring high levels of pellicin resistance. Phenotypic analysis of the bcsAA449T and bcsAA449V cultures revealed that the mutations affect cellulose synthesis rates and that cellulose crystallinity is affected in wet pellicles but not dry ones. Conclusions A449 is located in a predicted transmembrane domain of the BcsA protein suggesting that the structure of the transmembrane domain influences cellulose crystallization either by affecting the translocation of the nascent glucan chain or by allosterically altering protein-protein interactions.

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Missense mutations in a transmembrane domain of the Komagataeibacter xylinus BcsA lead to changes in cellulose synthesis

10.21203/rs.2.9716/v4 ◽

2019 ◽

Author(s):

Luis Salgado ◽

Silvia Blank ◽

Reza Alipour Moghadam Esfahani ◽

Janice L. Strap ◽

Dario Bonetta

Keyword(s):

Transmembrane Domain ◽

Bacterial Species ◽

Industrial Applications ◽

Forward Genetics ◽

Site Directed Mutagenesis ◽

Cellulose Synthesis ◽

Missense Mutations ◽

Cellulose Biosynthesis ◽

Phenotypic Analysis ◽

Komagataeibacter Xylinus

Abstract Background Cellulose is synthesized by an array of bacterial species. Komagataeibacter xylinus is the best characterized as it produces copious amounts of the polymer extracellularly. Despite many advances in the past decade, the mechanisms underlying cellulose biosynthesis are not completely understood. Elucidation of these mechanisms is essential for efficient cellulose production in industrial applications. Results In an effort to gain a better understanding of cellulose biosynthesis and its regulation, cellulose crystallization was investigated in K. xylinus mutants resistant to an inhibitor of cellulose I formation, pellicin. Through the use of forward genetics and site-directed mutagenesis, A449T and A449V mutations in the K. xylinus BcsA protein were found to be important for conferring high levels of pellicin resistance. Phenotypic analysis of the bcsAA449T and bcsAA449V cultures revealed that the mutations affect cellulose synthesis rates and that cellulose crystallinity is affected in wet pellicles but not dry ones. Conclusions A449 is located in a predicted transmembrane domain of the BcsA protein suggesting that the structure of the transmembrane domain influences cellulose crystallization either by affecting the translocation of the nascent glucan chain or by allosterically altering protein-protein interactions.

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Two novel mutations of the calcium-sensing receptor gene affecting the same amino acid position lead to opposite phenotypes and reveal the importance of p.N802 on receptor activity

Acta Endocrinologica ◽

10.1530/eje-12-0714 ◽

2013 ◽

Vol 168 (2) ◽

pp. K27-K34 ◽

Cited By ~ 13

Author(s):

Anne-Sophie Lia-Baldini ◽

Corinne Magdelaine ◽

Angélique Nizou ◽

Coraline Airault ◽

Jean-Pierre Salles ◽

...

Keyword(s):

Amino Acid ◽

Transmembrane Domain ◽

Direct Sequencing ◽

Receptor Gene ◽

Amino Acid Position ◽

Receptor Activity ◽

Site Directed Mutagenesis ◽

Missense Mutations ◽

Calcium Sensing Receptor ◽

Calcium Sensing

ObjectiveGain-of-function mutations of the calcium-sensing receptor (CASR) gene have been identified in patients with sporadic or familial autosomal dominant hypocalcemia (ADH). Inactivating mutations of the CASR gene cause familial hypocalciuric hypercalcemia (FHH). Here, we report two novel CASR mutations affecting the same amino acid (p.N802); one causes ADH and the other atypical FHH.Patients and methodsThe first patient, an 11-year-old girl suffering from hypocalcemia, developed nephrocalcinosis when she was only 5 years old. The second patient is a 30-year-old woman who presented with mild hypercalcemia. PCR amplification of CASR coding exons and direct sequencing of PCR products were used to identify mutations. Site-directed mutagenesis was used to generate mutated CASR cDNAs in an expression plasmid. Using the MAPK assay system and transient transfection of Cos-7 cells with wild-type (WT) and mutated CASR, we studied the responses of these mutated receptors to extracellular Ca2+ and to the negative allosteric CASR modulator, NPS2143.ResultsTwo heterozygous missense mutations (p.N802I and p.N802S) affecting a residue in the sixth transmembrane domain of CASR were identified. In functional tests, the response of the p.N802S mutant to calcium was typical of an inactivating mutation. However, the p.N802I mutant had 70% of the maximally stimulated WT receptor activity even in the absence of extracellular calcium. This constitutive activity was only partially inhibited by the inhibitor, NPS2143.ConclusionsThe asparagine at amino acid position 802 appears to be essential for the activity of the CASR protein and is implicated in the mechanism of CASR signaling.

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CelR, an Ortholog of the Diguanylate Cyclase PleD of Caulobacter, Regulates Cellulose Synthesis in Agrobacterium tumefaciens

Applied and Environmental Microbiology ◽

10.1128/aem.02148-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7188-7202 ◽

Cited By ~ 23

Author(s):

D. Michael Barnhart ◽

Shengchang Su ◽

Brenna E. Baccaro ◽

Lois M. Banta ◽

Stephen K. Farrand

Keyword(s):

Agrobacterium Tumefaciens ◽

Biofilm Formation ◽

Bacterial Species ◽

Cellulose Synthesis ◽

Diguanylate Cyclase ◽

Cellulose Biosynthesis ◽

Plant Host ◽

Cellulose Production ◽

Signal Molecule ◽

Content Type

ABSTRACTCellulose fibrils play a role in attachment ofAgrobacterium tumefaciensto its plant host. While the genes for cellulose biosynthesis in the bacterium have been identified, little is known concerning the regulation of the process. The signal molecule cyclic di-GMP (c-di-GMP) has been linked to the regulation of exopolysaccharide biosynthesis in many bacterial species, includingA. tumefaciens. In this study, we identified two putative diguanylate cyclase genes,celR(atu1297) andatu1060, that influence production of cellulose inA. tumefaciens. Overexpression of either gene resulted in increased cellulose production, while deletion ofcelR, but notatu1060, resulted in decreased cellulose biosynthesis.celRoverexpression also affected other phenotypes, including biofilm formation, formation of a polar adhesion structure, plant surface attachment, and virulence, suggesting that the gene plays a role in regulating these processes. Analysis ofcelRand Δcelmutants allowed differentiation between phenotypes associated with cellulose production, such as biofilm formation, and phenotypes probably resulting from c-di-GMP signaling, which include polar adhesion, attachment to plant tissue, and virulence. Phylogenetic comparisons suggest that species containing bothcelRandcelA, which encodes the catalytic subunit of cellulose synthase, adapted the CelR protein to regulate cellulose production while those that lackcelAuse CelR, called PleD, to regulate specific processes associated with polar localization and cell division.

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Endosidin20 is a broad-spectrum cellulose synthesis inhibitor with an herbicidal function

10.1101/2020.02.22.961094 ◽

2020 ◽

Author(s):

Lei Huang ◽

Chunhua Zhang

Keyword(s):

Cell Growth ◽

Plant Growth ◽

Broad Spectrum ◽

Plant Cell Wall ◽

Catalytic Domain ◽

Cellulose Synthesis ◽

Missense Mutations ◽

Cellulose Biosynthesis ◽

Synthesis Inhibitor ◽

Biosynthesis Inhibitor

AbstractCellulose is an important component of plant cell wall that controls anisotropic cell growth. Disruption of cellulose biosynthesis often leads to inhibited cell growth. Endosidin20 (ES20) was recently identified as a cellulose biosynthesis inhibitor (CBI) that targets the catalytic domain of Arabidopsis cellulose synthase 6 (CESA6) to inhibit plant growth. Here, we characterized the effects of ES20 on the growth of some other plant species and found that ES20 is a broad-spectrum plant growth inhibitor. We compared the inhibitory effects of ES20 and other CBIs on the growth of cesa6 plants that have reduced sensitivity to ES20. We found that most of the cesa6 with reduced sensitivity to ES20 show normal inhibited growth by other CBIs. ES20 also shows synergistic inhibitory effect on plant growth when applied together with other CBIs. We show ES20 has a different mode of action than tested CBIs isoxaben, indaziflam and C17. ES20 not only inhibits Arabidopsis growth under tissue culture condition, it inhibits plant growth under soil condition after direct spraying. We demonstrate that plants carrying two missense mutations can tolerate dual inhibition by ES20 and isoxaben.One sentence summaryCellulose biosynthesis inhibitor Endosidin20 has synergistic effect with other cellulose synthesis inhibitors and has the potential to be used as a spray herbicide.

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Acetan and Acetan-Like Polysaccharides: Genetics, Biosynthesis, Structure, and Viscoelasticity

Polymers ◽

10.3390/polym13050815 ◽

2021 ◽

Vol 13 (5) ◽

pp. 815

Author(s):

Janja Trček ◽

Iztok Dogsa ◽

Tomaž Accetto ◽

David Stopar

Keyword(s):

Bacterial Species ◽

Acetic Acid Bacteria ◽

Original Description ◽

Bioinformatic Analysis ◽

Industrial Sector ◽

Water Soluble ◽

Extracellular Polysaccharides ◽

Genetic Cluster ◽

Water Release ◽

Komagataeibacter Xylinus

Bacteria produce a variety of multifunctional polysaccharides, including structural, intracellular, and extracellular polysaccharides. They are attractive for the industrial sector due to their natural origin, sustainability, biodegradability, low toxicity, stability, unique viscoelastic properties, stable cost, and supply. When incorporated into different matrices, they may control emulsification, stabilization, crystallization, water release, and encapsulation. Acetan is an important extracellular water-soluble polysaccharide produced mainly by bacterial species of the genera Komagataeibacter and Acetobacter. Since its original description in Komagataeibacter xylinus, acetan-like polysaccharides have also been described in other species of acetic acid bacteria. Our knowledge on chemical composition of different acetan-like polysaccharides, their viscoelasticity, and the genetic basis for their production has expanded during the last years. Here, we review data on acetan biosynthesis, its molecular structure, genetic organization, and mechanical properties. In addition, we have performed an extended bioinformatic analysis on acetan-like polysaccharide genetic clusters in the genomes of Komagataeibacter and Acetobacter species. The analysis revealed for the first time a second acetan-like polysaccharide genetic cluster, that is widespread in both genera. All species of the Komagataeibacter possess at least one acetan genetic cluster, while it is present in only one third of the Acetobacter species surveyed.

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The Molecular Physiology of Sodium- and Proton-Coupled Solute Transporters

Physiology ◽

10.1152/physiologyonline.1998.13.3.123 ◽

1998 ◽

Vol 13 (3) ◽

pp. 123-131 ◽

Cited By ~ 3

Author(s):

Angela Steel ◽

Matthias A. Hediger

Keyword(s):

Xenopus Laevis ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Xenopus Laevis Oocytes ◽

Missense Mutations ◽

Molecular Physiology ◽

Inherited Diseases ◽

Biophysical Analysis ◽

Solute Transporters

The expression of cloned Na+- and H+-coupled solute transporters in Xenopus laevis oocytes has permitted detailed molecular and biophysical analysis and illuminated unique mechanistic features. The identification of missense mutations in inherited diseases and site-directed mutagenesis studies have enhanced our understanding of their roles in physiological and pathological processes.

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