Subunit Of Cellulose Synthase Research Articles

Organ-specific expression of genes associated with the UDP-glucose metabolism in sugarcane (Saccharum spp. hybrids)

BackgroundThe importance of uridine 5′-diphosphate glucose (UDP-G) synthesis and degradation on carbon (C) partitioning has been indicated in several studies of plant systems, whereby the kinetic properties and abundance of involved enzymes had a significant effect upon the volume of C moving into the hemicellulose, cellulose and sucrose pools. In this study, the expression of 136 genes belonging to 32 gene families related to UDP-G metabolism was studied in 3 major sugarcane organs (including leaf, internode and root) at 6 different developmental stages in 2 commercial genotypes.ResultsAnalysis of the genes associated with UDP-G metabolism in leaves indicated low expression of sucrose synthase, but relatively high expression of invertase genes, specifically cell-wall invertase 4 and neutral acid invertase 1–1 and 3 genes. Further, organs that are primarily responsible for sucrose synthesis or bioaccumulation, i.e., in source organs (mature leaves) and storage sink organs (mature internodes), had very low expression of sucrose, cellulose and hemicellulose synthesis genes, specifically sucrose synthase 1 and 2, UDP-G dehydrogenase 5 and several cellulose synthase subunit genes. Gene expression was mostly very low in both leaf and mature internode samples; however, leaves did have a comparatively heightened invertase and sucrose phosphate synthase expression. Major differences were observed in the transcription of several genes between immature sink organs (roots and immature internodes). Gene transcription favoured utilisation of UDP-G toward insoluble and respiratory pools in roots. Whereas, there was comparatively higher expression of sucrose synthetic genes, sucrose phosphate synthase 1 and 4, and comparatively lower expression of many genes associated with C flow to insoluble and respiratory pools including myo-Inositol oxygenase, UDP-G dehydrogenase 4, vacuolar invertase 1, and several cell-wall invertases in immature internodes.ConclusionThis study represents the first effort to quantify the expression of gene families associated with UDP-G metabolism in sugarcane. Transcriptional analysis displayed the likelihood that C partitioning in sugarcane is closely related to the transcription of genes associated with the UDP-G metabolism. The data presented may provide an accurate genetic reference for future efforts in altering UDP-G metabolism and in turn C partitioning in sugarcane.

Brittle culm 3, encoding a cellulose synthase subunit 5, is required for cell wall biosynthesis in barley (Hordeum vulgare L.).

The cell wall plays an important role in plant mechanical strength. Cellulose is the major component of plant cell walls and provides the most abundant renewable biomass resource for biofuels on earth. Mutational analysis showed that cellulose synthase (CESA) genes are critical in cell wall biosynthesis in cereal crops like rice. However, their role has not been fully elucidated in barley. In this study, we isolated a brittle culm mutant brittle culm 3 (bc3) derived from Yangnongpi 5 ethyl methanesulfonate (EMS) mutagenesis in barley. The bc3 mutants exhibited reduced mechanical strength of the culms due to impaired thickening of the sclerenchyma cell wall and reduced cellulose and hemicellulose content in the culms. Genetic analysis and map-based cloning revealed that the bc3 mutant was controlled by a single recessive gene and harbored a point mutation in the HvCESA5 gene, generating a premature stop codon near the N-terminal of the protein. Quantitative real-time PCR (qRT-PCR) analysis showed that the HvCESA5 gene is predominantly expressed in the culms and co-expressed with HvCESA4 and HvCESA8, consistent with the brittle culm phenotype of the bc3 mutant. These results indicate that the truncated HvCESA5 affects cell wall biosynthesis leading to a brittle culm phenotype. Our findings provide evidence for the important role of HvCESA5 in cell wall biosynthesis pathway and could be a potential target to modify cell wall in barley.

CRISPR/Cas9‐mediated P‐CR domain‐specific engineering of CESA4 heterodimerization capacity alters cell wall architecture and improves saccharification efficiency in poplar

SummaryCellulose is the most abundant unique biopolymer in nature with widespread applications in bioenergy and high‐value bioproducts. The large transmembrane‐localized cellulose synthase (CESA) complexes (CSCs) play a pivotal role in the biosynthesis and orientation of the para‐crystalline cellulose microfibrils during secondary cell wall (SCW) deposition. However, the hub CESA subunit with high potential homo/heterodimerization capacity and its functional effects on cell wall architecture, cellulose crystallinity, and saccharification efficiency remains unclear. Here, we reported the highly potent binding site containing four residues of Pro435, Trp436, Pro437, and Gly438 in the plant‐conserved region (P‐CR) of PalCESA4 subunit, which are involved in the CESA4‐CESA8 heterodimerization. The CRISPR/Cas9‐knockout mutagenesis in the predicted binding site results in physiological abnormalities, stunt growth, and deficient roots. The homozygous double substitution of W436Q and P437S and heterozygous double deletions of W436 and P437 residues potentially reduced CESA4‐binding affinity resulting in normal roots, 1.5–2‐fold higher plant growth and cell wall regeneration rates, 1.7‐fold thinner cell wall, high hemicellulose content, 37%–67% decrease in cellulose content, high cellulose DP, 25%–37% decrease in cellulose crystallinity, and 50% increase in saccharification efficiency. The heterozygous deletion of W436 increases about 2‐fold CESA4 homo/heterodimerization capacity led to the 50% decrease in plant growth and increase in cell walls thickness, cellulose content (33%), cellulose DP (20%), and CrI (8%). Our findings provide a strategy for introducing commercial CRISPR/Cas9‐mediated bioengineered poplars with promising cellulose applications. We anticipate our results could create an engineering revolution in bioenergy and cellulose‐based nanomaterial technologies.

Regulating the structure of bacterial cellulose by altering the expression of bcsD using CRISPR/dCas9

Gluconacetobacter xylinus is a primary strain producing bacterial cellulose (BC). In G. xylinus, BcsD is a subunit of cellulose synthase and is participated in the assembly process of BC. A series of G. xylinus with different expression levels of the bcsD gene were obtained by using the CRISPR/dCas9 technique. Analysis of the structural characteristics of BC showed that the crystallinity and porosity of BC changed with the expression of bcsD. The porosity varied from 59.95%-84.05%, and the crystallinity varied from 74.26%-93.75%, while the yield of BC did not decrease significantly upon changing the expression levels of bcsD. The results showed that the porosity of bacterial cellulose significantly increased, while the crystallinity was positively correlated with the expression of bcsD, when the expression level of bcsD was below 55.34%. By altering the expression level of the bcsD gene, obtaining BC with different structures but stable yield through a one-step fermentation of G. xylinus was achieved.

Rice Brittle Culm19 Encoding Cellulose Synthase Subunit CESA4 Causes Dominant Brittle Phenotype But has No Distinct Influence on Growth and Grain Yield

BackgroundMechanical strength is a crucial agronomic trait in rice (Oryza sativa), and brittle mutants are thought suitable materials to investigate the mechanism of cell wall formation. So far, almost all brittle mutants are recessive, and most of them are defected in multiple morphologies and/or grain yield, limiting their application in hybrid breeding and in rice straw recycling.ResultsWe identified a semi-dominant brittle mutant Brittle culm19 (Bc19) isolated from the japonica variety Nipponbare through chemical mutagenesis. The mutant showed the same apparent morphologies and grain yield to the wild type plant except for its weak mechanical strength. Its development of secondary cell wall in sclerenchyma cells was affected, along with reduced contents of cellulose, hemicellulose, lignin and sugars in culms and leaves. Positional cloning suggested that the Bc19 gene was allelic to OsCESA4, encoding one of the cellulose synthase A (CESA) catalytic subunits. In this mutant, a C-to-T substitution occurred in the coding sequence of BC19, causing the P507S missense mutation in its encoded product, which was located in the second cytoplasmic region of the OsCESA4 protein. Furthermore, introducing mutant gene Bc19 into the wild-type plant resulted in brittle plants, confirming that the P507S point mutation in OsCESA4 protein was responsible for the semi-dominant brittle phenotype of Bc19 mutant. Reverse correlation was revealed between cellulose contents and expression levels of mutant gene Bc19 among the homozygous mutant, the hybrid F1 plant, and the Bc19 overexpression transgenic plants, implying that gene Bc19 might affect cellulose synthesis in a dosage-dependent manner.ConclusionsBc19, a semi-dominant brittle mutant allele of gene OsCESA4, was identified using map-based cloning approach. The mutated protein of Bc19 possessing the P507S missense mutation behaved in a dosage-dependent semi-dominant manner. Unique brittle effect on phenotype and semi-dominant genetic quality of gene Bc19 indicated its potential application in grain-straw dual-purpose hybrid rice breeding.

Fiber Cell-Specific Expression of the VP16-Fused Ethylene Response Factor 41 Protein Increases Biomass Yield and Alters Lignin Composition.

Arabidopsis thaliana transcription factors belonging to the ERFIIId and ERFIIIe subclade (ERFIIId/e) of the APETALA 2/ethylene response factor (AP2/ERF) family enhance primary cell wall (PCW) formation. These transcription factors activate expression of genes encoding PCW-type cellulose synthase (CESA) subunits and other genes for PCW biosynthesis. In this study, we show that fiber-specific expression of ERF035-VP16 and ERF041-VP16, which are VP16-fused proteins of ERFIIId/e members, promote cell wall thickening in a wild-type background with a concomitant increase of alcohol insoluble residues (cell wall content) per fresh weight (FW) and monosaccharides related to the PCW without affecting plant growth. Furthermore, in the ERF041-VP16 lines, the total amount of lignin and the syringyl (S)/guaiacyl (G) ratio decreased, and the enzymatic saccharification yield of glucose from cellulose per fresh weight improved. In these lines, PCW-type CESA genes were upregulated and ferulate 5-hydropxylase1 (F5H1), which is necessary for production of the S unit lignin, was downregulated. In addition, various changes in the expression levels of transcription factors regulating secondary cell wall (SCW) formation were observed. In conclusion, fiber cell-specific ERF041-VP16 improves biomass yield, increases PCW components, and alters lignin composition and deposition and may be suitable for use in future molecular breeding programs of biomass crops.

Molecular Dynamics Simulation of Cellulose Synthase Subunit D Octamer with Cellulose Chains from Acetic Acid Bacteria: Insight into Dynamic Behaviors and Thermodynamics on Substrate Recognition.

The present study reports the building of a computerized model and molecular dynamics (MD) simulation of cellulose synthase subunit D octamer (CesD) from Komagataeibacter hansenii. CesD was complexed with four cellulose chains having DP = 12 (G12) by model building, which revealed unexpected S-shaped pathways with bending regions. Combined conventional and accelerated MD simulations of CesD complex models were carried out, while the pyranose ring conformations of the glucose residues were restrained to avoid undesirable deviations of the ring conformation from the 4C1 form. The N-terminal regions and parts of the secondary structures of CesD established appreciable contacts with the G12 chains. Hybrid quantum mechanical (QM) and molecular mechanical (MM) simulations of the CesD complex model were performed. Glucose residues located at the pathway bends exhibited reversible changes to the ring conformation into either skewed or boat forms, which might be related to the function of CesD in regulating microfibril production.

Local c-di-GMP Signaling in the Control of Synthesis of the E. coli Biofilm Exopolysaccharide pEtN-Cellulose

In many bacteria, the biofilm-promoting second messenger c-di-GMP is produced and degraded by multiple diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively. High target specificity of some of these enzymes has led to theoretical concepts of “local” c-di-GMP signaling. In Escherichia coli K-12, which has 12 DGCs and 13 PDEs, a single DGC, DgcC, is specifically required for the biosynthesis of the biofilm exopolysaccharide pEtN-cellulose without affecting the cellular c-di-GMP pool, but the mechanistic basis of this target specificity has remained obscure. DGC activity of membrane-associated DgcC, which is demonstrated in vitro in nanodiscs, is shown to be necessary and sufficient to specifically activate cellulose biosynthesis in vivo. DgcC and a particular PDE, PdeK (encoded right next to the cellulose operon), directly interact with cellulose synthase subunit BcsB and with each other, thus establishing physical proximity between cellulose synthase and a local source and sink of c-di-GMP. This arrangement provides a localized, yet open source of c-di-GMP right next to cellulose synthase subunit BcsA, which needs allosteric activation by c-di-GMP. Through mathematical modeling and simulation, we demonstrate that BcsA binding from the low cytosolic c-di-GMP pool in E. coli is negligible, whereas a single c-di-GMP molecule that is produced and released in direct proximity to cellulose synthase increases the probability of c-di-GMP binding to BcsA several hundred-fold. This local c-di-GMP signaling could provide a blueprint for target-specific second messenger signaling also in other bacteria where multiple second messenger producing and degrading enzymes exist.

The Escherichia coli cellulose synthase subunit G (BcsG) is a Zn2+-dependent phosphoethanolamine transferase

Bacterial biofilms are cellular communities that produce an adherent matrix. Exopolysaccharides are key structural components of this matrix and are required for the assembly and architecture of biofilms produced by a wide variety of microorganisms. The human bacterial pathogens Escherichia coli and Salmonella enterica produce a biofilm matrix composed primarily of the exopolysaccharide phosphoethanolamine (pEtN) cellulose. Once thought to be composed of only underivatized cellulose, the pEtN modification present in these matrices has been implicated in the overall architecture and integrity of the biofilm. However, an understanding of the mechanism underlying pEtN derivatization of the cellulose exopolysaccharide remains elusive. The bacterial cellulose synthase subunit G (BcsG) is a predicted inner membrane-localized metalloenzyme that has been proposed to catalyze the transfer of the pEtN group from membrane phospholipids to cellulose. Here we present evidence that the C-terminal domain of BcsG from E. coli (EcBcsGΔN) functions as a phosphoethanolamine transferase in vitro with substrate preference for cellulosic materials. Structural characterization of EcBcsGΔN revealed that it belongs to the alkaline phosphatase superfamily, contains a Zn2+ ion at its active center, and is structurally similar to characterized enzymes that confer colistin resistance in Gram-negative bacteria. Informed by our structural studies, we present a functional complementation experiment in E. coli AR3110, indicating that the activity of the BcsG C-terminal domain is essential for integrity of the pellicular biofilm. Furthermore, our results established a similar but distinct active-site architecture and catalytic mechanism shared between BcsG and the colistin resistance enzymes.

Seed-specific down-regulation of Arabidopsis CELLULOSE SYNTHASE 1 or 9 reduces seed cellulose content and differentially affects carbon partitioning.

Seed-specific down-regulation of AtCESA1 and AtCESA9, which encode cellulose synthase subunits, differentially affects seed storage compound accumulation in Arabidopsis. High amounts of cellulose can negatively affect crop seed quality, and, therefore, diverting carbon partitioning from cellulose to oil, protein and/or starch via molecular breeding may improve seed quality. To determine the effect of seed cellulose content reduction on levels of storage compounds, Arabidopsis thaliana CELLULOSE SYNTHASE1 (AtCESA1) and AtCESA9 genes, which both encode cellulose synthase subunits, were individually down-regulated using seed-specific intron-spliced hairpin RNA (hpRNAi) constructs. The selected seed-specific AtCESA1 and AtCESA9 Arabidopsis RNAi lines displayed reduced cellulose contents in seeds, and exhibited no obvious visual phenotypic growth defects with the exception of a minor effect on early root development in AtCESA1 RNAi seedlings and early hypocotyl elongation in the dark in both types of RNAi line. The seed-specific down-regulation of AtCESA9 resulted in a reduction in seed weight compared to empty vector controls, which was not observed in AtCESA1 RNAi lines. In terms of effects on carbon partitioning, AtCESA1 and AtCESA9 RNAi lines exhibited distinct effects. The down-regulation of AtCESA1 led to a ~ 3% relative increase in seed protein content (P = 0.04) and a ~ 3% relative decrease in oil content (P = 0.02), but caused no alteration in soluble glucose levels. On the contrary, AtCESA9 RNAi lines did not display a significant reduction in seed oil, protein or soluble glucose content. Taken together, our results indicate that the seed-specific down-regulation of AtCESA1 causes alterations in seed storage compound accumulation, while the effect of AtCESA9 on carbon partitioning is absent or minor in Arabidopsis.

Genome sequencing and phylogenetic analysis of K1G4: a new Komagataeibacter strain producing bacterial cellulose from different carbon sources.

The objective of this study was to evaluate the ability of a new Komagataeibacter xylinus strain in producing bacterial cellulose from glucose, mannitol and glycerol, and to assess the genome sequencing with special focus on bacterial cellulose related genes. Bacterial cellulose production during 9 days of cultivation was tested in glucose, mannitol and glycerol, respectively. Differences in the bacterial cellulose kinetic formation was observed, with a final yield of 9.47 g/L in mannitol, 8.30 g/L in glycerol and 7.57 g/L in glucose, respectively. The draft genome sequencing of K1G4 was produced, revealing a genome of 3.09 Mbp. Two structurally completed cellulose synthase operons and a third copy of the catalytic subunit of cellulose synthase were found. By using phylogenetic analysis, on the entire rRNA operon sequence, K1G4 was found to be closely related to Komagataeibacter xylinus LMG 1515T and K. xylinus K2G30. The different yields of bacterial cellulose produced on glucose, mannitol and glycerol can be correlated with the third copy of bcsAB operon harboured by K1G4, making it a versatile strain for industrial applications.

Genome-wide transcriptomic analysis during rhizome development of ginger (Zingiber officinale Roscoe.) reveals hormone and transcriptional regulation involved in cellulose production

Ginger is a popular vegetable crop primarily consumed in Chongqing and Sichuan, China. The rhizome becomes tough and fibrous rapidly with the tissue maturation, resulting in loss of edible quality. To characterize biochemical and molecular mechanisms of rhizome texture modification, we investigated the changes of the cell wall components, hormones and transcriptome profiles during rhizome development. With rhizomes maturation, the contents of cellulose and hemicellulose increased gradually, and no obvious change was observed in lignin accumulation. The levels of ABA and cytokinin exhibited a gradual decline during maturation of ginger rhizome, which were negatively correlated with cellulose content, while GA3 displayed an increasing trend and a positive correlation with cellulose production. Transcriptomic analysis identified candidate genes involved in cellulose biosynthesis including cellulose synthase subunit A (CesA) genes, hormone metabolism and signaling genes, and NAC-MYB-based transcription factors. Further analysis demonstrated ZoCESA7, ZoNAC16 and ZoMYB37/39/41, homologs of AtCESA7, AtVND6/7 and AtMYB46/83, respectively, positively correlated with cellulose contents, whereas ZoNAC3/9/13 and ZoMYB7/11/18, homologs of AtVND6/7 and AtMYB4, respectively, negatively correlated with cellulose levels, indicating their involvements in cellulose synthesis during rhizome development. These findings provide new insight into molecular events of cellulose accumulation mainly mediated by hormones and transcription factors in ginger rhizomes.

Cellulose defects in the Arabidopsis secondary cell wall promote early chloroplast development.

Lincomycin (LIN)-mediated inhibition of protein synthesis in chloroplasts prevents the greening of seedlings, represses the activity of photosynthesis-related genes in the nucleus, including LHCB1.2, and induces the phenylpropanoid pathway, resulting in the production of anthocyanins. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of LIN or other inhibitors of early chloroplast development. In a screen using concentrations of LIN lower than those employed to isolate gun mutants, we have identified happy on lincomycin (holi) mutants. Several holi mutants show an increased tolerance to LIN, exhibiting de-repressed LHCB1.2 expression and chlorophyll synthesis in seedlings. The mutations responsible were identified by whole-genome single-nucleotide polymorphism (SNP) mapping, and most were found to affect the phenylpropanoid pathway; however, LHCB1.2 expression does not appear to be directly regulated by phenylpropanoids, as indicated by the metabolic profiling of mutants. The most potent holi mutant is defective in a subunit of cellulose synthase encoded by IRREGULAR XYLEM3, and comparative analysis of this and other cell-wall mutants establishes a link between secondary cell-wall integrity and early chloroplast development, possibly involving altered ABA metabolism or sensing.

Overexpression of MusaNAC68 reduces secondary wall thickness of xylem tissue in banana

Secondary wall is an important component of xylem tissue, as it maintains the integrity of the vessel elements and provides mechanical strength for upward growth of plants. Enough prior evidence has linked secondary wall thickening with auxin-signaling pathway. Hence, plants showing auxin-mediated effects might demonstrate alteration of secondary wall depositions. GUS expression from promoter of NAC68 (pMusaNAC68) was observed in many organs with strong expression in vascular tissues. Transgenic banana plants overexpressing NAC68 transcription factor were analyzed for lignin and secondary wall depositions. Strong reduction of lignin in cross-sections of transgenic lines was observed by phloroglucinol-HCl and toluidine blue-O staining. The reduction of secondary wall thickness observed after calcofluor white staining was substantiated by data from scanning electron microscopy indicating reproducibly lower secondary wall depositions. A strong reduction in transcript levels of PAL, 4CL, C4H, COMT and CcOAMT was observed due to the overexpression of NAC68. Furthermore, substantial elevation of MYB transcription factors such as MYB4a-like and MYB4b-like which are probable repressors of phenylpropanoid biosynthesis pathway was also observed. Expression of MYB transcription factors, MYB85-like and MYB58-like which are probable activators of secondary wall depositions, was remarkably downregulated after NAC68 overexpression. Transgenic lines also showed altered expression of genes coding for cellulose synthase subunits with remarkable elevation in expression of a close homologue of Arabidopsis CesA8, which functions in cellulose deposition during secondary wall development. The study indicated a novel function of NAC68 transcription factor in regulating secondary wall thickening and will enhance our knowledge about crosstalk between auxin-signaling and secondary wall depositions.

Patterned Deposition of Xylan and Lignin is Independent from that of the Secondary Wall Cellulose of Arabidopsis Xylem Vessels.

The secondary cell wall (SCW) of xylem vessel cells provides rigidity and strength that enables efficient water conduction throughout the plant. To gain insight into SCW deposition, we mutagenized Arabidopsis thaliana VASCULAR-RELATED NAC-DOMAIN7-inducible plant lines, in which ectopic protoxylem vessel cell differentiation is synchronously induced. The baculites mutant was isolated based on the absence of helical SCW patterns in ectopically-induced protoxylem vessel cells, and mature baculites plants exhibited an irregular xylem (irx) mutant phenotype in mature plants. A single nucleic acid substitution in the CELLULOSE SYNTHASE SUBUNIT 7 (CESA7) gene in baculites was identified: while the mutation was predicted to produce a C-terminal truncated protein, immunoblot analysis revealed that cesa7bac mutation results in loss of production of CESA7 proteins, indicating that baculites is a novel cesa7 loss-of-function mutant. In cesa7bac , despite a lack of patterned cellulose deposition, the helically-patterned deposition of other SCW components, such as the hemicellulose xylan and the phenolic polymer lignin, was not affected. Similar phenotypes were found in another point mutation mutant cesa7mur10-2 , and an established knock-out mutant, cesa7irx3-4 Taken together, we propose that the spatio-temporal deposition of different SCW components, such as xylan and lignin, is not dependent on cellulose patterning.

Identifying Stable Fragments of Arabidopsis thaliana Cellulose Synthase Subunit 3 by Yeast Display.

Determining structures of large, complex proteins remains challenging, especially for transmembrane proteins, as the protein size increases. Arabidopsis thaliana cellulose synthesis complex is a large, multimeric complex located in the plant cell membrane that synthesizes cellulose microfibrils in the plant cell wall. Despite the biological and economic importance of cellulose and therefore cellulose synthesis, many aspects of the cellulase synthase complex (CSC) structure and function are still unknown. Here, yeast surface display (YSD) is used to determine the full-length expression of A. thaliana cellulose synthase 3 (AtCesA3) fragments. The level of stably-folded AtCesA3 fragments displayed on the yeast surface are determined using flow cytometric analysis of differential surface expression of epitopes flanking the AtCesA3 fragment. This technique provides a fast and simple method for examining folding and expression of protein domains and fragments of complex proteins.

Enhancement of crystallinity of cellulose produced by Escherichia coli through heterologous expression of bcsD gene from Gluconacetobacter xylinus.

To evaluate the crystallinity index of the cellulose produced by Escherichia coli Nissle 1917 after heterologous expression of the cellulose synthase subunit D (bcsD) gene of Gluconacetobacter xylinus BPR2001. The bcsD gene of G. xylinus BPR2001 was expressed in E. coli and its protein product was visualized using SDS-PAGE. FTIR analysis showed that the crystallinity index of the cellulose produced by the recombinants was 0.84, which is 17% more than that of the wild type strain. The increased crystallinity index was also confirmed by X-ray diffraction analysis. The cellulose content was not changed significantly after over-expressing the bcsD. The bcsD gene can improve the crystalline structure of the bacterial cellulose but there is not any significant difference between the amounts of cellulose produced by the recombinant and wild type E. coli Nissle 1917.

Biofilm formation and cellulose expression by Bordetella avium 197N, the causative agent of bordetellosis in birds and an opportunistic respiratory pathogen in humans

Although bacterial cellulose synthase (bcs) operons are widespread within the Proteobacteria phylum, subunits required for the partial-acetylation of the polymer appear to be restricted to a few γ-group soil, plant-associated and phytopathogenic pseudomonads, including Pseudomonas fluorescens SBW25 and several Pseudomonas syringae pathovars. However, a bcs operon with acetylation subunits has also been annotated in the unrelated β-group respiratory pathogen, Bordetella avium 197N. Our comparison of subunit protein sequences and GC content analyses confirms the close similarity between the B. avium 197N and pseudomonad operons and suggests that, in both cases, the cellulose synthase and acetylation subunits were acquired as a single unit. Using static liquid microcosms, we can confirm that B. avium 197N expresses low levels of cellulose in air–liquid interface biofilms and that biofilm strength and attachment levels could be increased by elevating c-di-GMP levels like the pseudomonads, but cellulose was not required for biofilm formation itself. The finding that B. avium 197N is capable of producing cellulose from a highly-conserved, but relatively uncommon bcs operon raises the question of what functional role this modified polymer plays during the infection of the upper respiratory tract or survival between hosts, and what environmental signals control its production.

Cellulosic fibres of flax recruit both primary and secondary cell wall cellulose synthases during deposition of thick tertiary cell walls and in the course of graviresponse.

Cellulose synthesising complex consists of cellulose synthase (CESA) subunits encoded by a multigene family; different sets of CESA genes are known to be expressed during primary and secondary cell wall formation. We examined the expression of LusCESAs in flax (Linum usitatissimum L.) cellulosic fibres at various stages of development and in the course of graviresponse by means of RNA-Seq and quantitative PCR. Transcripts for both primary and secondary cell wall-related CESAs were abundant in fibres depositing highly cellulosic tertiary cell walls. Gravistimulation of flax plants temporally increased the abundance of CESA transcripts, specifically in phloem fibres located at the pulling stem side. Construction of coexpression networks for LusCESAs revealed that both primary and secondary cell wall-related CESAs were involved in the joint coexpression group in fibres depositing tertiary cell walls, as distinct from other tissues, where these genes were within separate groups. The obtained data suggest that fibres depositing tertiary cell walls have a specific mechanism of cellulose biosynthesis and a specific way of its regulation.

Tissue-specific transcriptome analysis within the maturing sugarcane stalk reveals spatial regulation in the expression of cellulose synthase and sucrose transporter gene families.

Sugarcane (Saccharum spp. hybrids) accumulates high concentrations of sucrose in its mature stalk and a considerable portion of carbohydrate metabolism is also devoted to cell wall synthesis and fibre production. We examined tissue-specific expression patterns to explore the spatial deployment of pathways responsible for sucrose accumulation and fibre synthesis within the stalk. We performed expression profiling of storage parenchyma, vascular bundles and rind dissected from a maturing stalk internode of sugarcane, identifying ten cellulose synthase subunit genes and examining significant differences in the expression of their corresponding transcripts and those of several sugar transporters. These were correlated with differential expression patterns for transcripts of genes encoding COBRA-like proteins and other cell wall metabolism-related proteins. The sugar transporters genes ShPST2a, ShPST2b and ShSUT4 were significantly up-regulated in storage parenchyma while ShSUT1 was up-regulated in vascular bundles. Two co-ordinately expressed groups of cell wall related transcripts were also identified. One group, associated with primary cell wall synthesis (ShCesA1, ShCesA7, ShCesA9 and Shbk2l3), was up-regulated in parenchyma. The other group, associated with secondary cell wall synthesis (ShCesA10, ShCesA11, ShCesA12 and Shbk-2), was up-regulated in rind. In transformed sugarcane plants, the ShCesA7 promoter conferred stable expression of green fluorescent protein preferentially in the storage parenchyma of the maturing stalk internode. Our results indicate that there is spatial separation for elevated expression of these important targets in both sucrose accumulation and cell wall synthesis, allowing for increased clarity in our understanding of sucrose transport and fibre synthesis in sugarcane.