AcsA-AcsB: The core of the cellulose synthase complex from Gluconacetobacter hansenii ATCC23769.

The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA-AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA-AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA-AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx.

Isolation and characterization of two cellulose morphology mutants of Gluconacetobacter hansenii ATCC23769 producing cellulose with lower crystallinity.

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of ß-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.

Identification and characterization of non-cellulose-producing mutants of Gluconacetobacter hansenii generated by Tn5 transposon mutagenesis.

The acs operon of Gluconacetobacter is thought to encode AcsA, AcsB, AcsC, and AcsD proteins that constitute the cellulose synthase complex, required for the synthesis and secretion of crystalline cellulose microfibrils. A few other genes have been shown to be involved in this process, but their precise role is unclear. We report here the use of Tn5 transposon insertion mutagenesis to identify and characterize six non-cellulose-producing (Cel(-)) mutants of Gluconacetobacter hansenii ATCC 23769. The genes disrupted were acsA, acsC, ccpAx (encoding cellulose-complementing protein [the subscript "Ax" indicates genes from organisms formerly classified as Acetobacter xylinum]), dgc1 (encoding guanylate dicyclase), and crp-fnr (encoding a cyclic AMP receptor protein/fumarate nitrate reductase transcriptional regulator). Protein blot analysis revealed that (i) AcsB and AcsC were absent in the acsA mutant, (ii) the levels of AcsB and AcsC were significantly reduced in the ccpAx mutant, and (iii) the level of AcsD was not affected in any of the Cel(-) mutants. Promoter analysis showed that the acs operon does not include acsD, unlike the organization of the acs operon of several strains of closely related Gluconacetobacter xylinus. Complementation experiments confirmed that the gene disrupted in each Cel(-) mutant was responsible for the phenotype. Quantitative real-time PCR and protein blotting results suggest that the transcription of bglAx (encoding ß-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bglAx knockout mutant, generated via homologous recombination, produced only ∼16% of the wild-type cellulose level. Since the crp-fnr mutant did not produce any cellulose, Crp/Fnr may regulate the expression of other gene(s) involved in cellulose biosynthesis.

Processing of cellulose synthase (AcsAB) from Gluconacetobacter hansenii 23769.

The cellulose synthase protein (AcsAB) is encoded by a single gene in Gluconacetobacter hansenii ATCC 23769. We have examined the processing pattern of this enzyme and the localization of the cleavage products by heterologously expressing the truncated portions of the AcsAB protein and using specific antibodies generated against these regions. We found that the AcsAB protein is processed into three polypeptide subunits of molecular masses 46kDa, 34kDa and 95kDa. The 46kDa polypeptide (AcsA(cat)) harbors the conserved glycosyltransferase domain and hence contains the catalytic subunit of the enzyme. This polypeptide is localized in the cytoplasmic membrane. The 34kDa polypeptide (AcsA(reg)) is the regulatory subunit with the cyclic diGMP-binding PilZ domain. This polypeptide is largely cytoplasmic. The 95kDa subunit (AcsB) is of unknown function and contains a predicted signal peptide at its N-terminus. This subunit is localized in the outer membrane. In addition to this, we have also localized the AcsC protein in the outer membrane, confirming its predicted localization based on the OM-signal sequence at its N-terminus.

Genome sequence of a cellulose-producing bacterium, Gluconacetobacter hansenii ATCC 23769.

The Gram-negative bacterium Gluconacetobacter hansenii is considered a model organism for studying cellulose synthesis. We have determined the genome sequence of strain ATCC 23769.