Processing of cellulose synthase (AcsAB) from Gluconacetobacter hansenii 23769

Iyer, Prashanti R.; Liu, Yu An; Deng, Ying; McManus, J. Barry; Kao, Teh Hui

doi:10.1016/j.abb.2012.12.002

Cited by 11 publications

(12 citation statements)

References 34 publications

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“…Cleavage after an AxA motif suggests that a signal peptidase, found on the periplasmic side of the cytoplasmic membrane, is responsible for processing. The present results for AcsA are in contrast to our previous work showing that AcsA is further processed into two smaller fragments of 46 kDa and 34 kDa [29]. The weakness and variability of these two smaller bands on Western blots was the result of AcsA being unstable to boiling in the presence of SDS.…”

Section: Processing Of Acsa-acsbcontrasting

confidence: 86%

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

McManus

Deng

Nagachar

et al. 2016

Enzyme and Microbial Technology

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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.

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Section: Processing Of Acsa-acsbcontrasting

confidence: 86%

“…1B). The preparation and specificity of these antibodies had been documented in our previous paper [29]. The Western blot revealed an 85 kDa cross-reactive band when using anti-AcsA 1 and anti-AcsA 3 .…”

Section: Purification Of Acsa-acsb By Product Entrapmentmentioning

confidence: 98%

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

McManus

Deng

Nagachar

et al. 2016

Enzyme and Microbial Technology

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“…It has a PilZ domain that is responsive to c-di-GMP and, when activated, the AcsA protein changes conformation to allow for UDPG to access the catalytic site where polymerization commences (Ross et al 1987;Romling et al 2005;Morgan et al 2013). The AcsB protein has a C-terminal membrane spanning domain with the major part extending into the periplasm is thought to be weakly associated with the cytoplasmic membrane and extends into the periplasm where it is suggested to be tethered to the outer membrane (Iyer et al 2013;Morgan et al 2013). The function of AcsB is not known, but it is suggested that the cellulose binding domains found in the protein may support the unidirectional movement of the polymerized glucose chain from the cytoplasmic to the periplasmic space.…”

Section: Introductionmentioning

confidence: 96%

Characterization of an acsD disruption mutant provides additional evidence for the hierarchical cell-directed self-assembly of cellulose in Gluconacetobacter xylinus

2014

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The acsD gene is involved in cellulose biosynthesis among the Acetobacter species. In the current study, we created an acsD disruption mutant in the acsABCD cellulose synthase operon of Gluconacetobacter xylinus and characterized the resulting cellulose to aid in providing insight into the function of the acsD gene. Both the wild type G. xylinus AY201 (derivative of Gluconacetobacter hansenii ATCC 23769) and the acsD disruption mutant produced crystalline cellulose I microfibrils. The cellulose produced by both appeared to be synthesized from an aggregate of pores known as a linear terminal complex; however the total cellulose synthesized was 10 % that of the wild type G. xylinus AY201. TEM observations of the acsD disruption mutant confirmed that microfibrils and bundles of microfibrils were similar in size to the G. xylinus AY201 wild type; however, the final ribbon dimensions were narrower (53.4 ± 13.1 nm wt, vs. 28.2 ± 8.2 nm). Additional TEM observations of the mutant cells incubated at 4°C revealed an abnormal linear terminal complex orientation whereby the resulting band material could be observed in a transverse orientation as well as longitudinally to the long axis of the cell. Taken together, these data strongly suggest that acsD aids in the proper orientation of the linear terminal complexes along the longitudinal axis of the cell indicating the AcsD protein is involved in the final level of the hierarchical assembly of cellulose resulting in highly efficient cellulose synthesis.

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“…Electrophoresis was carried out at 200 V for ϳ50 min. The proteins were transferred onto polyvinylidene difluoride membranes, followed by blotting with anti-AcsA cat , -AcsB, -AcsC, and -AcsD antibodies (6) or an anti-Bgl Ax antibody. The details of these antibodies are listed in Table 1.…”

Section: Methodsmentioning

confidence: 99%

“…Their protein products have been proposed to form the CSC that synthesizes cellulose using UDP-glucose as substrate and secrets the cellulose microfibrils through the outer membrane. Some G. xylinus strains (e.g., ATCC 53582) produce separate AcsA and AcsB proteins, whereas G. hansenii ATCC 23769 synthesizes AcsA and AcsB as a fusion protein AcsAB (5), which is processed into three polypeptides of molecular masses 34, 46, and 95 kDa (6). Although the precise cleavage sites are unknown, the 46-kDa polypeptide was named AcsA cat since it contains a conserved "D,D,D,QXXRW" motif found in cellulose synthases or cellulose synthase-like proteins (7,8).…”

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confidence: 99%

Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn 5 Transposon Mutagenesis

et al. 2013

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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, ccp Ax (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 ccp Ax 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 bgl Ax (encoding ␤-glucosidase and located immediately downstream from acsD) was strongly dependent on Crp/Fnr. A bgl Ax knockout mutant, generated via homologous recombination, produced only ϳ16% 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.

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Processing of cellulose synthase (AcsAB) from Gluconacetobacter hansenii 23769

Cited by 11 publications

References 34 publications

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

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

Characterization of an acsD disruption mutant provides additional evidence for the hierarchical cell-directed self-assembly of cellulose in Gluconacetobacter xylinus

Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn 5 Transposon Mutagenesis

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