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

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Abstract

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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Authors and Affiliations

  1. Molecular Biosciences, College of Natural Sciences, The University of Texas at Austin, 100 West 24th Street, Austin, TX, 78712, USA

    Kalpa Mehta, Sarah Pfeffer & R. Malcolm Brown Jr.

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  1. Kalpa Mehta
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  2. Sarah Pfeffer
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  3. R. Malcolm Brown Jr.
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Correspondence to R. Malcolm Brown Jr..

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 17 kb)

Supplementary material 2 (PDF 12 kb)

Supplementary material 3 (PDF 19 kb)

The wild type G. xylinus AY201 (MPG 16850 kb)

Time lapse videos of the acsDdm mutant (MPG 12282 kb)

10570_2014_521_MOESM6_ESM.tif

Full color colony morphology photomicrographs of the G. xylinus AY201 wild type (a–d) and acsDdm mutant (e-h) under bright-field (a, b, e, and f), polarization extinction (c and g) and first order red polarization (d and h) with scale bar representing 100 μm (a and e) and 25 μm (b-d and f-g) (TIFF 1414 kb)

Full cell and ribbon images of the wild type G. xylinus AY201 (a and b) and the acsDdm mutant (c and d) (TIFF 317 kb)

10570_2014_521_MOESM8_ESM.tif

Negatively stained cellulose synthesized from individual cells with enlargements showing examples of the ribbons (a) and bundles of microfibrils within a ribbon (b) in the G. xylinus AY201 wild type; and the ribbons (c) and bundles of microfibrils within a ribbon (d) in the acsDdm mutant. Scale bar represents 25 nm (a and c) and 10 nm (b and c) (TIFF 526 kb)

10570_2014_521_MOESM9_ESM.tif

Examples of negatively stained cellulose synthesized by the G. xylinus AY201 wild type (a-h) and acsDdm mutant (i-p) that were used to obtain measurement data. Measurement bars were added for clarification. Scale bar represents 20 nm (a, b, e, f, 1, j, m, n) and 5 nm (c, d, g, h, k, o, l, and p) (TIFF 1154 kb)

10570_2014_521_MOESM10_ESM.tif

Negatively stained band material of the wild type G. xylinus AY201 all indicating linear TC orientation along the long axis of the cell (a-e). Arrows designate longitudinal orientation (b) on an enlarged version of the cell (a). Scale bar represents 0.5 μm (TIFF 665 kb)

10570_2014_521_MOESM11_ESM.tif

Negatively stained band material of the acsDdm mutant indicating various orientations of band material on the cell. Arrows designate transverse orientation on one cell (a); transverse and longitudinal orientation on the same cell (b); transverse only (c); transverse and longitudinal (d); and diagonal orientation (e). Scale bar represents 0.5 μm (TIFF 525 kb)

10570_2014_521_MOESM12_ESM.tif

Phase contrast of the wild type G. xylinus AY201 (a and b) and the acsDdm mutant (c and d) scale bar represents 10 um. Average cell dimensions (e) (TIFF 262 kb)

10570_2014_521_MOESM13_ESM.tif

Schematic illustrations of the observed movement due to possible linear TC orientation in the wild type G. xylinus AY201 (a) and the acsDdm mutant (b) (TIFF 144 kb)

10570_2014_521_MOESM14_ESM.tif

Schematic diagram of an example of linear TC arrangement which could result in reduced cellulose biosynthesis. Liner TC orientation along the longitudinal axis of the cell (a); linear TC orientation transverse to the long axis (b); movement of the cell parallel to the long axis (c); movement of the cell perpendicular to the long axis (d); and possible circular movement of the cell as both directional forces are combined (e) (TIFF 56 kb)

10570_2014_521_MOESM15_ESM.tif

Negatively stained band material synthesized at room temperature under agitated conditions by the wild type G. xylinus (a) and the acsDdm mutant (b). Scale bar represents 0.5 μm (TIFF 561 kb)

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Mehta, K., Pfeffer, S. & Brown, R.M. Characterization of an acsD disruption mutant provides additional evidence for the hierarchical cell-directed self-assembly of cellulose in Gluconacetobacter xylinus . Cellulose 22, 119–137 (2015). https://doi.org/10.1007/s10570-014-0521-y

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