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Microbial Ecology

Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degradation

Ace M. Baty, III, Callie C. Eastburn, Zhenjun Diwu, Somkiet Techkarnjanaruk, Amanda E. Goodman, Gill G. Geesey
Ace M. Baty
Department of Microbiology 1 and Center for Biofilm Engineering, 2 Montana State University, Bozeman, Montana 59717,
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Callie C. Eastburn
Department of Microbiology 1 and Center for Biofilm Engineering, 2 Montana State University, Bozeman, Montana 59717,
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Zhenjun Diwu
Molecular Probes, Inc., Eugene, Oregon 97402, 3 and
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Somkiet Techkarnjanaruk
Center for Biofilm Engineering, 2 Montana State University, Bozeman, Montana 59717,
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Amanda E. Goodman
School of Biological Sciences, The Flinders University of South Australia, Adelaide, South Australia 5001, Australia 4
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Gill G. Geesey
Department of Microbiology 1 and Center for Biofilm Engineering, 2 Montana State University, Bozeman, Montana 59717,
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DOI: 10.1128/AEM.66.8.3566-3573.2000
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    Fig. 1.

    Schematic representation of the experimental design showing LFCs, CSTR, and peristaltic pumps (⊗). The silicon and chitin LFCs were run in tandem. Epi, epifluorescence.

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    Fig. 2.

    Histograms of RFI based on chiA gene activity in a population of cells from a GlcNAc-grown batch culture, a glutamate-grown batch culture, a 400-h starved culture, or the effluent of LFCs containing a silicon or chitin substratum at 150 h postinoculation. Cells displaying RFIs between 1 and 11 were defined as down-expressed for chiA, while cells displaying RFIs between 12 and 1,000 were defined as up-expressed for chiA. Cell counts refer to the number of cells in the population displaying a particular RFI.

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    Fig. 3.

    Reflected DIC (A and C) and epifluorescence (B and D) micrographs of silicon (A and B) and chitin (C and D) surfaces at 150 h postinoculation. Shown are low-magnification (B and D) and high-magnification (E and F) reflected DIC-epifluorescence image overlays showing the tight association between chiAup-expressed cells (green) and chitinase activity as reported by cleavage of the ELF-97–N-acetyl-β-d-glucosaminide enzyme substrate (red) at the single-cell level. Overlap of chiAgene activity (green) and chitinase activity (red) appears as yellow.

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  • Table 1.

    Contribution of subpopulations to total cell population associated with chitin and silicon surfaces

    Surfacea% Subpopulation (mean ± SD)
    chiA positive-chitinase positivechiA positive-chitinase negativechiA negative-chitinase negativechiA negative-chitinase positiveCell negative-chitinase positive
    Chitin25.2 ± 7.131.8 ± 8.739.0 ± 12.41.33 ± 0.191.61 ± 0.29
    Silicon20.8 ± 4.644.2 ± 8.232.7 ± 6.11.25 ± 0.270.88 ± 0.26
    • ↵a A total of 1,780 and 1,149 cells were counted on the chitin and silicon surfaces, respectively.

  • Table 2.

    Distribution of chitinase activity among various compartments at 150 h postinoculation

    SurfaceRate of MUF-GlcNAc hydrolysis (μmol liter−1 h−1) (mean ± SD)
    Surface associatedSurface associated + free enzyme + free cellsSurface associated + free enzyme (<0.2-μm-pore-size filtrate)
    Chitin0.47 ± 0.050.50 ± 0.040.46 ± 0.05
    Silicon0.24 ± 0.030.21 ± 0.020.22 ± 0.02
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Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degradation
Ace M. Baty III, Callie C. Eastburn, Zhenjun Diwu, Somkiet Techkarnjanaruk, Amanda E. Goodman, Gill G. Geesey
Applied and Environmental Microbiology Aug 2000, 66 (8) 3566-3573; DOI: 10.1128/AEM.66.8.3566-3573.2000

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Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degradation
Ace M. Baty III, Callie C. Eastburn, Zhenjun Diwu, Somkiet Techkarnjanaruk, Amanda E. Goodman, Gill G. Geesey
Applied and Environmental Microbiology Aug 2000, 66 (8) 3566-3573; DOI: 10.1128/AEM.66.8.3566-3573.2000
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