Differentiation of chitinase-active and non-chitinase-active subpopulations of a marine bacterium during chitin degradation

Abstract

The ability of marine bacteria to adhere to detrital particulate organic matter and rapidly switch on metabolic genes in an effort to reproduce is an important response for bacterial survival in the pelagic marine environment. The goal of this investigation was to evaluate the relationship between chitinolytic gene expression and extracellular chitinase activity in individual cells of the marine bacterium Pseudoalteromonas sp. strain S91 attached to solid chitin. A green fluorescent protein reporter gene under the control of the chiA promoter was used to evaluate chiA gene expression, and a precipitating enzyme-linked fluorescent probe, ELF-97-N-acetyl-beta-D-glucosaminide, was used to evaluate extracellular chitinase activity among cells in the bacterial population. Evaluation of chiA expression and ELF-97 crystal location at the single-cell level revealed two physiologically distinct subpopulations of S91 on the chitin surface: one that was chitinase active and remained associated with the surface and another that was non-chitinase active and released daughter cells into the bulk aqueous phase. It is hypothesized that the surface-associated, non-chitinase-active population is utilizing chitin degradation products that were released by the adjacent chitinase-active population for cell replication and dissemination into the bulk aqueous phase.

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.

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.

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 chiA up-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 chiA gene activity (green) and chitinase activity (red) appears as yellow.