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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-␤-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.

Growth of the chitin-degrading marine bacterium S91 on solid surfaces under oligotrophic conditions was accompanied by the displacement of a large fraction of the surface-derived bacterial production into the flowing bulk aqueous phase, irrespective of the value of the surface as a nutrient source. Over a 200-h period of surface colonization, 97 and 75% of the bacterial biomass generated on biodegradable chitin and a nonnutritional silicon surface, respectively, detached to become part of the free-living population in the bulk aqueous phase. Specific surface-associated growth rates that included the cells that subsequently detached from the substrata varied depending on the nutritional value of the substratum and during the period of surface colonization. Specific growth rates of 3.79 and 2.83 day ؊1 were obtained when cells first began to proliferate on a pure chitin film and a silicon surface, respectively. Later, when cell densities on the surface and detached cells as CFU in the bulk aqueous phase achieved a quasi-steady state, specific growth rates decreased to 1.08 and 0.79 day ؊1 on the chitin and silicon surfaces, respectively. Virtually all of the cells that detached from either the chitin or the silicon surfaces and the majority of cells associated with the chitin surface over the 200-h period of surface colonization displayed no detectable expression of the chitin-degrading genes chiA and chiB. Cells displaying high levels of chiA-chiB expression were detected only on the chitin surface and then only clustered in discrete areas of the surface. Surface-associated, differential gene expression and displacement of bacterial production from surfaces represent adaptations at the population level that promote efficient utilization of limited resources and dispersal of progeny to maximize access to new sources of energy and maintenance of the population.In the marine environment, hydrolysis of particulate organic matter (POM) to low-molecular-weight dissolved organic matter (DOM) is mediated primarily by ectohydrolytic enzymes produced by particle-associated bacteria (25). While some of the DOM derived from POM hydrolysis is respired as CO 2 , a portion is used for new bacterial production (BP) (53). It has also been hypothesized that a significant portion of the DOM derived from enzymatic attack of POM by POM-associated bacteria supports maintenance and reproduction of free-living bacteria in the pelagic marine environment (3, 9). In fact, POM-associated bacteria are thought to provide more DOM for production of the free-living bacterial populations than for production of POM-associated populations (4, 20, 45, 51). However, detachment of POM-derived cells could lead to overestimation of dissolved organic carbon-derived, free-living BP. Jacobsen and Azam (23) reported that bacteria associated with copepod fecal pellets were displaced into the surrounding water during fecal pellet degradation. While these researchers recognized the potential importance of detachment as a process that contributes to free-livin...

A cluster of three closely linked chitinase genes organized in the order chiA, chiB and chic, with the same transcriptional direction, and two unlinked genes, chip and chiQ, involved in chitin degradation in Pseudoalteromnas sp. strain S91 were cloned, sequenced and characterized. The deduced amino acid sequences revealed that ChiA, Chis and Chic exhibited similarities to chitinases belonging to family 18 of the glycosyl hydrolases while Chip and ChiQ belonged to family 20. Chip and ChiQ showed different enzymic activities against fluorescent chitin analogues, but neither was able to degrade colloidal chitin. ChiA possessed chitinase activity but did not bind chitin; Chis bound chitin but had no chitinase activity; Chic possessed strong chitinase activity and also bound chitin. Production of Chic in 591 appeared to be controlled by chiA expression, since insertion of a transposon into the ORF of chiA resulted in the loss of chitinase activity as well as loss of Chic proteins in a chitinasenegative mutant. In Escherichia coli, Chic appeared to be expressed from its own promoter.

Two broad-host-range vectors previously constructed for use in soil bacteria (A. G. Matthysse, S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman, FEMS Microbiol. Lett. 145:87–94, 1996) were assessed by epifluorescence microscopy for use in tagging three marine bacterial species. Expression of gfp could be visualized in Vibrio sp. strain S141 cells at uniform levels of intensity from either the lac or thenpt-2 promoter, whereas expression of gfp could be visualized in Psychrobacter sp. strain SW5H cells at various levels of intensity only from the npt-2 promoter. Green fluorescent protein (GFP) fluorescence was not detected in the third species, Pseudoalteromonas sp. strain S91, when thegfp gene was expressed from either promoter. A new mini-Tn10-kan-gfp transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn10-kan-gfp generated random stable mutants at high frequencies with all three marine species. With this transposon, strongly and weakly expressed S91 promoters were isolated. Visualization of GFP by epifluorescence microscopy was markedly reduced when S91 (mini-Tn10-kan-gfp) cells were grown in rich medium compared to that when cells were grown in minimal medium. Mini-Tn10-kan-gfp was used to create an S91 chitinase-negative, GFP-positive mutant. Expression of the chi-gfp fusion was induced in cells exposed toN′-acetylglucosamine or attached to chitin particles. By laser scanning confocal microscopy, biofilms consisting of microcolonies of chi-negative, GFP+ S91 cells were found to be localized several microns from a natural chitin substratum. Tagging bacterial strains with GFP enables visualization of, as well as monitoring of gene expression in, living single cells in situ and in real time.

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