Characterization of extracellular chitinolytic activity in biofilms.

Extracellular enzymes produced by bacterial biofilms tend to become an integral, permanent part of the biofilm/substratum system. Thus, characterizing extracellular enzyme activity is an essential component of understanding biofilm ecology. Methods have been presented for characterizing three aspects of extracellular enzyme activity in biofilms: promoter activity of the structural gene, local catalytic activity, and kinetics of collective substrate degradation. The abundance of intracellular transcript derived from a structural gene is only indirectly related to the magnitude of catalytic activity of the corresponding enzyme. This relationship may be particularly tenuous in the case of extracellular enzymes, which must be transported out of the cell in order to become active. Fluorogenic substrates that allow direct detection of an increasingly greater variety of enzyme activities are becoming available. There are technical problems, originating from surface roughness and intrinsic fluorescence, associated with microscopic examination of biofilms on natural materials. Thin films provide one option for acquiring data about biofilms colonizing relevant materials.

Diethylene glycol removal by Echinodorus cordifolius (L.): the role of plant-microbe interactions.

This work presents the use of the plant Echinodorus cordifolius for remediating diethylene glycol (DEG) contaminated waters. The potential of this plant for treating DEG wastewater in a remediation system was observed. We found that E. cordifolius was able to remove DEG from wastewater, decrease the pH to neutral and remove approximately 95% of the chemical oxygen demand within 12 days. The plants can grow well in DEG wastewater, as indicated by their root and leaf biomass, which was found to be statistically similar to control. Wilting, chlorosis and necrosis were observed in DEG-treated plants, but the relative water content was not significantly different between control and treated plants, suggesting that the plants were able to take up and tolerate DEG present in the wastewater. Plant roots changed to black colour during experimental period. The fluorescence in situ hybridisation and bacterial enrichment confirmed that 4.30 × 10(5) cells/g of sulphate reducing bacteria and 9.30 × 10(8) cells/g of acid-producing bacteria were found associated with the plant roots. Furthermore, volatile fatty acids were found in non-sterile soil treatments, indicating that soil microorganisms are associated with DEG remediation. These results demonstrated that plants and bacteria have the ability to form a relationship to remove the organic contaminant DEG.

Use of a promoterless lacZ gene insertion to investigate chitinase gene expression in the marine bacterium Pseudoalteromonas sp. strain S9.

Sequence data for genes encoding 16S rRNA indicated that the marine strain previously named Pseudomonas sp. strain S9 would be better identified as a Pseudoalteromonas sp. By use of transposon mutagenesis, a chitinase-negative mutant of S9 with a lacZ reporter gene insertion was isolated. Part of the interrupted gene was cloned and sequenced. The deduced amino acid sequence had homology to sequences of bacterial chitinases. Expression of the chitinase gene promoter was quantified by measuring the lacZ reporter gene product, beta-galactosidase, beta-Galactosidase production was induced 10-fold by N-acetylglucosamine and 3-fold by chitin in minimal medium. Repression of beta-galactosidase synthesis was observed in rich medium either with or without chitin but was not observed in minimal medium containing glucose. The chitinase gene promoter was induced by starvation and higher-than-ambient levels of carbon dioxide but not by cadmium ion, heat or cold shock, or UV exposure.

Spatial and temporal variations in chitinolytic gene expression and bacterial biomass production during chitin degradation.

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.

Use of green fluorescent protein to tag and investigate gene expression in marine bacteria.

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 the npt-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 the gfp 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 to N'-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.

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

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.