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.

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.

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.

Comparison of microcolony formation between Vibrio sp. strain S141 and a flagellum-negative mutant developing on agar and glass substrata.

A flagellum-negative mutant, M8.2, of the marine bacterium Vibrio sp. S141 was produced by transposon mutagenesis. Time-lapse video imaging of surface colonisation behaviour and microcolony formation of S141 compared to M8.2 cells was carried out to investigate the role of the flagellum of Vibrio sp. S141 in microcolony formation on agar and glass substrata. On an agar surface, S141 cells formed a tetrad pattern after the first two cell divisions, during initial surface colonisation. Developed microcolonies consisted of tight circular arrangements of cells with infrequent branching of cells from the main body. In contrast, M8.2 cells did not form tetrad patterns and micro-colonies generally showed enhanced branching and did not develop circular arrangements of cells. On a glass surface under flow conditions, S141 cells displayed several types of movement behaviours at the surface which may have assisted microcolony formation. M8.2 cells appeared unable to develop micro-colonies, but rather displayed a behaviour which enabled them to spread out across the substratum. Laser scanning confocal microscopy revealed S141 mature biofilms consisted of characteristic towers of bacterial growth with scattered troughs. The flagellum-negative M8.2 biofilm did not form such architecture, displaying a homogeneous distribution of cells throughout the biofilm and across the entire substratum. Although not required for attachment to the glass substratum, the flagellum was required for alignment as well as specific movement behaviours by S141 cells.

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.

Carbon dioxide as a regulator of gene expression in microorganisms.

CO2 regulates gene expression across a diverse group of microorganisms including fungi, and both photosynthetic and non photosynthetic bacteria. The processes that CO2 regulates are diverse. Several CO2-responsive random promoter lacZ fusions of unknown function have been isolated from a marine Synechococcus and a Pseudoalteromonas sp., highlighting the wide effect of CO2 control in these organisms. Regulatory proteins have been described that mediate the CO2 response at transcription level in Bacillus anthracis, the group A streptococci and two Rhodobacter spp. These regulatory proteins include: AcpA and AtxA that are involved in CO2 control of B. anthracis capsule and toxin production; Mga that regulates surface associated virulence factors in the group A streptococci; and RegB/A, a two component signal transduction system that responds to environmental stimuli including CO2, to regulate photosynthetic apparatus and CO2 fixation enzyme synthesis in Rhodobacter spp.

Simultaneous determination of gene expression and bacterial identity in single cells in defined mixtures of pure cultures.

A protocol was developed to achieve the simultaneous determination of gene expression and bacterial identity at the level of single cells; a chromogenic beta-galactosidase activity assay was combined with in situ hybridization of fluorescently labelled oligonucleotide probes to rRNA. The method allows monitoring of gene expression and quantification of beta-galactosidase activity in single cells.

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.

Improved methods for in situ enzymatic amplification and detection of low copy number genes in bacteria.

We present alternative and improved protocols for in situ analysis of single copy genes in prokaryotes. Primed in situ amplification (PRINS) and cycle PRINS were used to detect, via the incorporation of a fluorescein labelled nucleotide, the presence of specific genes carried on both high and low copy number plasmids in individual cells of Escherichia coli and a marine bacterium, SW5. The optimised protocols described enabled a significant reduction in non-specific signals whilst maintaining high fluorescent activity via labelled nucleotide incorporation. In addition, nucleic acids were amplified linearly and were retained within the permeabilised microbial cells. These methods provide considerable advances in sensitivity, specificity and reliability compared to current protocols for bacterial in situ nucleic acid amplification.

rRNA sequences and evolutionary relationships among toxic and nontoxic cyanobacteria of the genus Microcystis.

A primary-structure analysis of the 16S rRNA gene was performed with 10 strains representing five described and one unidentified species of the genus Microcystis. The phylogenies determined illustrate the evolutionary affiliations among Microcystis strains, other cyanobacteria, and related plastids and bacteria. A cluster of 10 strains that included hepatotoxic isolates identified as Microcystis aeruginosa formed a monophyletic group. However, the genus Microcystis appeared to be polyphyletic and contained two strains that clustered with unicellular cyanobacteria belonging to the genus Synechococcus. The clustering of related Microcystis strains, including strains involved in the production of the cyclic peptide toxin microcystin, was consistent with cell morphology, gas vacuolation, and the low G + C contents of the genomes. The Microcystis lineage was also distinct from the lineage containing the unicellular genus Synechocystis and the filamentous, heterocyst-forming genus Nostoc. The secondary structure of a Microcystis 16S rRNA molecule was determined, and genus-specific sequence signatures were used to design primers that permitted identification of the potentially toxic cyanobacteria belonging to the genus Microcystis via DNA amplification.

Construction of GFP vectors for use in gram-negative bacteria other than Escherichia coli.

A set of vectors containing a mutated gfp gene was constructed for use with Gram-negative bacteria other than Escherichia coli. These constructs were: pTn3gfp for making random promoter probe gfp insertions into cloned DNA in E. coli for subsequent introduction into host strains; pUTmini-Tn5gfp for making random promoter probe gfp insertions directly into host strains; p519gfp and p519nfp, broad host range mob+ plasmids containing gfp expressed from a lac and an npt2 promoter, respectively.

Construction and use of a new vector/transposon, pLBT::mini-Tn10:lac:kan, to identify environmentally responsive genes in a marine bacterium.

The previously described pLOFKm transposon delivery plasmid (J.Bacteriol. (1990) 172, 6557-6567) was engineered such that a promotorless lacZ gene was cloned within the transposon cassette, generating the vector pLBT. Using pLBT, stable insertion mutations were generated at high frequencies in Vibrio sp. S141 and Pseudomonas sp. S91, and the interrupted genes could be monitored for their pattern of regulation. Genetic screens isolated mutants defective in a variety of activities. We describe the construction and use of pLBT as a tool for reporter gene mutant analysis in bacteria other than well-characterized laboratory strains.

Characterisation of carbon dioxide-inducible genes of the marine bacterium, Pseudomonas sp. S91.

Characterisation of two genes in Pseudomonas sp. S91 that are responsive to carbon dioxide is reported. These were identified by random transposon mutagenesis leading to fusion of the Escherichia coli lacZ reporter gene to the genes of interest. Expression of the genes' promoters was quantified by measuring the reporter gene product, beta-galactosidase. beta-Galactosidase synthesis was induced when cells were exposed to 10% CO2 on solid media or during growth in aqueous phase when the culture density was greater than 1 at 610 nm, in either rich or minimal media. Induction of beta-galactosidase synthesis was not due to: increased alkalinity, onset of stationary phase, build up of soluble metabolites in the culture supernatant, or cell density-dependent signalling. The CO2-inducible gene fusions were not induced by other environmental conditions that are known to stimulate global regulators of environmental gene expression. Benzoic acid (2 mM) induced beta-galactosidase synthesis in one of the mutants indicating the Co2 response may involve the intracellular CO2 partial pressure/bicarbonate ion concentration/pH equilibrium.

Genetic diversity and phylogeny of toxic cyanobacteria determined by DNA polymorphisms within the phycocyanin locus.

Cyanobacteria are a highly diverse group in relation to form, function, and habitat. Current cyanobacterial systematics relies on the observation of minor and plastic morphological characters. Accurate and reliable delineation of toxic and bloom-forming strains of cyanobacteria has not been possible by traditional methods. We have designed general primers to the phycocyanin operon (cpc gene) and developed a PCR which allows the amplification of a region of this gene, including a variable intergenic spacer sequence. Because of the specificity of this PCR for cyanobacterial isolates, the assay is appropriate for the rapid and reliable identification of strains in freshwater samples. Successive restriction endonuclease digestion of this amplification product, with a total of nine enzymes, yielded many identifying DNA profiles specific to the various taxonomic levels of cyanobacteria. The restriction enzyme profiles for MspI, RsaI, and TaqI were conserved for strains within each of the eight genera (40 strains) studied and clearly discriminated among these genera. Intrageneric delineation of strains was revealed by the enzymes AluI, CfoI, and HaeIII for members of the genus Microcystis, while strains of genus Anabaena were differentiated by the digestion patterns provided by AluI, CfoI, and ScrFI. Phenetic and cladistic analyses of the data were used to infer the genetic relatedness and evolution of toxic and bloom-forming cyanobacteria.

Rapid identification of Aeromonas species using 16S rDNA targeted oligonucleotide primers: a molecular approach based on screening of environmental isolates.

Published 16S rDNA sequencing data for Aeromonas species were analysed and the validity of signature sequences derived from our investigations of these sequences was examined by sequencing the corresponding 16S rDNA regions of 67 environmental isolates from sewage effluents and receiving waters around Sydney and one clinical isolate, all previously classified as Aeromonas species. Species-specific probes for Aer. hydrophila and Aer. veronii were designed and tested in PCR assays and clearly discriminated these species from the other Aeromonas isolates as identified by 16S rDNA sequences.

Substratum-induced morphological changes in a marine bacterium and their relevance to biofilm structure.

The effects of surfaces on the physiology of bacteria adhering to surfaces or immobilized within biofilms are receiving more interest. A study of the effects of hydrophobic and hydrophilic substrata on the colonization behavior of a marine bacterium, SW5, revealed major differences in the morphology of SW5 on these surfaces. Using epifluorescence, scanning confocal laser, and on-line visualization (time-lapse video) microscopy, the organisms at hydrophobic surfaces were characterized by the formation of tightly packed biofilms, consisting of single and paired cells, whereas those at hydrophilic surfaces exhibited sparse colonization and the formation of chains more than 100 microns long, anchored at the surface by the terminal (colonizing) cell. The results are discussed in terms of the possible factors inducing the observed morphological differences and the significance of these differences in terms of biofilm structure and plasmid transfer when SW5 is the recipient organism.

16S ribosomal RNA gene sequence and phylogeny of toxic Microcystis sp. (cyanobacteria).

The toxigenic and bloom-forming cyanobacterial genus Microcystis contains several ill-defined species. The 16S rDNA for two strains of toxic M. aeruginosa were sequenced and compared to available cyanobacterial, bacterial, and chloroplast 16S rRNA gene information. Phylogeny and the validity of a molecular taxonomy for the genus Microcystis is presented.

Conjugative Plasmid Transfer between Bacteria under Simulated Marine Oligotrophic Conditions.

Marine Vibrio S14 strains and an Escherichia coli strain were starved in artificial seawater (NSS) with no added carbon, nitrogen, or phosphorus. The broad-host-range plasmid RP1 was transferred between the starving S14 strains and also from the E. coli donor to the S14 recipient under oligotrophic conditions, in which mixtures of donor and recipient cells were held on Nuclepore filters either floated on NSS or held such that NSS flowed through the filter. Transconjugants were obtained from S14 donors and recipients starved for at least 15 days before being mixed together for conjugation, whereas transconjugants were recovered from the E. coli donor and S14 recipient for up to 3 days of prestarvation, but not after 5 days. Transconjugants were obtained when there were as few as about 10 and 10 cells of starving S14 donors and recipients, respectively, per ml held on the filters. Starved donor and recipient mixtures incubated at 4 or 26 degrees C, as well as those allowed to mate for 2, 5, or 24 h, all yielded numbers of transconjugants which were not significantly (P > 0.05) different.

Plasmid Transfer between Marine Bacteria in the Aqueous Phase and Biofilms in Reactor Microcosms.

Plasmid transfer of broad-host-range plasmid RP1 from marine Vibrio sp. strain S14 to marine strain SW5 under optimum conditions on the surface of nutrient plates was improved 2 orders of magnitude by using the plasmid transfer process to select an SW5 recipient more efficient than the wild type in receiving and/or maintaining the plasmid. This recipient strain, SW5H, was used to form biofilms under flow conditions on the surfaces of glass beads in reactors. The S142(RP1) donor strain was introduced to the reactors after either 48 or 170 h of biofilm formation, and production of transconjugants in the aqueous phases and biofilms without selection pressure was assessed. Plasmid transfer to the recipient cells in the biofilm was detected for biofilms formed for 170 h but not in those formed for 48 h. The plasmid transfer frequency was significantly higher (P < 0.05) among cells attached to the bead surfaces in the biofilm than among cells in the aqueous phase.

A strategy for obtaining active mammalian enzyme from a fusion protein expressed in bacteria using phospholipase A2 as a model.

An active preparation of human phospholipase A2 (PLA2) was made after expression as an insoluble fusion protein in Escherichia coli. The new key elements required for PLA2 isolation were the maintenance of the fusion protein in solution after the initial solubilization and the use of a tryptophan cleavage procedure for regeneration of native PLA2 from the fusion protein. The fusion protein was composed of a beta-galactosidase leader peptide incorporating six consecutive threonine residues to aid in insoluble inclusion body formation, followed by a tryptophan adjacent to the N-terminus of PLA2. The fusion protein was purified from cell lysates, and the leader peptide was cleaved on the C-terminal side of the tryptophan residue with N-chlorosuccinimide. The released PLA2 was refolded and renatured to produce an enzyme with activity comparable to that of other phospholipases A2.