Microcolony Development Research Articles

Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms

Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities1-7. Biofilm-forming bacteria such as the opportunistic pathogen7-10 Pseudomonas aeruginosa are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on factors impacting surface adhesion and it is known that P. aeruginosa secretes the Psl exopolysaccharide, which promotes surface attachment by acting as a ‘molecular glue’11-15. However, how individual surface-attached bacteria self-organize into microcolonies, the first step in communal biofilm organization, is not well understood. Here, we identify a new role for Psl in early biofilm development using a massively parallel cell-tracking algorithm to extract the motility history of every cell on a newly colonized surface via a search-engine based approach16. By combining these techniques with fluorescent Psl staining and computer simulations, we show that P. aeruginosa deposits a trail of Psl as it moves on a surface, which influences the surface motility of subsequent cells that encounter these trails and thus generate positive feedback. Both experiments and simulations indicate that the web of secreted Psl controls the distribution of surface visit frequencies, which can be approximated by a power law. This Zipf's Law17 indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system18, a ‘rich-get-richer’ mechanism of Psl accumulation that results in a small number of ‘elite’ cells extremely enriched in communally produced Psl. Using engineered strains with inducible Psl production, we show that local Psl levels determine post-division cell fates and that high local Psl levels ultimately allow ‘elite’ cells to serve as the founding population for initial microcolony development.

Kinetic development of biofilm on NF membranes at the Méry-sur-Oise plant, France

The kinetic formation of biofilms developing on nanofiltration (NF) membranes was studied for 2 years in the water production unit of Méry-sur-Oise, France. New membranes were set up in a pilot train integrated to the plant and autopsied after operation for 7, 80, 475 and 717 days. The biofouling layer was studied by confocal laser scanning microscope after 4′,6-diamidino-2-phenyindole dihydrochloride and lectin staining, and by attenuated total reflectance-Fourier transform infrared spectroscopy and rheology experiments. Three stages of biofilm growth were discriminated: (1) the presence of sessile microcolonies embedded in an exopolymeric matrix (after filtration for seven days); (2) membrane coverage expansion through microcolony development and biofilm growth in three dimensions (up to 80 days filtration); and (3) biofilm maturation by densification (after filtration for 80–717 days). Biofilm maturation resulted in total coverage of the membrane surface and matrix residue diversification, development of the polysaccharide network, and the strengthening of matrix cohesion through viscosity and elasticity increases. The wettability and permeability of the fouled NF membranes decreased quickly and continuously throughout the biofilm development process. The longitudinal pressure drop (LPD) increased only after the biofilm reached a quantitative threshold. The decline in membrane permeability may be the result of contributions from many fouling mechanisms but the LPD was more substantially influenced by biofilm development.

Microcultivation of anaerobic bacteria single cells entrapped in alginate microbeads

Alginate microbeads, produced by emulsion/internal gelation, were studied for the entrapment and microcultivation of microbial cells with biotechnological potential. An anaerobic consortium which was selected for its capacity to degrade complex carbohydrates, and a pure culture of cellulose degrading bacteria were used for entrapment studies. Optimization of conditions for the formation of spherical alginate microbeads in sizes between 20 and 80μm were examined. The best conditions were achieved by combining rapeseed methyl ester as oil phase and stirring at 100rpm using a rotation impeller. Calcium alginate microbeads produced under these conditions were shown to present morphological stability, with large pores in the internal matrix that favours microcolony development. Finally, single cells were observed inside the beads after the entrapment procedure and microcolony formation was confirmed after cultivation in cellobiose.

OPTIMISATION OF SOMATIC HYBRIDISATION IN CICHORIUM SPECIES

Improving the inulin production in the root of industrial chicory by conventional breeding has reached its limit. Therefore, we aimed to increase genetic variability in industrial chicory (Cichorium intybus) to enhance its inulin production. To this end, we executed symmetric protoplast fusions between industrial chicory on the one hand and endive or a wild type Cichorium intybus foliosum on the other hand. In order to produce plantlets, efficient protocols needed to be established for protoplast fusion and regeneration, and for the screening of the putative hybrids. We created a regeneration protocol by which we can produce plantlets out of protoplasts using low melting point agarose beads surrounded by liquid medium. Two weeks after protoplast isolation, 60% of the initial incubated protoplasts showed microcolony development in the beads. After transfer to solid medium, containing 0.5 mg L-1 IAA and 0.5 mg L-1 BAP, shoots were induced on the developing calli. Three months after protoplast isolation, the first plants were recovered. Protoplasts from different genotypes of C. intybus sativum and C. intybus foliosum could be regenerated. Symmetric protoplast fusion was performed using PEG-induced, chemical fusion. Industrial chicory (C. intybus sativum), endive (C. endivia crispum) and wild chicory (C. intybus foliosum) were used as fusion partners. Up to 30% of the regenerants after fusion between industrial chicory and endive were hybrids. The fusion of industrial chicory with wild chicory produced on average 4% hybrid regenerants. Microsatellite markers confirmed the hybridity of the putative fusion products. These findings enable us to produce, fuse and regenerate protoplasts of different Cichorium species in a rapid and efficient way. Moreover, the methods for symmetric protoplast fusion and regeneration presented in this study, can directly contribute to further research on asymmetric protoplast fusion experiments between these three species. This way, CMS could possibly be introduced by combining the nucleus of industrial chicory with the cytoplasm of other Cichorium types.

Transparent soil for imaging the rhizosphere.

Understanding of soil processes is essential for addressing the global issues of food security, disease transmission and climate change. However, techniques for observing soil biology are lacking. We present a heterogeneous, porous, transparent substrate for in situ 3D imaging of living plants and root-associated microorganisms using particles of the transparent polymer, Nafion, and a solution with matching optical properties. Minerals and fluorescent dyes were adsorbed onto the Nafion particles for nutrient supply and imaging of pore size and geometry. Plant growth in transparent soil was similar to that in soil. We imaged colonization of lettuce roots by the human bacterial pathogen Escherichia coli O157:H7 showing micro-colony development. Micro-colonies may contribute to bacterial survival in soil. Transparent soil has applications in root biology, crop genetics and soil microbiology.

Histological and cytological characterization of adult plant resistance to wheat stripe rust

Wheat cultivar Xingzi 9104 (XZ) possesses adult plant resistance (APR) to stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). In this study, histological and cytological experiments were conducted to elucidate the mechanisms of APR in XZ. The results of leaf inoculation experiments indicated that APR was initiated at the tillering stage, gradually increased as the plant aged and highly expressed after boot stage. The histology and oxidative burst in infected leaves of plants at seedling, tillering and boot stages were examined using light microscopic and histochemical methods. Subcellular changes in the host-pathogen interactions during the seedling and boot stages were analyzed by transmission electron microscopy. The results showed that haustorium formation was retarded in the adult plants and that the differentiation of secondary intercellular hyphae was significantly inhibited, which decreased the development of microcolonies in the adult plants, especially in plants of boot stage. The expression of APR to stipe rust during wheat development was clearly associated with extensive hypersensitive cell death of host cells and localized production of reactive oxygen species, which coincided with the restriction of fungal growth in infection sites in adult plants. At the same time, cell wall-related resistance in adult plants prevented ingression of haustorial mother cells into plant cells. Haustorium encasement was coincident with malformation or death of haustoria. The results provide useful information for further determination of mechanisms of wheat APR to stripe rust. The expression of APR to stipe rust in wheat cultivar Xingzi 9104 (XZ) was clearly associated with extensive hypersensitive cell death of host cells and the localized production of reactive oxygen species.

Can Simpson's paradox explain co-operation inPseudomonas aeruginosabiofilms?

Co-operative behaviours, such as the production of public goods, are commonly displayed by bacteria in biofilms and can enhance their ability to survive in environmental or clinical settings. Non-co-operative cheats commonly arise and should, theoretically, disrupt co-operative behaviour. Its stability therefore requires explanation, but no mechanisms to suppress cheating within biofilms have yet been demonstrated experimentally. Theoretically, repeated aggregation into groups, interleaved with dispersal and remixing, can increase co-operation via a 'Simpson's paradox'. That is, an increase in the global proportion of co-operators despite a decrease in within-group proportions, via differential growth of groups. We investigate the hypothesis that microcolony formation and dispersal produces a Simpson's paradox that explains bacterial co-operation in biofilms. Using the production of siderophores in Pseudomonas aeruginosa as our model system for co-operation, we use well-documented co-operator and siderophore-deficient cheat strains to measure the frequency of co-operating and cheating individuals, in-situ within-microcolony structures. We detected significant within-type negative density-dependant effects that vary over microcolony development. However, we find no evidence of Simpson's paradox. Instead, we see clear within-microcolony spatial structure (cheats occupying the interior portions of microcolonies) that may violate the assumption required for Simpson's paradox that group members share equally in the public good.

Evolution and adaptation in Pseudomonas aeruginosa biofilms driven by mismatch repair system-deficient mutators.

Pseudomonas aeruginosa is an important opportunistic pathogen causing chronic airway infections, especially in cystic fibrosis (CF) patients. The majority of the CF patients acquire P. aeruginosa during early childhood, and most of them develop chronic infections resulting in severe lung disease, which are rarely eradicated despite intensive antibiotic therapy. Current knowledge indicates that three major adaptive strategies, biofilm development, phenotypic diversification, and mutator phenotypes [driven by a defective mismatch repair system (MRS)], play important roles in P. aeruginosa chronic infections, but the relationship between these strategies is still poorly understood. We have used the flow-cell biofilm model system to investigate the impact of the mutS associated mutator phenotype on development, dynamics, diversification and adaptation of P. aeruginosa biofilms. Through competition experiments we demonstrate for the first time that P. aeruginosa MRS-deficient mutators had enhanced adaptability over wild-type strains when grown in structured biofilms but not as planktonic cells. This advantage was associated with enhanced micro-colony development and increased rates of phenotypic diversification, evidenced by biofilm architecture features and by a wider range and proportion of morphotypic colony variants, respectively. Additionally, morphotypic variants generated in mutator biofilms showed increased competitiveness, providing further evidence for mutator-driven adaptive evolution in the biofilm mode of growth. This work helps to understand the basis for the specific high proportion and role of mutators in chronic infections, where P. aeruginosa develops in biofilm communities.

Quorum Sensing between Pseudomonas aeruginosa Biofilms Accelerates Cell Growth

This manuscript describes the fabrication of arrays of spatially confined chambers embossed in a layer of poly(ethylene glycol) diacrylate (PEGDA) and their application to studying quorum sensing between communities of Pseudomonas aeruginosa. We hypothesized that biofilms may produce stable chemical signaling gradients in close proximity to surfaces, which influence the growth and development of nearby microcolonies into biofilms. To test this hypothesis, we embossed a layer of PEGDA with 1.5-mm wide chambers in which P. aeruginosa biofilms grew, secreted homoserine lactones (HSLs, small molecule regulators of quorum sensing), and formed spatial and temporal gradients of these compounds. In static growth conditions (i.e., no flow), nascent biofilms secreted N-(3-oxododecanoyl) HSL that formed a gradient in the hydrogel and was detected by P. aeruginosa cells that were ≤8 mm away. Diffusing HSLs increased the growth rate of cells in communities that were <3 mm away from the biofilm, where the concentration of HSL was >1 μM, and had little effect on communities farther away. The HSL gradient had no observable influence on biofilm structure. Surprisingly, 0.1-10 μM of N-(3-oxododecanoyl) HSL had no effect on cell growth in liquid culture. The results suggest that the secretion of HSLs from a biofilm enhances the growth of neighboring cells in contact with surfaces into communities and may influence their composition, organization, and diversity.

QTL identification and microphenotype characterisation of the developmentally regulated yellow rust resistance in the UK wheat cultivar Guardian.

Yellow rust (causal agent: Puccinia striiformis f.sp. tritici) resistance in the UK wheat cultivar Guardian is developmentally regulated, resistance increasing as the plant matures. Yellow rust resistance was assessed under field conditions on plants after ear emergence to ensure maximum expression of resistance. Three quantitative trait loci (QTL) for yellow rust resistance were identified, being located on chromosomes 1B (QPst.jic-1B), 2D (QPst.jic-2D) and 4B (QPst.jic-4B). The largest resistance effect, QPst.jic-1B located to the same position on the long arm of chromosome 1B as the known durable source of yellow rust resistance, Yr29. Microscopic studies were carried out to determine what effect the resistance in Guardian had on the development of P. striiformis f.sp. tritici. While the adult plant resistance in Guardian did not prevent germinated urediniospores from establishing an effective infection site, the growth of hyphae within flag leaf tissue was significantly inhibited, slowing the development of microcolonies. 3,3-diaminabenzadine (DAB) and trypan blue staining indicated that this inhibition of hyphal growth was not associated with hydrogen peroxide accumulation or extensive plant cell death.

Impact of the RNA chaperone Hfq on the fitness and virulence potential of uropathogenic Escherichia coli.

Hfq is a bacterial RNA chaperone involved in the posttranscriptional regulation of many stress-inducible genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Escherichia coli (UPEC) isolate UTI89 to effectively colonize the bladder and kidneys in a murine urinary tract infection model system. The disruption of hfq did not affect bacterial adherence to or invasion of host cells but did limit the development of intracellular microcolonies by UTI89 within the terminally differentiated epithelial cells that line the lumen of the bladder. In vitro, the hfq mutant was significantly impaired in its abilities to handle the antibacterial cationic peptide polymyxin B and reactive nitrogen and oxygen radicals and to grow in acidic medium (pH 5.0). Relative to the wild-type strain, the hfq mutant also had a substantially reduced migration rate on motility agar and was less prone to form biofilms. Hfq activities are known to impact the regulation of both the stationary-phase sigma factor RpoS (sigma(S)) and the envelope stress response sigma factor RpoE (sigma(E)). Although we saw similarities among hfq, rpoS, and rpoE deletion mutants in our assays, the rpoE and hfq mutants were phenotypically the most alike. Cumulatively, our data indicate that Hfq likely affects UPEC virulence-related phenotypes primarily by modulating membrane homeostasis and envelope stress response pathways.

Bet-hedging and epigenetic inheritance in bacterial cell development

Upon nutritional limitation, the bacterium Bacillus subtilis has the capability to enter the irreversible process of sporulation. This developmental process is bistable, and only a subpopulation of cells actually differentiates into endospores. Why a cell decides to sporulate or not to do so is poorly understood. Here, through the use of time-lapse microscopy, we follow the growth, division, and differentiation of individual cells to identify elements of cell history and ancestry that could affect this decision process. These analyses show that during microcolony development, B. subtilis uses a bet-hedging strategy whereby some cells sporulate while others use alternative metabolites to continue growth, providing the latter subpopulation with a reproductive advantage. We demonstrate that B. subtilis is subject to aging. Nevertheless, the age of the cell plays no role in the decision of its fate. However, the physiological state of the cell's ancestor (more than two generations removed) does affect the outcome of cellular differentiation. We show that this epigenetic inheritance is based on positive feedback within the sporulation phosphorelay. The extended intergenerational "memory" caused by this autostimulatory network may be important for the development of multicellular structures such as fruiting bodies and biofilms.

Dynamics of Neisseria gonorrhoeae Attachment: Microcolony Development, Cortical Plaque Formation, and Cytoprotection

Neisseria gonorrhoeae is the bacterium that causes gonorrhea, a major sexually transmitted disease and a significant cofactor for human immunodeficiency virus transmission. The retactile N. gonorrhoeae type IV pilus (Tfp) mediates twitching motility and attachment. Using live-cell microscopy, we reveal for the first time the dynamics of twitching motility by N. gonorrhoeae in its natural environment, human epithelial cells. Bacteria aggregate into microcolonies on the cell surface and induce a massive remodeling of the microvillus architecture. Surprisingly, the microcolonies are motile, and they fuse to form progressively larger structures that undergo rapid reorganization, suggesting that bacteria communicate with each other during infection. As reported, actin plaques form beneath microcolonies. Here, we show that cortical plaques comigrate with motile microcolonies. These activities are dependent on pilT, the Tfp retraction locus. Cultures infected with a pilT mutant have significantly higher numbers of apoptotic cells than cultures infected with the wild-type strain. Inducing pilT expression with isopropyl-beta-D-thiogalactopyranoside partially rescues cells from infection-induced apoptosis, demonstrating that Tfp retraction is intrinsically cytoprotective for the host. Tfp-mediated attachment is therefore a continuum of microcolony motility and force stimulation of host cell signaling, leading to a cytoprotective effect.

Evaluation of Adherence, Hydrophobicity, Aggregation, and Biofilm Development of Flavobacterium johnsoniae-Like Isolates

Flavobacterium spp. isolates have been identified in diverse biofilm structures, but the mechanism of adherence has not been elucidated. The absence of conventional biofilm-associated structures such as fimbriae, pili, and flagella suggest that surface hydrophobicity, and/or autoaggregation and coaggregation may play an important role in adherence and biofilm formation. The biofilm-forming capacity of 29 Flavobacterium johnsoniae-like isolates obtained from South African aquaculture systems was assessed using microtiter plate assays. The role of hydrophobicity [salting aggregation test (SAT) and bacterial adherence to hydrocarbons (BATH) assays], autoaggregation, and coaggregation on biofilm formation by Flavobacterium spp. was also investigated, while biofilm structure was examined using flow cells and microscopy. All isolates displayed a hydrophilic nature, but showed varying levels of adherence in microtiter assays. Significant negative correlations were observed between adherence and biofilm-forming capacity in nutrient-poor medium at 26 degrees C and BATH hydrophobicity and motility, respectively. Isolates displayed strain-to-strain variation in their autoaggregation indices and their abilities to coaggregate with various Gram-negative and Gram-positive organisms. Microcolony and/or biofilm development were observed microscopically, and flavobacterial isolates displayed stronger biofilm structures and interaction with a Vibrio spp. isolate than with an Aeromonas hydrophila isolate. The role of extracellular polysaccharides and specific outer membrane proteins will have to be examined to reveal mechanisms of adherence and coaggregation employed by biofilm-forming F. johnsoniae-like strains.

Phenotypic Diversification and Adaptation ofSerratia marcescensMG1 Biofilm-Derived Morphotypes

We report here the characterization of dispersal variants from microcolony-type biofilms of Serratia marcescens MG1. Biofilm formation proceeds through a reproducible process of attachment, aggregation, microcolony development, hollow colony formation, and dispersal. From the time when hollow colonies were observed in flow cell biofilms after 3 to 4 days, at least six different morphological colony variants were consistently isolated from the biofilm effluent. The timing and pattern of variant formation were found to follow a predictable sequence, where some variants, such as a smooth variant with a sticky colony texture (SSV), could be consistently isolated at the time when mature hollow colonies were observed, whereas a variant that produced copious amounts of capsular polysaccharide (SUMV) was always isolated at late stages of biofilm development and coincided with cell death and biofilm dispersal or sloughing. The morphological variants differed extensively from the wild type in attachment, biofilm formation, and cell ultrastructure properties. For example, SSV formed two- to threefold more biofilm biomass than the wild type in batch biofilm assays, despite having a similar growth rate and attachment capacity. Interestingly, the SUMV, and no other variants, was readily isolated from an established SSV biofilm, indicating that the SUMV is a second-generation genetic variant derived from SSV. Planktonic cultures showed significantly lower frequencies of variant formation than the biofilms (5.05 x 10(-8) versus 4.83 x 10(-6), respectively), suggesting that there is strong, diversifying selection occurring within biofilms and that biofilm dispersal involves phenotypic radiation with divergent phenotypes.

Temperature-Regulated Microcolony Formation byBurkholderia pseudomalleiRequirespilAand Enhances Association with Cultured Human Cells

Burkholderia pseudomallei is the causative agent of melioidosis, a potentially fatal disease that is endemic to Northern Australia and Southeast Asia and is acquired from soil or water. Adherence of B. pseudomallei 08 to cultured cells increases dramatically following prior growth at 30 degrees C or less compared to that following prior growth at 37 degrees C. Here, we show that this occurs almost entirely as the result of microcolony formation (bacterium-bacterium interactions) following growth at 27 degrees C but not at 37 degrees C, which considerably enhances bacterial association with eukaryotic cells. Further, we demonstrate that the type IVA pilin-encoding gene, pilA, is essential for microcolony development by B. pseudomallei 08, and thus optimum association with eukaryotic cells, but is not required for direct adherence (bacterium-cell interactions). In contrast, although the B. pseudomallei genome sequence strain, K96243, also contains transcriptionally active pilA, microcolony formation rarely occurs following growth at either 27 degrees C or 37 degrees C and cell association occurs significantly less than with strain 08. Analysis of pilA transcription in 08 identified that pilA is dramatically upregulated under microcolony-forming conditions, viz., growth at low temperature, and association with eukaryotic cells; the pattern of transcription of pilA in K96243 differed from that in 08. Our study also suggests that biofilm formation by B. pseudomallei 08 and K96243 on polyvinylchloride is not mediated by pilA. Adherence and microcolony formation, and pilA transcription, vary between strains, consistent with known genomic variation in B. pseudomallei, and these phenotypes may be relevant to colonization from the environment.

Architecture of a Nascent Sphingomonas sp. Biofilm under Varied Hydrodynamic Conditions

The architecture of a Sphingomonas biofilm was studied during early phases of its formation, using strain L138, a gfp-tagged derivative of Sphingomonas sp. strain LB126, as a model organism and flow cells and confocal laser scanning microscopy as experimental tools. Spatial and temporal distribution of cells and exopolymer secretions (EPS) within the biofilm, development of microcolonies under flow conditions representing varied Reynolds numbers, and changes in diffusion length with reference to EPS production were studied by sequential sacrificing of biofilms grown in multichannel flow cells and by time-lapse confocal imaging. The area of biofilm in terms of microscopic images required to ensure representative sampling varied by an order of magnitude when area of cell coverage (2 x 10(5) microm(2)) or microcolony size (1 x 10(6) microm(2)) was the biofilm parameter under investigation. Hence, it is necessary to establish the inherent variability of any biofilm metric one is attempting to quantify. Sphingomonas sp. strain L138 biofilm architecture consisted of microcolonies and extensive water channels. Biomass and EPS distribution were maximal at 8 to 9 mum above the substratum, with a high void fraction near the substratum. Time-lapse confocal imaging and digital image analysis showed that growth of the microcolonies was not uniform: adjacently located colonies registered significant growth or no growth at all. Microcolonies in the biofilm had the ability to move across the attachment surface as a unit, irrespective of fluid flow direction, indicating that movement of microcolonies is an inherent property of the biofilm. Width of water channels decreased as EPS production increased, resulting in increased diffusion distances in the biofilm. Changing hydrodynamic conditions (Reynolds numbers of 0.07, 52, and 87) had no discernible influence on the characteristics of microcolonies (size, shape, or orientation with respect to flow) during the first 24 h of biofilm development. Inherent factors appear to have overriding influence, vis-a-vis environmental factors, on early stages of microcolony development under these laminar flow conditions.

Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation

Quorum sensing is a process by which bacteria communicate by using secreted chemical signaling molecules called autoinducers. Many bacterial species modulate the expression of a wide variety of physiological functions in response to changes in population density by this mechanism. In this study, the opportunistic pathogen Klebsiella pneumoniae was observed to secrete type 2 signaling molecules. A homologue of luxS, the gene required for AI-2 synthesis in Vibrio harveyi, was isolated from the K. pneumoniae genome. A V. harveyi bioassay showed the luxS functionality in K. pneumoniae and its ability to complement the luxS-negative phenotype of Escherichia coli DH5alpha. Autoinducer activity was detected in the supernatant, and maximum expression of specific messengers detected by quantitative reverse transcription-PCR analysis occurred during the late exponential phase. The highest levels of AI-2 were observed in minimal medium supplemented with glycerol. To determine the potential role of luxS in colonization processes, a K. pneumoniae luxS isogenic mutant was constructed and tested for its capacity to form biofilms in vitro on an abiotic surface and to colonize the intestinal tract in a murine model. No difference was observed in the level of intestinal colonization between the wild-type strain and the luxS mutant. Microscopic analysis of biofilm structures revealed that the luxS mutant was able to form a mature biofilm but with reduced capacities in the development of microcolonies, mostly in the early steps of biofilm formation. These data suggest that a LuxS-dependent signal plays a role in the early stages of biofilm formation by K. pneumoniae.

Geomicrobiology of carbonate–silicate microbialites from Hawaiian basaltic sea caves

Modern microbial mats and microbialites are described from basaltic sea caves on the island of Kauai, HI. The mats grow on the ceilings and walls in the photic zone of several open caves where fresh water seeps out of the rock. Scanning (SEM) and transmission electron microscopy (TEM) showed that the active mats are dominated by filamentous and nonfilamentous cyanobacteria in the surface layers and heterotrophic bacteria in deeper layers. Energy dispersive X-ray analysis revealed that copious amounts of extracellular polymeric substances (EPS) are rich in Mg, Si, O, and Ca, likely concentrated from solution. Petrographic microscopy and electron microprobe analysis of the mineralized microbialites showed textures reminiscent of stromatolitic laminations, consisting mainly of alternating calcium carbonate (calcite and aragonite) and magnesium-rich silicate (kerolite). Thin coatings rich in magnesite, hydromagnesite and monohydrocalcite surround the microbialites on the rock surfaces and are likely inorganic in origin. Within the mats, minerals tend to form and concentrate within, or around, dense matrices of EPS. Microenvironments with geochemical conditions favorable for mineral crystallization likely develop in the mats as a result of the mucilaginous extracellular material and the development of bacterial microcolonies. In addition, copious amounts of extracellular polymers bind ions from solution and provide nucleation sites for mineral crystallization and growth. This combination of biological and inorganic processes can explain the occurrence of the secondary minerals in these caves, as well as the stromatolitic textures of the microbialites.

Osmotic stimulation of microcolony development by Nitrosomonas europaea

In liquid cultures low levels of salt (0.1 M NaCl) had no effect on the growth rate of proliferating cells of Nitrosomonas europaea (NCIMB 11850), while at higher salt concentrations the growth rate was progressively reduced. A technique which enabled enumeration of microcolony-forming cells after just a few days incubation on the surface of membrane filters was subsequently used to investigate the effects of medium osmolarity and osmoprotectants on the ability to initiate cell division in single cells from liquid cultures. Typically, only ∼50% of the cells from early stationary phase cultures in basal medium was able to initiate cell division and form microcolonies during subsequent incubation on filters on the same medium. However, the fraction of cells forming microcolonies was stimulated by low levels of salt, while higher concentrations were inhibitory. A range of bacterial compatible solutes (betaine, trehalose, proline, MOPS, taurine and GABA) were unable to enhance microcolony formation at inhibitory salt concentrations. Other solutes (KCl, LiCl, mannitol, sucrose) also stimulated microcolony development, demonstrating that the effect was osmotic. A low salt (0.1 M NaCl) shock prior to incubation at inhibitory salt levels dramatically increased the number of cells forming microcolonies. A comparative study of several Nitrosospira spp. indicated that osmotic stimulation of microcolony development may be specific to N. europaea, although the proportion of cells able to form microcolonies was much smaller in the Nitrosospira spp. examined. Taken together these results suggest that while inhibition of growth in N. europaea by high osmolarity may be similar to that observed in other bacteria, the osmotic stimulation observed for initiation of cell division may have important implications for the successful establishment and recovery of this nitrifier in biofilms and in soil.