Biofilms exist in a variety of habitats that are routinely or periodically not saturated with water, and residents must integrate cues on water abundance (matric stress) or osmolarity (solute stress) into lifestyle strategies. Here we examine this hypothesis by assessing the extent to which alginate production by Pseudomonas putida strain mt-2 and by other fluorescent pseudomonads occurs in response to water limitations and how the presence of alginate in turn influences biofilm development and stress tolerance. Total exopolysaccharide (EPS) and alginate production increased with increasing matric, but not solute, stress severity, and alginate was a significant component, but not the major component, of EPS. Alginate influenced biofilm architecture, resulting in biofilms that were taller, covered less surface area, and had a thicker EPS layer at the air interface than those formed by an mt-2 algD mutant under water-limiting conditions, properties that could contribute to less evaporative water loss. We examined this possibility and show that alginate reduces the extent of water loss from biofilm residents by using a biosensor to quantify the water potential of individual cells and by measuring the extent of dehydration-mediated changes in fatty acid composition following a matric or solute stress shock. Alginate deficiency decreased survival of desiccation not only by P. putida but also by Pseudomonas aeruginosa PAO1 and Pseudomonas syringae pv. syringae B728a. Our findings suggest that in response to water-limiting conditions, pseudomonads produce alginate, which influences biofilm development and EPS physiochemical properties. Collectively these responses may facilitate the maintenance of a hydrated microenvironment, protecting residents from desiccation stress and increasing survival.

The physiological mechanisms utilized by soil bacteria for acclimation to sudden increases in soil water potential are poorly understood. In this study, we examined the physiological responses of soil isolates of Pseudomonas chlororaphis, P. fluorescens, Bacillus pumulis, and Streptomyces griseus to a sudden increase in solution water potential (dilution). Bacterial isolates were cultured at a low solute water potential (−3.0 MPa) and subjected to rapid water potential increases of 0.5 to 2.0 MPa. The small amount of protein and DNA released by a 2.0 MPa dilution suggests that water potential increases up to 2.0 MPa did not cause significant cell lysis. In response to dilution, intracellular solutes were released into the extracellular environment rather than polymerized into osmotically less‐active compounds or catabolized to CO2 In general, the Gram‐positive isolates B. pumulis and S. griseus were more tolerant to dilution than the Pseudomonas spp., since dilution had no effect on culturability, and the amount of solutes released was small (<10% of the intracellular solute pool). The Pseudomonas spp. released a maximum of 22 to 26% of their amino acid pool and 54 to 60% of their low molecular weight neutral sugar pool. The amounts of amino acids and low molecular weight carbohydrates released and the reduction in culturability was, in general, proportional to the magnitude of dilution. Pseudomonas fluorescens tolerated a 0.5 MPa water potential increase, but water potential shocks of greater magnitude resulted in a large reduction in culturability and an increase in the amount of solutes released. These results suggest that a potential source of mineralizable C following the wetting of dry soils is the release of organic compatible solutes from the microbial community.

Temperature is one of the most important environmental factors affecting the growth and survival of microorganisms and in light of current global patterns is of particular interest. Here, we highlight studies revealing how vitamin B 12 (cobalamin)-producing bacteria increase the fitness of the unicellular alga Chlamydomonas reinhardtii following an increase in environmental temperature. Heat stress represses C. reinhardtii cobalamin-independent methionine synthase (METE) gene expression coinciding with a reduction in METE-mediated methionine synthase activity, chlorosis and cell death during heat stress. However, in the presence of cobalamin-producing bacteria or exogenous cobalamin amendments C. reinhardtii cobalamin-dependent methionine synthase METHmediated methionine biosynthesis is functional at temperatures that result in C. reinhardtii death in the absence of cobalamin. Artificial microRNA silencing of C. reinhardtii METH expression leads to nearly complete loss of cobalamin-mediated enhancement of thermal tolerance. This suggests that methionine biosynthesis is an essential cellular mechanism for adaptation by C. reinhardtii to thermal stress. Increased fitness advantage of METH under environmentally stressful conditions could explain the selective pressure for retaining the METH gene in algae and the apparent independent loss of the METE gene in various algal species. Our results show that how an organism acclimates to a change in its abiotic environment depends critically on co-occurring species, the nature of that interaction, and how those species interactions evolve.

A green fluorescent protein-based Pseudomonas fluorescens strain A506 biosensor was constructed and characterized for its potential to measure benzene, toluene, ethylbenzene, and related compounds in aqueous solutions. The biosensor is based on a plasmid carrying the toluene-benzene utilization (tbu) pathway transcriptional activator TbuT from Ralstonia pickettii PKO1 and a transcriptional fusion of its promoter PtbuA1 with a promoterless gfp gene on a broad-host-range promoter probe vector. TbuT was not limiting, since it was constitutively expressed by being fused to the neomycin phosphotransferase (nptII) promoter. The biosensor cells were readily induced, and fluorescence emission after induction periods of 3 h correlated well with toluene, benzene, ethylbenzene, and trichloroethylene concentrations. Our experiments using flow cytometry show that intermediate levels of gfp expression in response to toluene reflect uniform induction of cells. As the toluene concentration increases, the level of gfp expression per cell increases until saturation kinetics of the TbuTPtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with K app values that ranged from 3.3 ؎ 1.8 M for toluene to 35.6 ؎ 16.6 M for trichloroethylene (means ؎ standard errors of the means), and maximal fluorescence response. The fluorescence response was specific for alkyl-substituted benzene derivatives and branched alkenes (di-and trichloroethylene, 2-methyl-2-butene). The biosensor responded in an additive fashion to the presence of multiple inducers and was unaffected by the presence of compounds that were not inducers, such as those present in gasoline. Flow cytometry revealed that, in response to toxic concentrations of gasoline, there was a small uninduced population and another larger fully induced population whose levels of fluorescence corresponded to the amount of effectors present in the sample. These results demonstrate the potential for green fluorescent protein-based bacterial biosensors to measure environmental contaminants.

We examined the effect of reduced water availability on the fatty acid composition of Pseudomonas putida strain mt-2 grown in a defined medium in which the water potential was lowered with the permeating solutes NaCl or polyethylene glycol (PEG) with a molecular weight of 200 (PEG 200) or the nonpermeating solute PEG 8000. Transmission electron microscopy showed that ؊1.0-MPa PEG 8000-treated cells had convoluted outer membranes, whereas ؊1.0-MPa NaCl-treated or control cells did not. At the range of water potential (؊0.25 to ؊1.5 MPa) that we examined, reduced water availability imposed by PEG 8000, but not by NaCl or PEG 200, significantly altered the amounts of trans and cis isomers of monounsaturated fatty acids that were present in whole-cell fatty acid extracts. Cells grown in basal medium or under the ؊0.25-MPa water potential imposed by NaCl or PEG 200 had a higher trans:cis ratio than ؊0.25-MPa PEG 8000-treated cells. As the water potential was lowered further with PEG 8000 amendments, there was an increase in the amount of trans isomers, resulting in a higher trans:cis ratio. Similar results were observed in cells grown physically separated from PEG 8000, indicating that these changes were not due to PEG toxicity. When cells grown in ؊1.5-MPa PEG 8000 amendments were exposed to a rapid water potential increase of 1.5 MPa or to a thermodynamically equivalent concentration of the permeating solute, NaCl, there was a decrease in the amount of trans fatty acids with a corresponding increase in the cis isomer. The decrease in the trans/cis ratio following hypoosomotic shock did not occur in the presence of the lipid synthesis inhibitor cerulenin or the growth inhibitors chloramphenicol and rifampicin, which indicates a constitutively operating enzyme system. These results indicate that thermodynamically equivalent concentrations of permeating and nonpermeating solutes have unique effects on membrane fatty acid composition.

Pseudomonas putida strain mt-2 unsaturated biofilm formation proceeds through three distinct developmental phases, culminating in the formation of a microcolony. The form and severity of reduced water availability alter cell morphology, which influences microcolony size and ultrastructure. The dehydration (matric stress) treatments resulted in biofilms comprised of smaller cells, but they were taller and more porous and had a thicker extracellular polysaccharide layer at the air interface. In the solute stress treatments, cell filamentation occurred more frequently in the presence of high concentrations of ionic (but not nonionic) solutes, and these filamented cells drastically altered the biofilm architecture.In terrestrial habitats, bacteria reside on soil matrices or plant surfaces as aggregates of cells, or microcolonies, that are frequently enmeshed in exopolymeric substances of their own making and can be described as biofilms (1,5,19,23). These biofilms are commonly unsaturated, although water films, which vary in thickness depending on the environmental conditions, surround them. In a saturated system, the water potential is comprised almost exclusively of the solute potential (24). However, under unsaturated conditions, water availability is influenced by both the solute and matric potentials (24). The difference between these two stresses is that with a solute stress, bacteria are bathed in water of diminished activity but that with a matric stress, bacteria are dehydrated due to low water contents and the availability of the water is reduced through its interaction with the matrix. Under normal conditions, soil bacteria experience significant matric stress (12).In aquatic systems, channels in the biofilm act as conduits for waste removal and nutrient supply (34). Under low-shear laminar flow, Pseudomonas aeruginosa PAO1 biofilms consist of a monolayer of cells with mound-shaped, circular microcolonies, but under high-shear turbulent flow, PAO1 biofilms consist of filamentous streamers (8,14,25). The development of this complex architecture is influenced by hydrodynamics, nutrient composition, and biological properties such as quorum sensing, extracellular polysaccharide (EPS) production, and motility (8). In unsaturated habitats, the lack of laminar fluid flow dramatically influences nutrient availability patterns, metabolic-waste accumulation and disposal, and the accumulation of cell-signaling molecules and, consequently, biofilm architecture.Atomic-force microscopy of fresh and desiccated Pseudomonas putida biofilms revealed that drying had little effect on physical morphology and surface properties (1). However, those studies focused on fully developed, mature biofilms that were then dried. In many ways, the growth of bacteria on agar surfaces better approximates the conditions that bacteria experience in many unsaturated habitats; they are able to acquire nutrients from the underlying matrix and from a relatively thin water film covering the biofilm. Examination of the organization of cells withi...

SummaryBacteria in terrestrial habitats frequently reside as biofilm communities on surfaces that are unsaturated, i.e. biofilms are covered in water films varying in thickness depending on the environmental conditions. Water availability in these habitats is influenced by the osmolarity of the water (solute stress) and by cellular dehydration imposed by matric stress, which increases as water content decreases. Unfortunately, we understand relatively little about the molecular mechanisms required for bacterial growth in lowwater-content habitats. Here, we describe the use of mini-Tn 5 -¢ ¢ ¢ ¢ pho A to identify genes in Pseudomonas putida that are matric water stress controlled and to generate mutants defective in desiccation tolerance. We identified 20 genes that were induced by a matric stress but not by a thermodynamically equivalent solute stress, 11 genes were induced by both a matric and a solute stress, three genes were induced by a solute stress and three genes were repressed by a matric stress. Their patterns of expression were analysed in laboratory media, and their contribution to desiccation tolerance was evaluated. Twenty-six genes were homologous to sequences present in the completed P. putida KT2440 genome sequence or plasmid pWWO sequence that are involved in protein fate, nutrient or solute acquisition, energy generation, motility, alginate biosynthesis or cell envelope structure, and the function of five could not be predicted from the sequence. Together, these genes and their importance to desiccation tolerance provide a view of the environment perceived by bacteria in low-watercontent habitats, and suggest that the mechanisms for adaptation for growth in low-water-content habitats are different from those for growth in highosmolarity habitats.

Despite the vast surface area of terrestrial plant leaves and the large microbial communities they support, little is known of the ability of leaf-associated microorganisms to access and degrade airborne pollutants. Here, we examined bacterial acquisition and degradation of phenol on leaves by an introduced phenol degrader and by natural phyllosphere communities. Whole-cell gfp-based Pseudomonas fluorescens bioreporter cells detected phenol on leaves that had previously been transiently exposed to gaseous phenol, indicating that leaves accumulated phenol; moreover, they accumulated it in sites that were accessible to epiphytic bacteria and to concentrations that were at least 10-fold higher than those in the air. After inoculated leaves were exposed to gaseous 14C-phenol, leaves harbouring the phenol-degrading Pseudomonas sp. strain CF600 released eight times more 14CO2 than did leaves harbouring a non-degrading mutant, demonstrating that CF600 actively mineralized phenol on leaves. We evaluated phenol degradation by natural microbial communities on green ash leaves that were collected from a field site rich in airborne organic pollutants. We found that significantly more phenol was mineralized by these leaves when the communities were present than by these leaves following surface sterilization. Thus, phenol-degrading organisms were present in these natural communities and were metabolically capable of phenol degradation. Collectively, these results provide the first direct evidence that bacteria on leaves can degrade an organic pollutant from the air, and indicate that bacteria on leaves could potentially contribute to the natural attenuation of organic air pollutants.