Larry J. Halverson

Alginate Production by Pseudomonas putida Creates a Hydrated Microenvironment and Contributes to Biofilm Architecture and Stress Tolerance under Water-Limiting Conditions

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.

Development and Characterization of a Green Fluorescent Protein-Based Bacterial Biosensor for Bioavailable Toluene and Related Compounds

ABSTRACT 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 TbuT-PtbuA1 system are observed. Each inducer had a unique minimum concentration that was necessary for induction, with Kapp values that ranged from 3.3 ± 1.8 μM for toluene to 35.6 ± 16.6 μM 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.

Reduced Water Availability Influences the Dynamics, Development, and Ultrastructural Properties of Pseudomonas putida Biofilms

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.

Cell envelope components contributing to biofilm growth and survival of Pseudomonas putida in low-water-content habitats.

Bacteria 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 low‐water‐content habitats. Here, we describe the use of mini‐Tn5‐′phoA 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‐water‐content habitats, and suggest that the mechanisms for adaptation for growth in low‐water‐content habitats are different from those for growth in high‐osmolarity habitats.

Differential Effects of Permeating and Nonpermeating Solutes on the Fatty Acid Composition of Pseudomonas putida

ABSTRACT 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 oftrans 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 highertrans: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 oftrans fatty acids with a corresponding increase in thecis 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.

Cell–cell and cell–surface interactions mediated by cellulose and a novel exopolysaccharide contribute to Pseudomonas putida biofilm formation and fitness under water‐limiting conditions

The composition of the exopolysaccharide matrix of Pseudomonas putida mt2 biofilms is relatively undefined as well as the contributions of each polymer to ecological fitness. Here, we describe the role of two putative exopolysaccharide gene clusters, putida exopolysaccharide A (pea) and bacterial cellulose (bcs) in biofilm formation and stability, rhizosphere colonization and matrix hydration under water-limiting conditions. Our findings suggest that pea is involved in the production of a novel glucose, galactose, and mannose-rich polymer that contributes to cell-cell interactions necessary for pellicle and biofilm formation and stability. In contrast, Bcs plays a minor role in biofilm formation and stability, although it does contribute to rhizosphere colonization based on a competition assay. We show that pea expression is highly induced transiently under water-limiting conditions but only slightly by high osmolarity, as determined by qRT-PCR. In contrast, both forms of water stress highly induced bcs expression. Cells deficient in making one or more exopolysaccharide experienced greater dehydration-mediated cell-envelope stress, leading to increased alginate promoter activity. However, this did not lead to increased exopolysaccharide production, except in bcs or pea mutants unable to produce alginate, indicating that P. putida compensates by producing, presumably more Pea or Bcs exopolysaccharides, to facilitate biofilm hydration. Collectively, the data suggest that Pea and Bcs contribute to biofilm formation and in turn their presence contributes to fitness under water-limiting conditions, but not to the extent of alginate.

Identification and Genetic Characterization of Phenol-Degrading Bacteria from Leaf Microbial Communities

Microbial communities on aerial plant leaves may contribute to the degradation of organic air pollutants such as phenol. Epiphytic bacteria capable of phenol degradation were isolated from the leaves of green ash trees grown at a site rich in airborne pollutants. Bacteria from these communities were subjected, in parallel, to serial enrichments with increasing concentrations of phenol and to direct plating followed by a colony autoradiography screen in the presence of radiolabeled phenol. Ten isolates capable of phenol mineralization were identified. Based on 16S rDNA sequence analysis, these isolates included members of the genera Acinetobacter, Alcaligenes, and Rhodococcus. The sequences of the genes encoding the large subunit of a multicomponent phenol hydroxylase (mPH) in these isolates indicated that the mPHs of the gram-negative isolates belonged to a single kinetic class, and that is one with a moderate affinity for phenol; this affinity was consistent with the predicted phenol levels in the phyllosphere. PCR amplification of genes for catechol 1,2-dioxygenase (C12O) and catechol 2,3-dioxygenase (C23O) in combination with a functional assay for C23O activity provided evidence that the gram-negative strains had the C12O−, but not the C23O−, phenol catabolic pathway. Similarly, the Rhodococcus isolates lacked C23O activity, although consensus primers to the C12O and C23O genes of Rhodococcus could not be identified. Collectively, these results demonstrate that these leaf surface communities contained several taxonomically distinct phenol-degrading bacteria that exhibited diversity in their mPH genes but little diversity in the catabolic pathways they employ for phenol degradation.

Chlamydomonas reinhardtii thermal tolerance enhancement mediated by a mutualistic interaction with vitamin B12-producing bacteria.

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 B12 (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 METH-mediated 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.

Influence of water limitation on endogenous oxidative stress and cell death within unsaturated Pseudomonas putida biofilms

Here we examined how water limitation (matric stress) and high osmolarity (solute stress) influence the extent of endogenous oxidative stress and cell death patterns within Pseudomonas putida biofilms. The temporal dynamics and spatial organization of reactive oxygen species (ROS) accumulation and dead cells in biofilms developed under water-replete and solute stress conditions were similar to each other. Arrays of dead cells, typically one cell width in diameter, were distributed throughout the biofilm and occasionally they spanned the entire depth of the biofilm. These arrays of dead cells were not observed under water-limiting conditions, although the extent of ROS accumulation and cell death was substantially greater. Despite the greater death rate under water-limiting conditions, culturable population sizes were transiently maintained at levels comparable to those under water-replete and solute stress conditions. There was greater spatial stratification of dead cells under water-limiting than water-replete conditions with viable cells primarily located at the air interface, which could facilitate cell dispersal following a wetting event. Under water-limiting conditions, ROS accumulation is greater in an DeltaalgD mutant compared with the wild type, suggesting that the exopolysaccharide alginate attenuates the extent of dehydration-mediated oxidative stress. We conclude that endogenous ROS accumulation is correlated with cell death within P. putida biofilms, although mechanisms contributing to their accumulation may differ under water-replete and water-limiting conditions.

Minimization of treatment time for in vitro 1.1 MHz destruction of Pseudomonas aeruginosa biofilms by high-intensity focused ultrasound.

Medical implants are prone to colonization by bacterial biofilms. Normally, surgery is required to replace the infected implant. One promising noninvasive modality is to destroy biofilms with high-intensity focused ultrasound. In our study, Pseudomonas aeruginosa biofilms were grown on implant-mimicking graphite disks in a flow chamber for 3 days prior to exposing them to ultrasound pulses. Exposure time at each treatment location was varied between 5, 15 and 30s. Burst period was varied between 1, 3, 6 and 12 milliseconds (ms). The pulses were 20 cycles in duration at 1.1 MHz from a spherically focused transducer (f/1, 63 mm focal length), creating peak compressional and rarefactional pressures at the graphite disk surface of 30 and 13 MPa, respectively. P. aeruginosa were tagged with green fluorescent protein, and killed cells were visualized using propidium iodide before determining the extent of biofilm destruction. The exposure-induced temperature rise was measured to be less than 0.2°C at the focus, namely the interface between graphite disk and water. Then, the temperature rise was measured at the focus between the graphite disk and a tissue-mimicking phantom to evaluate therapy safety. Two thresholds, of bacteria destruction increase and of complete bacteria removal, respectively, were identified to divide our eight exposure conditions. Results indicated that 30-s exposure and 6-ms pulse period were sufficient to destroy the biofilms. However, the 15-s exposure and 3-ms pulse period were viewed as optimum when considering exposure time, efficacy, and safety.