Single-Walled Carbon Nanotube Probes for the Characterization of Biofilm-Degrading Enzymes Demonstrated against Pseudomonas aeruginosa Extracellular Matrices.

Hydrolase co-therapies that degrade biofilm extracellular polymeric substances (EPS) allow for a better diffusion of antibiotics and more effective treatment; current methods for quantitatively measuring the enzymatic degradation of EPS are not amendable to high-throughput screening. Herein, we present biofilm EPS-functionalized single-walled carbon nanotube (SWCNT) probes for rapid screening of hydrolytic enzyme selectivity and activity on EPS. The extent of biofilm EPS degradation is quantified by monitoring the quenching of the SWCNT fluorescence. We used this platform to screen 16 hydrolases with varying bond breaking selectivity against a panel of wild-type Pseudomonas aeruginosa and mutants deficient or altered in one or more EPS. Next, we performed concentration-dependent studies of six enzymes on two common strains found in cystic fibrosis (CF) environments and, for each enzyme, extracted three first-order rate constants and their relative contributions by fitting a parallel, multi-site degradation model, with a good model fit (R2 from 0.65 to 0.97). Reaction rates (turnover rates) are dependent on the enzyme concentration and range from 6.67 × 10-11 to 2.80 × 10-3 *s-1 per mg/mL of enzymes. Lastly, we confirmed findings from this new assay using an established crystal-violet staining assay for a subset of hydrolase panels. In summary, our work shows that this modular sensor is amendable to the high-throughput screening of EPS degradation, thereby improving the rate of discovery and development of novel hydrolases.

Environment dependent microbial co-occurrences across a cyanobacterial bloom in a freshwater lake.

Microbial taxon-taxon co-occurrences may directly or indirectly reflect the potential relationships between the members within a microbial community. However, to what extent and the specificity by which these co-occurrences are influenced by environmental factors remains unclear. In this report, we evaluated how the dynamics of microbial taxon-taxon co-occurrence is associated with the changes of environmental factors in Nan Lake at Wuhan city, China with a Modified Liquid Association method. We were able to detect more than 1000 taxon-taxon co-occurrences highly correlated with one or more environmental factors across a phytoplankton bloom using 16S rRNA gene amplicon community profiles. These co-occurrences, referred to as environment dependent co-occurrences (ED_co-occurrences), delineate a unique network in which a taxon-taxon pair exhibits specific, and potentially dynamic correlations with an environmental parameter, while the individual relative abundance of each may not. Microcystis involved ED_co-occurrences are in important topological positions in the network, suggesting relationships between the bloom dominant species and other taxa could play a role in the interplay of microbial community and environment across various bloom stages. Our results may broaden our understanding of the response of a microbial community to the environment, particularly at the level of microbe-microbe associations.

High-throughput fluorescence-activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants.

The genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low-lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence-activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high-lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid-induction periods.

Quantitative proteomics insights into Chlamydomonas reinhardtii thermal tolerance enhancement by a mutualistic interaction with Sinorhizobium meliloti.

Interactions between photosynthetic microalgae and bacteria impact the physiology of both partners, which influence the fitness and ecological trajectories of each partner in an environmental context-dependent manner. Thermal tolerance of Chlamydomonas reinhardtii can be enhanced through a mutualistic interaction with vitamin B12 (cobalamin)-producing Sinorhizobium meliloti. Here, we used label-free quantitative proteomics to reveal the metabolic networks altered by the interaction under normal and high temperatures. We created a scenario where the growth of Sinorhizobium requires carbon provided by Chlamydomonas for growth in co-cultures, and survival of Chlamydomonas under high temperatures relies on cobalamin and possibly other metabolites produced by Sinorhizobium. Differential abundance analysis identified proteins produced by each partner in co-cultures compared to mono-cultures at each temperature. Proteins involved in cobalamin production by Sinorhizobium increased in the presence of Chlamydomonas under elevated temperatures, whereas in Chlamydomonas, there was an increase in cobalamin-dependent methionine synthase and certain proteins associated with methylation reactions. Co-cultivation and heat stress strongly modulated the central metabolism of both partners as well as various transporters that could facilitate nutrient cross-utilization. Co-cultivation modulated expression of various components of two- or one-component signal transduction systems, transcriptional activators/regulators, or sigma factors, suggesting complex regulatory networks modulate the interaction in a temperature-dependent manner. Notably, heat and general stress-response and antioxidant proteins were upregulated in co-cultures, suggesting that the interaction is inherently stressful to each partner despite the benefits of mutualism. Our results shed insight into the metabolic tradeoffs required for mutualism and how metabolic networks are modulated by elevated temperature. IMPORTANCE: Photosynthetic microalgae are key primary producers in aquatic ecosystems, playing an important role in the global carbon cycle. Nearly every alga lives in association with a diverse community of microorganisms that influence each other and their metabolic activities or survival. One chemical produced by bacteria that influence algae is vitamin B12, an enzyme cofactor used for a variety of metabolic functions. The alga Chlamydomonas reinhardtii benefits from vitamin B12 produced by Sinorhizobium meliloti by producing the amino acid methionine under high temperatures which are required for Chlamydomonas thermotolerance. Yet, our understanding of this interaction under normal and stressful temperatures is poor. Here, we used quantitative proteomics to identify differentially expressed proteins to reveal metabolic adjustments made by Chlamydomonas and Sinorhizobium that could facilitate this mutualism. These findings will enhance our understanding of how photosynthetic algae and their associated microbiomes will respond as global temperatures increase.

Nutrient-dependent cross-kingdom interactions in the hyphosphere of an arbuscular mycorrhizal fungus.

Introduction: The hyphosphere of arbuscular mycorrhizal (AM) fungi is teeming with microbial life. Yet, the influence of nutrient availability or nutrient forms on the hyphosphere microbiomes is still poorly understood. Methods: Here, we examined how the microbial community (prokaryotic, fungal, protistan) was affected by the presence of the AM fungus Rhizophagus irregularis in the rhizosphere and the root-free zone, and how different nitrogen (N) and phosphorus (P) supplements into the root-free compartment influenced the communities. Results: The presence of AM fungus greatly affected microbial communities both in the rhizosphere and the root-free zone, with prokaryotic communities being affected the most. Protists were the only group of microbes whose richness and diversity were significantly reduced by the presence of the AM fungus. Our results showed that the type of nutrients AM fungi encounter in localized patches modulate the structure of hyphosphere microbial communities. In contrast we did not observe any effects of the AM fungus on (non-mycorrhizal) fungal community composition. Compared to the non-mycorrhizal control, the root-free zone with the AM fungus (i.e., the AM fungal hyphosphere) was enriched with Alphaproteobacteria, some micropredatory and copiotroph bacterial taxa (e.g., Xanthomonadaceae and Bacteroidota), and the poorly characterized and not yet cultured Acidobacteriota subgroup GP17, especially when phytate was added. Ammonia-oxidizing Nitrosomonas and nitrite-oxidizing Nitrospira were significantly suppressed in the presence of the AM fungus in the root-free compartment, especially upon addition of inorganic N. Co-occurrence network analyses revealed that microbial communities in the root-free compartment were complex and interconnected with more keystone species when AM fungus was present, especially when the root-free compartment was amended with phytate. Conclusion: Our study showed that the form of nutrients is an important driver of prokaryotic and eukaryotic community assembly in the AM fungal hyphosphere, despite the assumed presence of a stable and specific AM fungal hyphoplane microbiome. Predictable responses of specific microbial taxa will open the possibility of using them as co-inoculants with AM fungi, e.g., to improve crop performance.

Machine Learning Prediction of Nitrification From Ammonia- and Nitrite-Oxidizer Community Structure.

Accurately modeling nitrification and understanding the role specific ammonia- or nitrite-oxidizing taxa play in it are of great interest and importance to microbial ecologists. In this study, we applied machine learning to 16S rRNA sequence and nitrification potential data from an experiment examining interactions between cropping systems and rhizosphere on microbial community assembly and nitrogen cycling processes. Given the high dimensionality of microbiome datasets, we only included nitrifers since only a few taxa are capable of ammonia and nitrite oxidation. We compared the performance of linear and nonlinear algorithms with and without qPCR measures of bacterial and archaea ammonia monooxygenase subunit A (amoA) gene abundance. Our feature selection process facilitated the identification of taxons that are most predictive of nitrification and to compare habitats. We found that Nitrosomonas and Nitrospirae were more frequently identified as important predictors of nitrification in conventional systems, whereas Thaumarchaeota were more important predictors in diversified systems. Our results suggest that model performance was not substantively improved by incorporating additional time-consuming and expensive qPCR data on amoA gene abundance. We also identified several clades of nitrifiers important for nitrification in different cropping systems, though we were unable to detect system- or rhizosphere-specific patterns in OTU-level biomarkers for nitrification. Finally, our results highlight the inherent risk of combining data from disparate habitats with the goal of increasing sample size to avoid overfitting models. This study represents a step toward developing machine learning approaches for microbiome research to identify nitrifier ecotypes that may be important for distinguishing ecotypes with defining roles in different habitats.

Farming on Mars: Treatment of basaltic regolith soil and briny water simulants sustains plant growth.

A fundamental challenge in human missions to Mars is producing consumable foods efficiently with the in situ resources such as soil, water, nutrients and solar radiation available on Mars. The low nutrient content of martian soil and high salinity of water render them unfit for direct use for propagating food crops on Mars. It is therefore essential to develop strategies to enhance nutrient content in Mars soil and to desalinate briny water for long-term missions on Mars. We report simple and efficient strategies for treating basaltic regolith simulant soil and briny water simulant for suitable resources for growing plants. We show that alfalfa plants grow well in a nutrient-limited basaltic regolith simulant soil and that the alfalfa biomass can be used as a biofertilizer to sustain growth and production of turnip, radish and lettuce in the basaltic regolith simulant soil. Moreover, we show that marine cyanobacterium Synechococcus sp. PCC 7002 effectively desalinates the briny water simulant, and that desalination can be further enhanced by filtration through basalt-type volcanic rocks. Our findings indicate that it is possible to grow food crops with alfalfa treated basaltic regolith martian soil as a substratum watered with biodesalinated water.

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.

Agricultural Management Affects the Active Rhizosphere Bacterial Community Composition and Nitrification.

Cropping system diversity provides yield benefits that may result from shifts in the composition of root-associated bacterial and fungal communities, which either enhance nutrient availability or limit nutrient loss. We investigated whether temporal diversity of annual cropping systems (four versus two crops in rotation) influences the composition and metabolic activities of root-associated microbial communities in maize at a developmental stage when the peak rate of nitrogen uptake occurs. We monitored total (DNA-based) and potentially active (RNA-based) bacterial communities and total (DNA-based) fungal communities in the soil, rhizosphere, and endosphere. Cropping system diversity strongly influenced the composition of the soil microbial communities, which influenced the recruitment of the resident microbial communities and, in particular, the potentially active rhizosphere and endosphere bacterial communities. The diversified cropping system rhizosphere recruited a more diverse bacterial community (species richness), even though there was little difference in soil species richness between the two cropping systems. In contrast, fungal species richness was greater in the conventional rhizosphere, which was enriched in fungal pathogens; the diversified rhizosphere, however, was enriched in Glomeromycetes. While cropping system influenced endosphere community composition, greater correspondence between DNA- and RNA-based profiles suggests a higher representation of active bacterial populations. Cropping system diversity influenced the composition of ammonia oxidizers, which coincided with diminished potential nitrification activity and gross nitrate production rates, particularly in the rhizosphere. The results of our study suggest that diversified cropping systems shift the composition of the rhizosphere's active bacterial and total fungal communities, resulting in tighter coupling between plants and microbial processes that influence nitrogen acquisition and retention. IMPORTANCE Crops in simplified, low-diversity agroecosystems assimilate only a fraction of the inorganic nitrogen (N) fertilizer inputs. Much of this N fertilizer is lost to the environment as N oxides, which degrade water quality and contribute to climate change and loss of biodiversity. Ecologically inspired management may facilitate mutualistic interactions between plant roots and microbes to liberate nutrients when plants need them, while also decreasing nutrient loss and pathogen pressure. In this study, we investigate the effects of a conventional (2-year rotation, inorganic fertilization) and a diversified (4-year rotation, manure amendments) cropping system on the assembly of bacterial and fungal root-associated communities, at a maize developmental stage when nitrogen demand is beginning to increase. Our results indicate that agricultural management influences the recruitment of root-associated microbial communities and that diversified cropping systems have lower rates of nitrification (particularly in the rhizosphere), thereby reducing the potential for loss of nitrate from these systems.

Transient alginate gene expression by Pseudomonas putida biofilm residents under water-limiting conditions reflects adaptation to the local environment.

Under water-limiting conditions Pseudomonas putida produces the exopolysaccharide alginate, which influences biofilm development and facilitates maintaining a hydrated microenvironment. Since alginate is a minor biofilm matrix component it is important to determine whether alginate production occurs by all or a subset of residents, and when and to what extent cells contribute to alginate production. To address these questions we employed stable and unstable fluorescent reporters to measure alginate biosynthesis (algD) operon expression and metabolic activity in vivo quantitatively by flow cytometry and visually by microscopy. Here we report that during growth under water-limiting conditions and when biofilms become dehydrated most residents transiently express the alginate biosynthesis genes leading to distinct spatial patterns as the biofilm ages. Transient alginate gene expression was not a consequence of decreased metabolic activity, since metabolic reporters were still expressed, nor was it likely due to transient cytosolic availability of the alternative sigma factor AlgT, based on qRT-PCR. Our findings also indicate that one or more biofilm attribute, other than alginate, provides protection from desiccation stress. Collectively, our findings suggest that differentiated cells dedicated to alginate production are not part of the P. putida biofilm lifestyle under water-limiting conditions. Alternatively, P. putida biofilm cells may be responding to their own local environment, producing alginate because of the fitness advantage it confers under those particular conditions.

Ozonolysis of Alkynes-A Flexible Route to Alpha-Diketones: Synthesis of AI-2.

A mild procedure for the low-temperature conversion of alkynes to diketones has been developed and employed in the synthesis of AI-2.

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.

Impact of discontinued obstetrical services in rural Missouri: 1990-2002.

PURPOSE: This study examines the potential relationship between loss of local obstetrical services and pregnancy outcomes. METHODS: Missouri Hospital Association and Missouri Department of Health birth certificate records were used as sources of information. All member hospitals of the Missouri Hospital Association that were located in cities of 10,000 or less were identified and surveyed by telephone. RESULTS: Frequency of low birth weight babies originating from service areas where hospitals closed services was statistically increased in the first year after service closures. This effect was transient. CONCLUSIONS: Transient increases in the rate of lower birth weights may reveal difficulties in service access after closure. These outcomes merit further investigation into the consequences of disruptions in access to maternity care in rural communities.

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.

A residency clinic chronic condition management quality improvement project.

BACKGROUND AND OBJECTIVES: Quality improvement in chronic disease management is a major agenda for improving health and reducing health care costs. A six-component chronic disease management model can help guide this effort. Several characteristics of the "new model" of family medicine described by the Future of Family Medicine (FFM) Project Leadership Committee are promulgated to foster practice changes that improve quality. Our objective was to implement and assess a quality improvement project guided by the components of a chronic disease management model and FFM new model characteristics. METHODS: Diabetes was selected as a model chronic disease focus. Multiple practice changes were implemented. A mature electronic medical record facilitated data collection and measurement of quality improvement progress. RESULTS: Data from the diabetes registry demonstrates that our efforts have been effective. Significant improvement occurred in five out of six quality indicators. CONCLUSIONS: Multidisciplinary teamwork in a model residency practice guided by chronic disease management principles and the FFM new model characteristics can produce significant management improvements in one important chronic disease.

Integrated Microfluidic Flow-Through Microbial Fuel Cells.

This paper reports on a miniaturized microbial fuel cell with a microfluidic flow-through configuration: a porous anolyte chamber is formed by filling a microfluidic chamber with three-dimensional graphene foam as anode, allowing nutritional medium to flow through the chamber to intimately interact with the colonized microbes on the scaffolds of the anode. No nutritional media flow over the anode. This allows sustaining high levels of nutrient utilization, minimizing consumption of nutritional substrates, and reducing response time of electricity generation owing to fast mass transport through pressure-driven flow and rapid diffusion of nutrients within the anode. The device provides a volume power density of 745 µW/cm3 and a surface power density of 89.4 µW/cm2 using Shewanella oneidensis as a model biocatalyst without any optimization of bacterial culture. The medium consumption and the response time of the flow-through device are reduced by 16.4 times and 4.2 times, respectively, compared to the non-flow-through counterpart with its freeway space volume six times the volume of graphene foam anode. The graphene foam enabled microfluidic flow-through approach will allow efficient microbial conversion of carbon-containing bioconvertible substrates to electricity with smaller space, less medium consumption, and shorter start-up time.

Shoulder joint capsule distension (hydroplasty): a case series of patients with "frozen shoulders" treated in a primary care office.

"Frozen shoulder," most often caused by adhesive capsulitis, is frequently treated with intra-articular steroid injections, physical therapy, and surgical manipulation under anesthesia. These therapies provide limited benefits. Hydraulic distension of the shoulder joint capsule (hydroplasty) has potential to provide rapid relief of pain and immediate improvement of shoulder function for patients with adhesive capsulitis. We performed 21 hydroplasty procedures on 16 patients over a 4-year period. Ninety-four percent (17/18) of the procedures improved patient's measured mobility immediately after the procedure. Fifty-three percent (10/19) of the procedures produced immediate, short term, and sustained improvement in comfort and function. No significant complications of the procedure were detected. Our series suggests that the hydroplasty procedure should be further evalutated.

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.