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At least 91 records · Page 5

Identification of Critical Surface Parameters Driving Lectin-Mediated Capture of Bacteria from Solution

Lectin-functional interfaces are useful for isolation of bacteria from solution because they are low-cost and allow nondestructive, reversible capture. This study provides a systematic investigation of physical and chemical surface parameters that influence bacteria capture over lectin-functionalized polymer interfaces and then applies these findings to construct surfaces with significantly enhanced bacteria capture. The designer block copolymer poly(glycidyl methacrylate)-block-poly(vinyldimethyl azlactone) was used as a lectin attachment layer, and lectin coupling into the polymer film through azlactone–lectin coupling reactions was first characterized. Here, experimental parameters including polymer areal chain density, lectin molecular weight, and lectin coupling buffer were systematically varied to identify parameters driving highest azlactone conversions and corresponding lectin surface densities. To introduce physical nanostructures into the attachment layer, nanopillar arrays (NPAs) of varied heights (300 and 2100 nm) were then used to provide an underlying surface template for the functional polymer layer. Capture of Escherichia coli on lectin–polymer surfaces coated over both flat and NPA surfaces was then investigated. For flat polymer interfaces, bacteria were detected on the surface after incubation at a solution concentration of 10 3 cfu/mL, and a corresponding detection limit of 1.7 × 10 3 cfu/mL was quantified. This detection limit was 1 order of magnitude lower than control lectin surfaces functionalized with standard, carbodiimide coupling chemistry. NPA surfaces containing 300 nm tall pillars further improved the detection limit to 2.1 × 10 2 cfu/mL, but also reduced the viability of captured cells. Finally, to investigate the impact of cell surface parameters on capture, we used Agrobacterium tumefaciens cells genetically modified to allow manipulation of exopolysaccharide adhesin production levels. Statistical analysis of surface capture levels revealed that lectin surface density was the primary factor driving capture, as opposed to exopolysaccharide adhesin expression. These findings emphasize the critical importance of the synthetic interface and the development of surfaces that combine high lectin densities with tailored physical features to drive high levels of capture. These insights will aid in design of biofunctional interfaces with physicochemical surface properties favorable for capture and isolation of bacteria cells from solutions.

59 BASIC BIOLOGICAL SCIENCES↗

Elevated temperature increases carbon and nitrogen fluxes between phytoplankton and heterotrophic bacteria through physical attachment

Quantifying the contribution of marine microorganisms to carbon and nitrogen cycles and their response to predicted ocean warming is one of the main challenges of microbial oceanography. Here we present a single-cell NanoSIMS isotope analysis to quantify C and N uptake by free-living and attached phytoplankton and heterotrophic bacteria, and their response to short-term experimental warming of 4 °C. Elevated temperature increased total C fixation by over 50%, a small but significant fraction of which was transferred to heterotrophs within 12 h. Cell-to-cell attachment doubled the secondary C uptake by heterotrophic bacteria and increased secondary N incorporation by autotrophs by 68%. Warming also increased the abundance of phytoplankton with attached heterotrophs by 80%, and promoted C transfer from phytoplankton to bacteria by 17% and N transfer from bacteria to phytoplankton by 50%. Lastly, our results indicate that phytoplankton-bacteria attachment provides an ecological advantage for nutrient incorporation, suggesting a mutualistic relationship that appears to be enhanced by temperature increases.

54 ENVIRONMENTAL SCIENCES↗

Unusual biology across a group comprising more than 15% of domain Bacteria

All rights reserved. A prominent feature of the bacterial domain is a radiation of major lineages that are defined as candidate phyla because they lack isolated representatives. Bacteria from these phyla occur in diverse environments and are thought to mediate carbon and hydrogen cycles. Genomic analyses of a few representatives suggested that metabolic limitations have prevented their cultivation. Here we reconstructed 8 complete and 789 draft genomes from bacteria representing >35 phyla and documented features that consistently distinguish these organisms from other bacteria. We infer that this group, which may comprise >15% of the bacterial domain, has shared evolutionary history, and describe it as the candidate phyla radiation (CPR). All CPR genomes are small and most lack numerous biosynthetic pathways. Owing to divergent 16S ribosomal RNA (rRNA) gene sequences, 50-100% of organisms sampled from specific phyla would evade detection in typical cultivation-independent surveys. CPR organisms often have self-splicing introns and proteins encoded within their rRNA genes, a feature rarely reported in bacteria. Furthermore, they have unusual ribosome compositions. All are missing a ribosomal protein often absent in symbionts, and specific lineages are missing ribosomal proteins and biogenesis factors considered universal in bacteria. This implies different ribosome structures and biogenesis mechanisms, and underlines unusual biology across a large part of the bacterial domain.

59 BASIC BIOLOGICAL SCIENCES↗

Structure-function studies reveal ComEA contains an oligomerization domain essential for transformation in gram-positive bacteria

An essential step in bacterial transformation is the uptake of DNA into the periplasm, across the thick peptidoglycan cell wall of Gram-positive bacteria, or the outer membrane and thin peptidoglycan layer of Gram-negative bacteria. ComEA, a DNA-binding protein widely conserved in transformable bacteria, is required for this uptake step. Here we determine X-ray crystal structures of ComEA from two Gram-positive species, Bacillus subtilis and Geobacillus stearothermophilus, identifying a domain that is absent in Gram-negative bacteria. X-ray crystallographic, genetic, and analytical ultracentrifugation (AUC) analyses reveal that this domain drives ComEA oligomerization, which we show is required for transformation. We use multi-wavelength AUC (MW-AUC) to characterize the interaction between DNA and the ComEA DNA-binding domain. Finally, we present a model for the interaction of the ComEA DNA-binding domain with DNA, suggesting that ComEA oligomerization may provide a pulling force that drives DNA uptake across the thick cell walls of Gram-positive bacteria.

59 BASIC BIOLOGICAL SCIENCES↗

Genome-resolved metagenomics reveals site-specific diversity of episymbiotic CPR bacteria and DPANN archaea in groundwater ecosystems

Abstract Candidate phyla radiation (CPR) bacteria and DPANN archaea are unisolated, small-celled symbionts that are often detected in groundwater. The effects of groundwater geochemistry on the abundance, distribution, taxonomic diversity and host association of CPR bacteria and DPANN archaea has not been studied. Here, we performed genome-resolved metagenomic analysis of one agricultural and seven pristine groundwater microbial communities and recovered 746 CPR and DPANN genomes in total. The pristine sites, which serve as local sources of drinking water, contained up to 31% CPR bacteria and 4% DPANN archaea. We observed little species-level overlap of metagenome-assembled genomes (MAGs) across the groundwater sites, indicating that CPR and DPANN communities may be differentiated according to physicochemical conditions and host populations. Cryogenic transmission electron microscopy imaging and genomic analyses enabled us to identify CPR and DPANN lineages that reproducibly attach to host cells and showed that the growth of CPR bacteria seems to be stimulated by attachment to host-cell surfaces. Our analysis reveals site-specific diversity of CPR bacteria and DPANN archaea that coexist with diverse hosts in groundwater aquifers. Given that CPR and DPANN organisms have been identified in human microbiomes and their presence is correlated with diseases such as periodontitis, our findings are relevant to considerations of drinking water quality and human health.

59 BASIC BIOLOGICAL SCIENCES↗

Improved methods to assess the effect of bacteria on germination of fungal spores

Abstract Bacterial-fungal interactions (BFI) play a major role on ecosystem functioning and might be particularly relevant at a specific development stage. For instance, in the case of biological control of fungal pathogens by bacteria, a highly relevant kind of BFI, in-vitro experiments often assess the impact of a bacterium on the inhibition of actively growing mycelia. However, this fails to consider other stages of plant infection such as the germination of a spore or a sclerotium. This study aims to present novel experimental platforms for in-vitro experiments with fungal spores, in order to assess the effect of bacteria on germination and fungal growth control, to recover the metabolites produced in the interaction, and to enhance direct visualisation of BFI. Botrytis cinerea, a phytopathogenic fungus producing oxalic acid (OA) as pathogenicity factor, was used as model. Given that oxalotrophic bacteria have been shown previously to control the growth of B. cinerea, the oxalotrophic bacteria Cupriavidus necator and Cupriavidus oxalaticus were used as models. The experiments performed demonstrated the suitability of the methods and confirmed that both bacteria were able to control the growth of B. cinerea, but only in media in which soluble OA was detected by the fungus. The methods presented here can be easily performed in any microbiology laboratory and are not only applicable to screen for potential biocontrol agents, but also to better understand BFI.

Estoppey, Aislinn↗

Activation of intact bacteria and bacterial fragments mixed with agar as cloud droplets and ice crystals in cloud chamber experiments

Biological particles, including bacteria and bacterial fragments, have beenof much interest due to the special ability of some to nucleate ice atmodestly supercooled temperatures. This paper presents results from a recentstudy conducted on two strains of cultivated bacteria which suggest thatbacterial fragments mixed with agar, and not whole bacterial cells, serve ascloud condensation nuclei (CCN). Due to the absence of whole bacteria cellsin droplets, they are unable to serve as ice nucleating particles (INPs) inthe immersion mode under the experimental conditions. Experiments wereconducted at the Aerosol Interaction and Dynamics in the Atmosphere (AIDA)cloud chamber at the Karlsruhe Institute of Technology (KIT) by injectingbacteria-containing aerosol samples into the cloud chamber and inducing cloudformation by expansion over a temperature range of -5 to -12 ° C.Cloud droplets and ice crystals were sampled through a pumped counterflowvirtual impactor inlet (PCVI) and their residuals were characterized with asingle particle mass spectrometer (miniSPLAT). The size distribution of theoverall aerosol was bimodal, with a large particle mode composed of intactbacteria and a mode of smaller particles composed of bacterial fragmentsmixed with agar that were present in higher concentrations. Results fromthree expansions with two bacterial strains indicate that the cloud dropletresiduals had virtually the same size distribution as the smaller particlesize mode and had mass spectra that closely matched those of bacterialfragments mixed with agar. The characterization of ice residuals that weresampled through an ice-selecting PCVI (IS-PCVI) also shows that the sameparticles that activate to form cloud droplets, bacteria fragments mixed withagar, were the only particle type observed in ice residuals. These resultsindicate that the unavoidable presence of agar or other growth media inall laboratory studies conducted on cultivated bacteria can greatlyaffect the results and needs to be considered when interpreting CCN and INactivation data.

54 ENVIRONMENTAL SCIENCES↗

Enrichments for phototrophic bacteria and characterization by morphology and pigment analysis

The purpose of this investigation was to examine several sulfide containing environments for the presence of phototrophic bacteria and to obtain enriched cultures of some of the bacteria present. The field sites were Alum Rock State Park, the Palo Alto salt marsh, the bay area salt ponds, and Big Soda Lake (near Fallon, Nevada). Bacteria from these sites were characterized by microscopic examination, measurement of in vitro absorption spectra, and analysis of carotenoid pigments. Field observations at one of the bay area salt ponds, in which the salt concentration was saturating (about 30 percent NaCl) and the sediments along the shore of the pond covered with a gypsum crust, revealed a layer of purple photosynthetic bacteria under a green layer in the gypsum crust. Samples of this gypsum crust were taken to the laboratory to measure light transmission through the crust and to try to identify the purple photosynthetic bacteria present in this extremely saline environment.

Brune, D.↗

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.

NASA Discipline Number 04-10↗

Characterization of viable bacteria from Siberian permafrost by 16S rDNA sequencing

Viable bacteria were found in permafrost core samples from the Kolyma-Indigirka lowland of northeast Siberia. The samples were obtained at different depths; the deepest was about 3 million years old. The average temperature of the permafrost is -10 degrees C. Twenty-nine bacterial isolates were characterized by 16S rDNA sequencing and phylogenetic analysis, cell morphology, Gram staining, endospore formation, and growth at 30 degrees C. The majority of the bacterial isolates were rod shaped and grew well at 30 degrees C; but two of them did not grow at or above 28 degrees C, and had optimum growth temperatures around 20 degrees C. Thirty percent of the isolates could form endospores. Phylogenetic analysis revealed that the isolates fell into four categories: high-GC Gram-positive bacteria, beta-proteobacteria, gamma-proteobacteria, and low-GC Gram-positive bacteria. Most high-GC Gram-positive bacteria and beta-proteobacteria, and all gamma-proteobacteria, came from samples with an estimated age of 1.8-3.0 million years (Olyor suite). Most low-GC Gram-positive bacteria came from samples with an estimated age of 5,000-8,000 years (Alas suite).

Non-NASA Center↗

Using Fluorescent Viruses for Detecting Bacteria in Water

A method of detecting water-borne pathogenic bacteria is based partly on established molecular-recognition and fluorescent-labeling concepts, according to which bacteria of a species of interest are labeled with fluorescent reporter molecules and the bacteria can then be detected by fluorescence spectroscopy. The novelty of the present method lies in the use of bacteriophages (viruses that infect bacteria) to deliver the fluorescent reporter molecules to the bacteria of the species of interest.

Tabacco, Mary Beth↗

Standardizing experimental approaches to investigate interactions between bacteria and ectomycorrhizal fungi

Bacteria and ectomycorrhizal fungi (EcMF) represent two of the most dominant plant root-associated microbial groups on Earth, and their interactions continue to gain recognition as significant factors that shape forest health and resilience. Yet, we currently lack a focused review that explains the state of bacteria-EcMF interaction research in the context of experimental approaches and technological advancements. To these ends, we illustrate the utility of studying bacteria-EcMF interactions, detail outstanding questions, outline research priorities in the field, and provide a suite of approaches that can be used to promote experimental reproducibility, field advancement, and collaboration. Though this review centers on the ecology of bacteria, EcMF, and trees, it by default offers experimental and conceptual insights that can be adapted to various subfields of microbiology and microbial ecology.

59 BASIC BIOLOGICAL SCIENCES↗

Interactions between ammonia and nitrite oxidizing bacteria in co-cultures: Is there evidence for mutualism, commensalism, or competition?

Nitrification is a two-step environmental microbial process in the nitrogen cycle in which ammonia is oxidized to nitrate. Ammonia-oxidizing bacteria and archaea oxidize ammonia to nitrite and nitrite is oxidized to nitrate by nitrite-oxidizing bacteria. These microorganisms, which likely act in concert in a microbial community, play critical roles in the movement of inorganic N in soils, sediments and waters and are essential to the balance of the nitrogen cycle. Anthropogenic activity has altered the balance of the nitrogen cycle through agriculture practices and organic waste byproducts. Through their influence on available N for plant growth, nitrifying microorganisms influence plant productivity for food and fiber production and the associated carbon sequestration. N Fertilizer production, primarily as ammonia, requires large inputs of natural gas and hydrogen. In croplands fertilized with ammonia-based fertilizers, nitrifiers contribute to the mobilization of this N by producing nitrate (NO3-), wasting the energy used in the production and application of ammonia-based fertilizer. The resulting nitrate is readily leached from these soils, oxidized to gaseous N oxides (greenhouse gases), and denitrified to N2 (which is no longer available as a plant N source). Still, ammonia oxidizers are beneficial in the treatment of wastewater and they also show potential to contribute to microbial bioremediation strategies for clean up of environments contaminated with chlorinated hydrocarbons. Mitigation of the negative effects and exploitation of the beneficial effects of nitrifiers will be facilitated by a systems-level understanding of the interactions of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria with the environment and with each other.

54 ENVIRONMENTAL SCIENCES↗

Optimization of Molecular Methods for Detecting Duckweed-Associated Bacteria

The bacterial colonization dynamics of plants can differ between phylogenetically similar bacterial strains and in the context of complex bacterial communities. Quantitative methods that can resolve closely related bacteria within complex communities can lead to a better understanding of plant–microbe interactions. However, current methods often lack the specificity to differentiate phylogenetically similar bacterial strains. In this study, we describe molecular strategies to study duckweed–associated bacteria. We first systematically optimized a bead-beating protocol to co-isolate nucleic acids simultaneously from duckweed and bacteria. We then developed a generic fingerprinting assay to detect bacteria present in duckweed samples. To detect specific duckweed–bacterium associations, we developed a genomics-based computational pipeline to generate bacterial strain-specific primers. These strain-specific primers differentiated bacterial strains from the same genus and enabled the detection of specific duckweed–bacterium associations present in a community context. Moreover, we used these strain-specific primers to quantify the bacterial colonization of duckweed by normalization to a plant reference gene and revealed differences in colonization levels between strains from the same genus. Lastly, confocal microscopy of inoculated duckweed further supported our PCR results and showed bacterial colonization of the duckweed root–frond interface and root interior. The molecular methods introduced in this work should enable the tracking and quantification of specific plant-microbe associations within plant-microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗

Characterization of phthalate-degrading bacteria from Asian carp microbiomes and riverine sediments

Phthalates are ubiquitous in the environment due to widespread production and distribution. The carcinogenic compounds dimethyl phthalate (DMP), diethyl phthalate (DEP), and dibutyl phthalate (DBP) are non-covalently bonded to plastics; thus prone to dispersal in various environments. Phthalates not only sorb to riverine sediments, but are also taken up by a variety of aquatic organisms. Asian carp species silver (Hypophthalmichthys molitrix) and bighead (Hypophthaltnichthys nobilis) are exposed to phthalates by ingestion and absorption.,The biodegradation of phthalates has been extensively investigated; however, no studies have isolated phthalate degrading bacteria from aquatic species. The purpose of this study was to characterize the growth and biodegradation kinetics of phthalate-degrading bacteria isolated from the gut, gill, and scale microbiomes of Asian carp, and riverine sediments. 16S rRNA sequencing identified similar genera in sediment and H. molitrix gut microbiome inoculated phthalate enrichments. Achromobacter aegrifaciens strain SKTGEO1 and Pseudomonas japonica strain SKEO1 were enriched from sediments; Bacillus subtilis strain SK18, Pseudomonas putida strain SKTG1, and Consortium SK-1 were enriched from Asian carp microbiomes. Each bacteria isolated was shown to eliminate phthalates from experimental systems. This is the first study documenting the biodegradation of phthalates by bacteria isolated from Asian carp gut and H. molitrix scale microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗

Effects of intestinal bacteria-derived p-cresyl sulfate on Th1-type immune response in vivo and in vitro

Protein fermentation by intestinal bacteria generates various compounds that are not synthesized by their hosts. An example is p-cresol, which is produced from tyrosine. Patients with chronic kidney disease (CKD) accumulate high concentrations of intestinal bacteria-derived p-cresyl sulfate (pCS), which is the major metabolite of p-cresol, in their blood, and this accumulation contributes to certain CKD-associated disorders. Immune dysfunction is a CKD-associated disorder that frequently contributes to infectious diseases among CKD patients. Although some studies imply pCS as an etiological factor, the relation between pCS and immune systems is poorly understood. In the present study, we investigated the immunological effects of pCS derived from intestinal bacteria in mice. For this purpose, we fed mice a tyrosine-rich diet that causes the accumulation of pCS in their blood. The mice were shown to exhibit decreased Th1-driven 2, 4-dinitrofluorobenzene-induced contact hypersensitivity response. The concentration of pCS in blood was negatively correlated with the degree of the contact hypersensitivity response. In contrast, the T cell-dependent antibody response was not influenced by the accumulated pCS. We also examined the in vitro cytokine responses by T cells in the presence of pCS. The production of IFN-γ was suppressed by pCS. Further, pCS decreased the percentage of IFN-γ-producing Th1 cells. Our results suggest that intestinal bacteria-derived pCS suppressesTh1-type cellular immune responses. - Highlights: • Mice fed a tyrosine-rich diet accumulated p-cresyl sulfate in their blood. • p-Cresyl sulfate negatively correlated with contact hypersensitivity response. • The in vitro production of IFN-γ was suppressed by p-cresyl sulfate. • p-Cresyl sulfate decreased the percentage of IFN-γ-producing Th1 cells in vitro.

60 APPLIED LIFE SCIENCES↗

Bacteria attenuation by iron electrocoagulation governed by interactions between bacterial phosphate groups and Fe(III) precipitates

Iron electrocoagulation (Fe-EC) is a low-cost process in which Fe(II) generated from an Fe(0) anode reacts with dissolved O 2 to form (1) Fe(III) precipitates with an affinity for bacterial cell walls and (2) bactericidal reactive oxidants. Previous work suggests that Fe-EC is a promising treatment option for groundwater containing arsenic and bacterial contamination. However, the mechanisms of bacteria attenuation and the impact of major groundwater ions are not well understood. In this work, using the model indicator Escherichia coli (E. coli), we show that physical removal via enmeshment in EC precipitate flocs is the primary process of bacteria attenuation in the presence of HCO 3 - , which significantly inhibits inactivation, possibly due to a reduction in the lifetime of reactive oxidants. Here, we demonstrate that the adhesion of EC precipitates to cell walls, which results in bacteria encapsulation in flocs, is driven primarily by interactions between EC precipitates and phosphate functional groups on bacteria surfaces. In single solute electrolytes, both P (0.4 mM) and Ca/Mg (1-13 mM) inhibited the adhesion of EC precipitates to bacterial cell walls, whereas Si (0.4 mM) and ionic strength (2-200 mM) did not impact E. coli attenuation. Interestingly, P (0.4 mM) did not affect E. coli attenuation in electrolytes containing Ca/Mg, consistent with bivalent cation bridging between bacterial phosphate groups and inorganic P sorbed to EC precipitates. Finally, we found that EC precipitate adhesion is largely independent of cell wall composition, consistent with comparable densities of phosphate functional groups on Gram-positive and Gram-negative cells. Our results are critical to predict the performance of Fe-EC to eliminate bacterial contaminants from waters with diverse chemical compositions.

59 BASIC BIOLOGICAL SCIENCES↗

Cytoprotective metal-organic frameworks for anaerobic bacteria

We report a strategy to uniformly wrap Morella thermoacetica bacteria with a metal-organic framework (MOF) monolayer of nanometer thickness for cytoprotection in artificial photosynthesis. The catalytic activity of the MOF enclosure toward decomposition of reactive oxygen species (ROS) reduces the death of strictly anaerobic bacteria by fivefold in the presence of 21% O 2 , and enables the cytoprotected bacteria to continuously produce acetate from CO 2 fixation under oxidative stress. The high definition of the MOF–bacteria interface involving direct bonding between phosphate units on the cell surface and zirconium clusters on MOF monolayer, provides for enhancement of life throughout reproduction. The dynamic nature of the MOF wrapping allows for cell elongation and separation, including spontaneous covering of the newly grown cell surface. Finally, the open-metal sites on the zirconium clusters lead to 600 times more efficient ROS decomposition compared with zirconia nanoparticles.

59 BASIC BIOLOGICAL SCIENCES↗