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PubMed2006

Microbial diversity in natural environments: focusing on fundamental questions.

Interactions with Gijs Kuenen and other Dutch scientists have led my lab to fundamental insights into the composition, structure and function of a hot spring cyanobacterial mat community that should influence our thinking about all microbial communities. By focusing on the distribution of molecular sequence variants of predominant mat phototrophs, we have discovered that small-scale sequence variation can be ecologically meaningful. By applying novel cultivation approaches, we have been able to obtain genetically relevant community members and thus to test the hypothesis that closely related sequence variants arose via adaptive evolutionary radiation. By applying the analytical tools of organic geochemistry we have gained insight into the metabolisms of major phototrophic members of the mat community as well as interactions between phototrophic guilds. These observations challenge traditional paradigms about prokaryotic species and cause us to consider evolutionary ecology theory as we develop genome-based methods for high-resolution analysis of the species-like fundamental units comprising microbial communities, and for investigating how such units coordinate the physiological activities within guilds of the community.

Autotrophic Processes↗
PubMed1989

Virulence of Mycobacterium avium complex strains from acquired immune deficiency syndrome patients: relationship with characteristics of the parasite and host.

The virulence of 24 strains of Mycobacterium avium complex (MAC) isolated from patients with acquired immune deficiency syndrome (AIDS) was assessed using the beige mouse model. Most changes in colony forming unit (cfu) counts in spleen and lungs, and spleen weights occurred between days 1 and 14, with comparatively smaller changes 14-28 days postinfection. The virulence was assessed by a score formulated from the four most useful parameters: mortality, spleen cfu, lung cfu and spleen weights at 28 days. The scores of the 24 strains showed a normal distribution; four strains falling above one standard deviation from the mean were classified as high virulent, those four falling below one standard deviation as low virulent, and the remaining 16 as of intermediate virulence. Virulence was associated with the total number of plasmids and the occurrence of large plasmids (greater than 100 MDa) in the MAC strains. There was an inverse correlation between virulence and the organism's capacity to trigger the release of oxygen metabolites from peritoneal macrophages. Macrophages from mice infected with the MAC strains of different degrees of virulence released superoxide anion (O2-) with a peak at two weeks, the peak levels bearing an inverse correlation to virulence. No association was seen between virulence and source of specimens, biochemical characteristics, drug susceptibility, serotypes or phage types.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Importance of extra- and intracellular domains of TLR1 and TLR2 in NFkappa B signaling.

Recognition of ligands by toll-like receptor (TLR) 2 requires interactions with other TLRs. TLRs form a combinatorial repertoire to discriminate between the diverse microbial ligands. Diversity results from extracellular and intracellular interactions of different TLRs. This paper demonstrates that TLR1 and TLR2 are required for ara-lipoarabinomannan- and tripalmitoyl cysteinyl lipopeptide-stimulated cytokine secretion from mononuclear cells. Confocal microscopy revealed that TLR1 and TLR2 cotranslationally form heterodimeric complexes on the cell surface and in the cytosol. Simultaneous cross-linking of both receptors resulted in ligand-independent signal transduction. Using chimeric TLRs, we found that expression of the extracellular domains along with simultaneous expression of the intracellular domains of both TLRs was necessary to achieve functional signaling. The domains from each receptor did not need to be contained within a single contiguous protein. Chimeric TLR analysis further defined the toll/IL-1R domains as the area of crucial intracellular TLR1-TLR2 interaction.

Antibodies, Monoclonal↗
PubMed2025

Archaea produce peptidoglycan hydrolases that kill bacteria.

The social life of archaea is poorly understood. In particular, even though competition and conflict are common themes in microbial communities, there is scant evidence documenting antagonistic interactions between archaea and their abundant prokaryotic brethren: bacteria. Do archaea specifically target bacteria for destruction? If so, what molecular weaponry do they use? Here, we present an approach to infer antagonistic interactions between archaea and bacteria from genome sequence. We show that a large and diverse set of archaea encode peptidoglycan hydrolases, enzymes that recognize and cleave a structure-peptidoglycan-that is a ubiquitous component of bacterial cell walls but absent from archaea. We predict the bacterial targets of archaeal peptidoglycan hydrolases using a structural homology approach and demonstrate that the predicted target bacteria tend to inhabit a similar niche to the archaeal producer, indicative of ecologically relevant interactions. Using a heterologous expression system, we demonstrate that two peptidoglycan hydrolases from the halophilic archaeaon Halogranum salarium B-1 kill the halophilic bacterium Halalkalibacterium halodurans, a predicted target, and do so in a manner consistent with peptidoglycan hydrolase activity. Our results suggest that, even though the tools and rules of engagement remain largely unknown, archaeal-bacterial conflicts are likely common, and we present a roadmap for the discovery of additional antagonistic interactions between these two domains of life. Our work has implications for understanding mixed microbial communities that include archaea and suggests that archaea might represent a large untapped reservoir of novel antibacterials.

N-Acetylmuramoyl-L-alanine Amidase↗
PubMedDate not supplied

Leucine-rich repeats in host-pathogen interactions.

Leucine-rich repeats (LRRs) are versatile binding motifs found in a variety of proteins and are involved in protein-protein interactions. The LRR domain is composed of repeats forming a characteristic solenoid horse-shoe structure, which provides a scaffold for numerous insertions involved in binding to pathogen-associated molecular patterns and surface receptors. LRRs have been shown to be involved in the host defense systems of both plants (resistance genes) and mammals (Toll-like receptors and nucleotide-binding oligomerisation domain proteins), where they sense specific pathogen-associated molecules and activate the innate immune system. Paradoxically, LRRs have also been shown to be part of microbial virulence factors involved in the interaction with host cells and establishment of infection. The potential of LRRs to bind a vast array of structurally unrelated ligands and their well-documented involvement in microbial pathogenesis make them a potential target for vaccines and new drugs. The recent identification of LRRs in the obligate intracellular protozoan parasite Leishmania and their participation in the macrophage-parasite interaction have added new insight into the role of LRRs in the host cell invasion.

Amino Acid Sequence↗
PubMed2003

The damage-response framework of microbial pathogenesis.

The late twentieth century witnessed the emergence of numerous infectious diseases that are caused by microorganisms that rarely cause disease in normal, healthy immunocompetent hosts. The emergence of these diseases shows that the existing concepts of pathogenicity and virulence do not take into account the fact that both the microorganism and the host contribute to microbial pathogenesis. To address this impediment to studies of host-microorganism interactions, we propose a new theoretical approach to understanding microbial pathogenesis, known as the 'damage-response' framework.

Animals↗
PubMed2006

Impact of electrokinetic remediation on microbial communities within PCP contaminated soil.

Electrokinetic techniques have been used to stimulate the removal of organic pollutants within soil, by directing contaminant migration to where remediation may be more easily achieved. The effect of this and other physical remediation techniques on the health of soil microbial communities has been poorly studied and indeed, largely ignored. This study reports the impact on soil microbial communities during the application of an electric field within ex situ laboratory soil microcosms contaminated with pentachlorophenol (PCP; 100mg kg(-1) oven dry soil). Electrokinetics reduced counts of culturable bacteria and fungi, soil microbial respiration and carbon substrate utilisation, especially close to the acidic anode where PCP accumulated (36d), perhaps exacerbated by the greater toxicity of PCP at lower soil pH. There is little doubt that a better awareness of the interactions between soil electrokinetic processes and microbial communities is key to improving the efficacy and sustainability of this remediation strategy.

Electrolysis↗
PubMed2006

Phage display in the study of infectious diseases.

Microbial infections are dependent on the panoply of interactions between pathogen and host and identifying the molecular basis of such interactions is necessary to understand and control infection. Phage display is a simple functional genomic methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping, antibody engineering and screening for receptor agonists or antagonists. Phage display is also used widely in various forms, including the use of fragment libraries of whole microbial genomes, to identify peptide-ligand and protein-ligand interactions that are of importance in infection. In particular, this technique has proved successful in identifying microbial adhesins that are vital for colonization.

Adhesins, Bacterial↗
PubMed1987

Diel interactions of oxygenic photosynthesis and n(2) fixation (acetylene reduction) in a marine microbial mat community.

Diel variations in N(2) fixation (acetylene reduction), CO(2) fixation, and oxygen concentrations were measured, on three separate occasions, in a marine microbial mat located on Shackleford Banks, North Carolina. Nitrogenase activity (NA) was found to be inversely correlated with CO(2) fixation and, in two of the three diel periods studied, was higher at night than during the day. Oxygen concentrations within the top 3 mm of the mat ranged from 0 to 400 muM on a diel cycle; anaerobic conditions generally persisted below 4 mm. NA in the mat was profoundly affected by naturally occurring oxygen concentrations. Experimentally elevated oxygen concentrations resulted in a significant depression of NA, whereas the addition of the Photosystem II inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea decreased oxygen concentrations within the mat and resulted in a significant short-term enhancement of NA. Mat N(2)-fixing microorganisms include cyanobacteria and heterotrophic, photoautotrophic, and chemolithotrophic eubacteria. Measured (whole-mat) NA is probably due to a combination of the NA of each of these groups of organisms. The relative contributions of each group to whole-mat NA probably varied during diel and seasonal (successional) cycles. Reduced compounds derived from photosynthetic CO(2) fixation appeared to be an important source of energy for NA during the day, whereas heterotrophic or chemolithotrophic utilization of reduced compounds appeared to be an important source of energy for NA at night, under reduced ambient oxygen concentrations. Previous estimates of N(2) fixation calculated on the basis of daytime measurements may have seriously underestimated diel and seasonal nitrogen inputs in mat systems.

Journal Article↗
PubMed1999

Estimation of microbial nitrogen flow to the duodenum of cattle based on dry matter intake and diet composition.

The objectives of this study were: 1) to evaluate the National Research Council equation used to predict microbial N flow to the duodenum in lactating cows, and 2) to determine whether improved equations could be developed by using dietary parameters used in the field. Treatment means from 55 trials with lactating and nonlactating cattle with duodenal cannulas were subjected to the backward elimination procedure of multiple regression. Variation within and among trials was accounted for by weighting the observations and including trial effects in all models. The equations to predict microbial N flow based on net energy for lactation (NEL) intake were different from the equation based on NEL intake used by the dairy National Research Council. Dry matter intake (DMI) estimated microbial N flow as well as did NEL intake, indicating that DMI drives predictions based on NEL intake. When multiple dietary factors [i.e., DMI; dietary percentages of crude protein, forage, and neutral detergent fiber; and all two-way interactions] were included, the resulting equation [microbial N (grams per day) = 16.1 + 22.9 x DMI (kilograms per day) - 0.365 x DMI2 - 1.74 x dietary neutral detergent fiber (percentage of dry matter)] tended to fit the data better than the equations based on NEL intake but not better than the equation based on DMI alone. The multiple-factor equation appeared to be the best overall equation for prediction; in contrast to the equation based on DMI, this equation is sensitive to diet composition. An asymptotic multiple-factor equation was developed, which may be more appropriate when extrapolating beyond the data range.

Animal Feed↗
PubMed1996

Factors influencing microbial growth and the efficiency of microbial protein synthesis: a review.

The possibilities of influencing microbial growth and the efficiency of microbial protein synthesis are reviewed. The authors present a detailed discussion of the effects exerted by the level of feed intake, the feeding frequency, the concentrate to forage ratio, the quality of the carbohydrate and protein source, and the interaction of carbohydrate and protein on microbial protein synthesis and, consequently, on the flow of microbial protein. An in-depth knowledge of the above factors influencing bacterial growth and microbial fermentation is essential for the well-balanced feeding of ruminants (primarily dairy cows) of high production level.

Ammonia↗
PubMed2026

Amplicon and metagenomic sequencing reveal thifluzamide drive rhizosphere microbial structural shifts and functional adaption.

Thifluzamide (TF) is a widely used phenyl urea fungicide in rice production; however, its impacts on the structural composition and functional dynamics of the rhizosphere microbiome remain poorly understood. Here, we systematically investigated the effects of TF on the structure, interactions, and functional potential of the rice (Oryza sativa L.) rhizosphere microbiome using integrated amplicon sequencing and metagenomic approaches. TF application significantly altered both bacterial and fungal community composition, bacterial diversity was markedly reduced, whereas fungal diversity increased. With bacterial diversity markedly reduced while fungal diversity increased. Beta-diversity analyses revealed strong treatment-driven community separation, indicating pronounced TF-induced microbial restructuring. Co-occurrence network analysis demonstrated reduced complexity and connectivity in bacterial networks but increased negative co-occurrence patterns within fungal communities, suggesting contrasting stability responses between microbial kingdoms. Metagenomic profiling further revealed substantial functional shifts, including the differential enrichment of KEGG and COG pathways associated with xenobiotic metabolism. Notably, while total ARG abundance remained stable, TF exposure altered the resistome profile by selectively enriching specific classes of antibiotic resistance genes (ARGs), biocide resistance genes (BRGs), and mobile genetic elements (MGEs). Strong positive correlations between MGEs and ARGs highlighted an elevated potential for horizontal gene transfer. Metagenome-assembled genome (MAG) analysis identified specific TF-enriched bacterial taxa, including Methylophilus, Sulfurospirillum, and Azospirillum, which harbored genes involved in pesticide degradation and xenobiotic transformation. Collectively, these findings demonstrate that TF profoundly reshapes the rice rhizosphere microbiome by altering microbial diversity, interaction networks, resistance gene profiles, and functional capacities. This study provides genomic insights into fungicide-microbiome interactions, underscoring the potential ecological implications associated with TF application, while identifying candidate microbial taxa that may contribute to pesticide degradation and rhizosphere microecology resilience.

Rhizosphere↗
PubMed1998

Modeling ruminal digestibility of carbohydrates and microbial protein flow to the duodenum.

Carbohydrates are the major source of energy for dairy cows and for microbial protein synthesis in the rumen. The prediction of ruminal carbohydrate digestibility and of the flow of microbial protein to the small intestine is difficult because of the variability among various feeds in the kinetics of digestion and passage of neutral detergent fiber and starch. Disappearance of fiber and starch in vitro or in situ and gas production in vitro have been extensively evaluated, improved, and reviewed. Similarly, markers and models to measure ruminal passage rate have been extensively researched and improved. Sources of variation and decreased accuracy for these techniques are discussed. Variation and potential errors also remain for the prediction of microbial protein flow to the duodenum using in vivo procedures. However, when in vivo results were accumulated into a database, microbial N flow to the duodenum over a wide range of conditions could be predicted accurately by intake of net energy for lactation or by dry matter intake and percentage of neutral detergent fiber in the diet. Although evaluation of feeding interactions and specific dietary limitations for microbial protein production in the rumen are possible with some models but not with this regression approach, mechanistic models need further validation and more accurate rate constants for improved accuracy over a wide range of conditions.

Animals↗
PubMed2000

Measuring the forces involved in polyvalent adhesion of uropathogenic Escherichia coli to mannose-presenting surfaces.

Mechanisms of bacterial pathogenesis have become an increasingly important subject as pathogens have become increasingly resistant to current antibiotics. The adhesion of microorganisms to the surface of host tissue is often a first step in pathogenesis and is a plausible target for new antiinfective agents. Examination of bacterial adhesion has been difficult both because it is polyvalent and because bacterial adhesins often recognize more than one type of cell-surface molecule. This paper describes an experimental procedure that measures the forces of adhesion resulting from the interaction of uropathogenic Escherichia coli to molecularly well defined models of cellular surfaces. This procedure uses self-assembled monolayers (SAMs) to model the surface of epithelial cells and optical tweezers to manipulate the bacteria. Optical tweezers orient the bacteria relative to the surface and, thus, limit the number of points of attachment (that is, the valency of attachment). Using this combination, it was possible to quantify the force required to break a single interaction between pilus and mannose groups linked to the SAM. These results demonstrate the deconvolution and characterization of complicated events in microbial adhesion in terms of specific molecular interactions. They also suggest that the combination of optical tweezers and appropriately functionalized SAMs is a uniquely synergistic system with which to study polyvalent adhesion of bacteria to biologically relevant surfaces and with which to screen for inhibitors of this adhesion.

Bacterial Adhesion↗
PubMed1998

Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology.

Studying the cross talk between nonpathogenic organisms and their mammalian hosts represents an experimental challenge because these interactions are typically subtle and the microbial societies that associate with mammalian hosts are very complex and dynamic. A large, functionally stable, climax community of microbes is maintained in the murine and human gastrointestinal tracts. This open ecosystem exhibits not only regional differences in the composition of its microbiota but also regional differences in the differentiation programs of its epithelial cells and in the spatial distribution of its component immune cells. A key experimental strategy for determining whether "nonpathogenic" microorganisms actively create their own regional habitats in this ecosystem is to define cellular function in germ-free animals and then evaluate the effects of adding single or several microbial species. This review focuses on how gnotobiotics-the study of germ-free animals-has been and needs to be used to examine how the gastrointestinal ecosystem is created and maintained. Areas discussed include the generation of simplified ecosystems by using genetically manipulatable microbes and hosts to determine whether components of the microbiota actively regulate epithelial differentiation to create niches for themselves and for other organisms; the ways in which gnotobiology can help reveal collaborative interactions among the microbiota, epithelium, and mucosal immune system; and the ways in which gnotobiology is and will be useful for identifying host and microbial factors that define the continuum between nonpathogenic and pathogenic. A series of tests of microbial contributions to several pathologic states, using germ-free and ex-germ-free mice, are proposed.

Animals↗
PubMed2005

Invasion processes of pathogenic Escherichia coli.

Pathogenic Escherichia coli causes extraintestinal infections such as urinary tract infection and meningitis, which are prevalent and associated with considerable morbidity. Previous investigations have identified common strategies evolved by pathogenic E. coli to exploit host cell function and cause extraintestinal infections, which include the invasion into non-phagocytic eukaryotic cells such as epithelial and endothelial cells and associated host cell actin cytoskeletal rearrangements. However, the mechanisms involved in pathogenic E. coli invasion of eukaryotic cells are shown to differ depending upon types of host tissues and microbial determinants. In this mini-review, invasion processes of pathogenic E. coli are discussed using E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC) as a paradigm. E. coli K1 is the most common Gram-negative organism causing neonatal meningitis, and E. coli invasion of HBMEC is shown to be a prerequisite for E. coli traversal of the blood-brain barrier in vivo. Previous studies have demonstrated that E. coli translocation of the blood-brain barrier is the result of specific E. coli host interactions including specific signal transduction pathways and modulation of endocytic pathways. Recent studies using functional genomics have identified additional microbial determinants contributing to E. coli K1 invasion of HBMEC. Complete understanding of microbial-host interactions that are involved in E. coli K1 invasion of HBMEC should help in the development of new strategies to prevent E. coli meningitis.

Animals↗
PubMed2006

Interactive cellular and physiological responses of the freshwater crab Potamonautes warreni to cadmium and microbial gill infestations.

Cellular and physiological responses of the freshwater crab, Potamonautes warreni, to cadmium and microbial gill infestations were investigated. Infested crabs were collected from the Mooi River, at Noordbrug, Northwest Province, South Africa in the autumn of 1995 and exposed to 0.2mg Cd(2+)L(-1) or kept unexposed under constant laboratory conditions for 7, 14 and 21 days at 24 degrees C. Cellular lesions with microbial gill infestations were persistent over 21 days and showed a similar species diversity and density as recorded in the previous summer [Schuwerack et al., 2001a. Pathological and physiological changes in the South African freshwater crab, P. warreni Calman induced by microbial gill infestations. J. Invertebrate Pathol. 77(4), 259-269]. With Cd exposure the microbial community disappeared and crabs showed a higher mean but wider range in ammonia excretion and lower mean O:N ratio after 7 days. After 14 days exposure to Cd, time- and stressor-specific cellular and physiological responses included degeneration of oxygen and gas exchange gill tissues and a mean reduced growth rate, ion exchange, a significantly reduced O:N ratio (P<or=0.05) and induced hemolymph pH (P<or=0.05). Both stressors, i.e. gill infestation and Cd, produce a barrier, which obstructs pathways in ion exchange and oxygen uptake, resulting in NH(3)/NH(4)(+) retention and similar physiological responses after 21 days. The interactive cellular and physiological mechanisms in P. warreni exposed to multiple stress are discussed.

Adaptation, Physiological↗