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PubMed2005

Analysis of microbial gene transcripts in environmental samples.

We analyzed gene expression in marine and freshwater bacterioplankton communities by the direct retrieval and analysis of microbial transcripts. Environmental mRNA, obtained from total RNA by subtractive hybridization of rRNA, was reverse transcribed, amplified with random primers, and cloned. Approximately 400 clones were analyzed, of which approximately 80% were unambiguously mRNA derived. mRNAs appeared to be from diverse taxonomic groups, including both Bacteria (mainly alpha- and gamma-Proteobacteria) and Archaea (mainly Euryarchaeota). Many transcripts could be linked to environmentally important processes such as sulfur oxidation (soxA), assimilation of C1 compounds (fdh1B), and acquisition of nitrogen via polyamine degradation (aphA). Environmental transcriptomics is a means of exploring functional gene expression within natural microbial communities without bias toward known sequences, and provides a new approach for obtaining community-specific variants of key functional genes.

Archaea↗
PubMed2005

Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli.

Flavonols are plant polyphenolic compounds that belong to the class of molecules collectively known as flavonoids. Because of their demonstrated health benefits towards a wide array of human pathological conditions, a great interest has emerged for their biosynthesis from well-characterized microbial hosts. We present the functional expression in Escherichia coli of a plant P450 flavonoid 3', 5'-hydroxylase (F3'5'H) as a fusion protein with a P450 reductase. This expression allowed metabolic engineering of E. coli to produce the flavonol kaempferol and the 3', 4' B-ring hydroxylated flavonol quercetin from the p-coumaric acid precursor by simultaneously co-expressing the fusion protein with 4-coumaroyl:CoA-ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3beta-hydroxylase (FHT) and flavonol synthase (FLS). Biosynthesis of the B-ring tri-hydroxylated flavonol myricetin from the engineered strains was accomplished when flavanones rather than phenylpropanoid acids were used as precursor molecules. Cultivation of the recombinant strains in rich medium increased the synthesis of all flavonoids with the exception of myricetin. The present work opens the possibility of the future production of several other hydroxylated flavonoid molecules in E. coli.

Cytochrome P-450 Enzyme System↗
PubMed1990

Lactic acid and pH as indicators of spoilage for vacuum-packed cooked ring sausages.

Lactic acid production and pH changes of 206 vacuum-packed cooked ring sausages stored at 2, 4 and 12 degrees C from 21 different production runs were monitored as a function of time and of microbial growth. The total lactic acid concentrations and pH values were first at a constant level, starting to increase sharply after the lactobacilli count reached about 5 x 10(7) or 6 x 10(7) cfu/g, respectively. The lactic acid and pH changes as a function of the lactobacilli count were similar at 4 and 12 degrees C. The sharp increase at high lactobacilli counts was observed in both L-lactic acid and D-lactic acid. The variation was lesser and the increase greater in D-lactic acid formation than in L-lactic acid. Above a level of 3-4 mg lactic acid/g most of the samples were deemed unfit. The pH started to decrease from a level of approx. 6.3; below 5.8-5.9 the samples were deemed unfit. The lowest pH value observed was 4.58. Both a high lactic acid content and a low pH indicated that the sausage was spoiled. These changes, however, took place at later stages of storage, and do not give information about the early phase of spoilage.

Animals↗
PubMed1994

Bacteriophages presence in human faeces of healthy subjects and patients with gastrointestinal disturbances.

The variation of the content of enteric bacteria and their bacteriophages in faeces from the different types of diarrhoeal processes has been studied. A total of 122 samples of human faeces from both healthy individuals and patients with diarrhoeal diseases of functional or infectious origin were tested. Detection rates for all microbial parameters tested decreased in the faeces of individuals with functional gastrointestinal disturbances. On the contrary, no significant differences of the microbial detection frequency was observed in faeces containing pathogenic microorganisms compared to faeces of healthy subjects. Human faeces were a poor source of F-specific, Salmonella, and Bacteroides bacteriophages, whereas specific Escherichia coli phages were isolated in most samples tested. Coliphage concentrations in faeces of healthy individuals were not directly correlated with levels of faecal coliforms. On the basis of their high correlation, faecal streptococci and coliphages were the most adequate indicators of the intestinal ecosystem variations in subjects with diarrhoeal processes.

Bacteriophages↗
PubMed2025

Functional metaproteomics for enzyme discovery.

Discovery of microbial biocatalysts traditionally relied on activity screening of isolated bacterial strains. However, since most microorganisms cannot be cultivated in the lab, such an approach leaves the majority of the microbial enzyme diversity untapped. Metagenomic approaches, in which the DNA from a microbial community is directly isolated and then used either for the creation of an expression library or for sequencing and metagenome annotation have alleviated this shortcoming to an extent, but have their own limitations: the generation of large expression libraries is time-consuming and their screening is costly, while metagenome annotation can infer biocatalytic function only from prior knowledge. We have thus developed a functional metaproteomic approach, which combines the immediacy of traditional activity screening with the comprehensiveness of a meta-omics approach. Briefly, the whole metaproteome of an environmental sample is separated on a 2-D gel, biocatalytically active proteins are visualized in-gel through zymography, and those candidate biocatalysts are then identified through mass spectrometry, searching against a metagenome-derived database obtained from the very same environmental sample. Here we explain the process in detail, with a focus on esterases, and give guidelines on how to develop a functional metaproteomic workflow for enzyme discovery.

Proteomics↗
PubMed2006

Sarcopenia in the Caenorhabditis elegans pharynx correlates with muscle contraction rate over lifespan.

In muscles, sarcopenia, the loss of muscle mass, is the major cause of aging-related functional decline and frailty. Several factors are correlated with sarcopenia during aging, including contraction-related cellular injury, oxidative stress, endocrine changes and reduced regenerative potential. However the involvement of these factors has not been experimentally investigated. Here, we report that contraction-related injury may significantly promote the progression of sarcopenia in the pharynx of the nematode, Caenorhabditis elegans, a model of aging in non-regenerative tissues. Both functional and structural declines in the pharynx during aging were significantly delayed in mutants with reduced muscle contraction rates. We also examined the role of bacteria in pharynx muscle decline during aging, as previous studies reported that antimicrobial treatments could extend C. elegans lifespan. Although microbial infection may have enhanced functional decline in the pharynx during aging, it was not the sole cause of decreased pumping rates in old animals. This study identifies contraction-related injury as a factor affecting the initiation and progression of sarcopenia during aging. Further, characterization of the specific types of damage induced by muscle contraction will be helpful for understanding the underlying causes of sarcopenia.

Aging↗
PubMed2004

Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors.

We have identified, expressed and characterized two genes from Arabidopsis thaliana (AtPMEI-1 and AtPMEI-2) encoding functional inhibitors of pectin methylesterases. AtPMEI-1 and AtPMEI-2 are cell wall proteins sharing many features with the only pectin methylesterase inhibitor (PMEI) characterized so far from kiwi fruit. Both Arabidopsis proteins interact with and inhibit plant-derived pectin methylesterases (PMEs) but not microbial enzymes. The occurrence of functional PMEIs in Arabidopsis indicates that a mechanism of controlling pectin esterification by inhibition of endogenous PMEs is present in different plant species.

Amino Acid Sequence↗
PubMed1998

Different mathematical approaches to estimating microbial protein supply in ruminants.

Many of the amino acids that are available for absorption in ruminants are derived from microbial protein that has been synthesized in the reticulorumen. This paper focuses on the prediction of the microbial protein supply and evaluates different approaches to represent mathematically the process of microbial protein synthesis. In current protein evaluation systems for ruminants, the microbial protein supply is predicted using empirical equations that relate microbial protein production to the amounts of ruminally available energy and nitrogen. In contrast, mechanistic models of rumen function endeavor to describe quantitatively the microbial protein production that is based on underlying identifiable processes. A brief description is presented of two culture techniques used to examine microbial ecosystems, namely, batch culture and chemostat culture. The mathematical equations describing these cultures are helpful in understanding key parameters of microbial production for inclusion in models, including specific growth rate, growth yield, and substrate affinity. The availability of carbohydrates is a primary determinant of microbial protein production in the rumen, and the adequacy of mathematical representations of this relationship in empirical and mechanistic models is assessed. The representation of substrate utilization for nongrowth processes and the relationship between microbial protein production and the availability of various nitrogen sources are discussed. A variable part of the synthesized microbial protein does not reach the duodenum but is degraded in the rumen, and its representation is examined. The prediction of microbial protein supply should be based on a sound representation of the underlying mechanisms, including the interactions among microbes and between microbial activity and substrate degradation.

Animals↗
PubMed1987

Sequential process of protective immunity against microbial infections in mice.

The sequence of host-defense against microbial invasion consists of nonspecific active substances which are distributed diffusely in the body fluids and exert their functions quickly to eliminate microorganisms, nonspecific humoral elements capable of exerting their functions selectively at the site of microbial invasion, neutrophils, scavenger macrophages, protection at an early stage by IgM antibody or a type of cell-mediated immunity (primitive T cell response) and protection at a late stage by typical immunities. The nature of primitive T cell response distinctive from those of typical immunities are described and its role in the host-defense is discussed.

Animals↗
PubMed2002

Infectomics: genomics and proteomics of microbial infections.

The completion of genomic sequences is the greatest triumph of molecular reductionism since the discovery of the DNA double helix in 1953. However, the utility of reductionism is becoming limited and holistic approaches, including theories and techniques, are desperately needed in the postgenomic era. In the field of infectious diseases there is an urgent need for global approaches that can efficiently, precisely and integratively study structural and functional genomics and proteomics of microbial infections (infectomics). The combination of new (e.g. DNA and protein microarrays) and traditional approaches (e.g. cloning, PCR, gene knockout and knockin, and antisense) will help overcome the challenges we are facing today. We assume that the global phenotypic changes (infectomes) in microbes and their host during infections are encoded by the genomes of microbial pathogens and their hosts, expressed in certain environmental conditions devoted to specific microbe-host interactions. Global drug responses (pharmacomes) in microbes and their host can be detected by genomic and proteomic approaches. Genome-wide approaches to genotyping and phenotyping or expression profiling will eventually lead to global dissection of microbial pathogenesis, efficient and rapid diagnosis of infectious diseases, and the development of novel strategies to control infections. The key fundamental issue of infectious diseases is how to globally and integratively understand the interactions between microbial pathogens and their hosts by using infectomics. In this review, we focus on the events that are considered important in infectomics.

Animals↗
PubMed1997

Antimicrobial peptides of phagocytes and epithelia.

Human and other vertebrate leukocytes contain multiple distinct antimicrobial (poly)peptides. Of these, BPI is a LPB protein active against gram-negative bacteria, PLA2 specifically cleaves bacterial phospholipids, while defensins and cathelicidins are broad spectrum antimicrobials that preferentially permeabilize microbial membranes. These and other polypeptides function in both phagocytic and extracellular killing of microbes, attacking multiple molecular targets to cooperatively penetrate and disrupt the microbial surfaces and membrane barriers. Such antimicrobial substances are of interest not only for students of phagocytic and epithelial host defenses but may lead to the development of novel pharmaceuticals for the treatment of infections and their sequellae.

Animals↗
PubMed2001

The immunomodulatory effects of gentamicin, imipenem, piperacillin and amphotericin B on LAK effector function in vitro.

An understanding of the immunomodulating effects of anti-microbial regimens on recombinant interleukin-2 (rIL-2) induced peripheral leukocyte function, i.e. lymphokine-activated killer (LAK)-cell efficacy, would be clinically useful in the selection of commonly employed bone marrow transplantation (BMT) antibiotics to avoid post-transplant complications and optimize anti-microbial, anti-viral, anti-tumor therapies. In this report we evaluated the modulatory effects of a number of antibiotics used in BMT on LAK-cell cytotoxicities, in vitro. Our data showed that, even at serum trough titer, amphotericin B was significantly (P < or =0.05) immunostimulatory, whereas gentamicin, imipenem, and piperacillin, individually, were significantly (P < or =0.05) immunosuppressive. Statistical analysis detected no modulation due to aztreonam, amikacin, cotrimoxazole, or ceftazidime, or any of the six cephalosporins tested at molar equivalent concentration. We conclude that certain antibiotics may be more suitable for infection prone BMT hosts.

Adjuvants, Immunologic↗
PubMed2003

Differentiation of effector/memory Vdelta2 T cells and migratory routes in lymph nodes or inflammatory sites.

Vdelta2 T lymphocytes recognize nonpeptidic antigens without presentation by MHC molecules and mount both immediate effector functions and memory responses after microbial infection. However, how Vdelta2 T cells mediate different facets of a memory response remains unknown. Here, we show that the expression of CD45RA and CD27 antigens defines four subsets of human Vdelta2 T cells with distinctive compartmentalization routes. Naive CD45RA+CD27+ and memory CD45RA-CD27+ cells express lymph node homing receptors, abound in lymph nodes, and lack immediate effector functions. Conversely, memory CD45RA-CD27- and terminally differentiated CD45RA+CD27- cells, which express receptors for homing to inflamed tissues, are poorly represented in the lymph nodes while abounding at sites of inflammation, and display immediate effector functions. These observations and additional in vitro experiments indicate a lineage differentiation pattern for human Vdelta2 T cells that generates naive cells circulating in lymph nodes, effector/memory cells patrolling the blood, and terminally differentiated effector cells residing in inflamed tissues.

Animals↗
PubMed2026

Cross-domain cooperation drives nutrient acquisition and metabolism in the bark beetle holobiont.

Microbial symbiosis underpins host adaptation, yet mechanisms of metabolic integration in holobionts remain unclear. Using metatranscriptomics, genomics, and metabolic assays, we investigated gut microbiome interactions in the European spruce bark beetle (Ips typographus). We observed metabolic complementarity among symbionts and host, forming cross-domain networks that support nutrient acquisition. Nitrogen recycling revealed strong interdependence: no single partner possessed a complete uric acid degradation pathway, but combined evidence supports a distributed pathway spanning beetle, Bacteria, and fungi. Additionally, bacterial nitrate reduction to ammonia indicates a potential nitrogen influx, making otherwise inaccessible inorganic nitrogen available to the host. Shaped by microbial interactions, symbionts also likely supply specific amino acids, while vitamin metabolism showed cross-domain co-metabolism, with Bacteria as main producers of B vitamins, while host and fungi modulated interconversion. Carbohydrate degradation was highly partitioned; bacteria target xylan and pectin, while fungi contribute to glucan breakdown. Crucially, our data provide indirect evidence that the beetle may contribute to complete cellulose degradation, highlighting an underappreciated host role in lignocellulose processing. In terms of enzymatic functional diversity, the bacteriome emerged as the most important microbiome component-an observation that contrasts with the traditional focus on fungi and underscores the need to consider bacterial contributions in insect symbioses. Despite life-stage variation, core metabolic functions remained stable. Overall, metabolic interdependence, rather than microbial composition alone, structures holobiont function. These results highlight functional redundancy and ecological resilience, emphasizing the importance of microbial cooperation and host-microbe metabolic evolution.

Bark beetle↗
PubMed2003

[Biological processes of the human environment regeneration within the Martian crew life support systems].

Five ground-based experiments at RF SRC-IBMP had the purpose to make a thorough investigation of a model of the human-unicellular algae-mineralization life support system. The system measured 15 m3 and contained 45 liters of alga suspension; the dry alga density was 10 to 12 g/l and water volume (including the alga suspension) amounted to 59 l. More sophisticated LSS models where algae were substituted by higher plants (crop area in the greenhouse equaled 15 m2) were investigated in three experiments from 1.5 mos. to 2 mos. in duration. It was found that the alga containing LSS was able to fulfill not only the macrofunction (air and water regeneration) but also several additional functions (air purification, establishment of microbial cenosis etc.) providing an adequate human environment. This polyfunctionality of the biological regenerative processes is a weighty argument for their integration into space LSSs. Another important aspect is that the unicellular algae containing systems are highly reliable owing to a huge number of species-cells which will be quickly recovered in case of the death of a part of the population and, consequently, functionality of the LSS autotrophic component will be restored before long. For an extended period of time the Martian crew will have no communication with the Earth's biosphere which implies that LSS should be absolutely reliable and redundant. Redundancy can be achieved through installation aboard the vehicle of two systems constructed on different principles of regeneration, i.e. physical-chemical and biological. Each of the LSSs should have the power to satisfy all needs of the crew. The best option is when two systems are functioning in parallel sharing the responsibility for the human environment. Redundancy in this case will mean that in the event of failure or a drastic decrease in performance of one system the other one will make up for the loss by increasing its share in the overall regeneration process.

Biomass↗
PubMed1992

Defense system in the biliary tract against bacterial infection.

Bacteria can invade the biliary tract by ascending from the duodenum and via the hematogenous route from the hepatic portal venous blood. The sphincter of Oddi, situated at the junction of the biliary tract and the upper gastrointestinal tract, forms an effective mechanical barrier to duodenal reflex and ascending bacterial infection. Conversely, Kupffer cells and the tight junctions between hepatocytes help prevent bacteria and toxic metabolites from entering the hepatobiliary system from the portal circulation. The continuous flushing action of bile and the bacteriostatic effects of bile salts keeps the biliary tract sterile under normal conditions. Secretory immunoglobulin A (sIgA), the predominant immunoglobulin in the bile, and mucus excreted by the biliary epithelium probably function as antiadherence factors, preventing microbial colonization. When barrier mechanisms break down, as in surgical or endoscopic sphincterotomy and with insertion of biliary stents, pathogenic bacteria enter the biliary system at high concentrations and take up residence on any foreign bodies. Intrabiliary pressure is a key factor in the development of cholangitis. Chronic biliary obstruction raises the intrabiliary pressure. This adversely influences the defensive mechanisms such as the tight junctions, Kupffer cell functions, bile flow, and sIgA production in the system, resulting in a higher incidence of septicemia and endotoxemia in these patients. Knowledge of biliary defense against infection is still quite primitive. Unclear are the roles of sIgA in the bile, mechanism of bacterial adhesion to the biliary epithelium, Kupffer cell function in biliary obstruction, and the antimicrobial activity of bile salts.

Animals↗
PubMed2001

Importance of a proline-rich sequence in the amino-terminal region for correct folding of mitochondrial and soluble microbial p450s.

All microsomal P450s have a proline-rich sequence (PR) in the amino-terminal region that is needed for proper folding [Kusano, K., Sakaguchi, M., Kagawa, N., Waterman, M.R. and Omura, T. (2001) J. Biochem., 129, 259-269]. There are also multiple proline residues near the amino-termini of the mature forms of all mitochondrial P450s and the amino-termini of soluble microbial P450s. To examine the functional significance of the PR in mitochondrial P450s, we expressed human P450c27 (CYP27) and bovine P450scc (CYP11A1) in an Escherichia coli heterologous expression system, and found that in each one specific proline residue is important for correct folding. Deletions from the amino-terminus further indicated the importance of the PR for the expression of a spectrally normal P450c27. Essentially the same results were obtained with two soluble microbial P450s, P450cam (CYP101) and P450nor, in each of which a PR is important for proper folding. We conclude that in all P450s (mitochondrial, microbial and microsomal P450s), a proline-rich sequence located in the amino-terminal region is important for proper folding. Furthermore, we predict that the importance of the PR in P450 folding is to reduce the tendency of the polypeptide to misfold prior to heme binding.

Amino Acid Sequence↗
PubMed2026

Integrating multi-omics technologies to decipher microbiome functions.

Multi-omics approaches have revolutionized our understanding of microbial communities by enabling simultaneous interrogation of genomic, transcriptomic, proteomic, and metabolomic data. The systematic integration and analysis of these deep datasets help decipher the functional roles of microbiomes, providing critical insights into microbial activities, interactions, and dynamics across diverse environments. Biological complexity makes multi-omics analysis of a single, isolated organism demanding but highly informative, yet this complexity increases further when samples comprise hundreds to thousands of individual species. As microbiome research continues to expand into clinical, environmental, and engineered systems, standardized workflows, benchmarked datasets, and community-driven initiatives are essential to ensure reproducibility, standardization and interpretability. Establishing and disseminating best practices for experimental design, data processing, and integrative analyses will be critical for maximizing comparability and scientific rigor across studies. This perspective highlights recent advances in multi-omics microbiome research, outlines key obstacles in data integration and metadata harmonization, and proposes a collaborative roadmap for scalable, FAIR-compliant multi-omics investigations and potentially disruptive Artificial Intelligence (AI) advances comparable to those of AlphaFold in the field of microbiome science.

Multiomics↗