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At least 973 records · Page 54Linked to original sources
PubMed2005

Imidazole antibiotics inhibit the nitric oxide dioxygenase function of microbial flavohemoglobin.

Flavohemoglobins metabolize nitric oxide (NO) to nitrate and protect bacteria and fungi from NO-mediated damage, growth inhibition, and killing by NO-releasing immune cells. Antimicrobial imidazoles were tested for their ability to coordinate flavohemoglobin and inhibit its NO dioxygenase (NOD) function. Miconazole, econazole, clotrimazole, and ketoconazole inhibited the NOD activity of Escherichia coli flavohemoglobin with apparent K(i) values of 80, 550, 1,300, and 5,000 nM, respectively. Saccharomyces cerevisiae, Candida albicans, and Alcaligenes eutrophus enzymes exhibited similar sensitivities to imidazoles. Imidazoles coordinated the heme iron atom, impaired ferric heme reduction, produced uncompetitive inhibition with respect to O(2) and NO, and inhibited NO metabolism by yeasts and bacteria. Nevertheless, these imidazoles were not sufficiently selective to fully mimic the NO-dependent growth stasis seen with NOD-deficient mutants. The results demonstrate a mechanism for NOD inhibition by imidazoles and suggest a target for imidazole engineering.

Anti-Bacterial Agents↗
PubMed2004

Genomic approaches to antibacterial discovery.

This chapter describes two key strategies for the discovery of new antibacterial agents and illustrates the critical role played by genomics in each. The first approach is genomic target-based screening. Comparative genomics and bioinformatics are used to identify novel, selective antibacterial targets of the appropriate antibacterial spectrum. Genetic technologies integral for the success of this approach, such as essentiality testing, are also described. An unprecedented number of novel targets have been discovered via this approach, and a plethora of examples are discussed. This section concludes with the case history of a target successfully progressed from identification by genomics, to high-throughput screening, and onto proof of concept in curing experimental infections. The second approach is based on screening for compounds with antibacterial activity and then employing a broad variety of newer technologies to identify the molecular target of the antibacterial agent. The advantage of this approach is that compounds already possess antibacterial activity, which is a property often challenging to engineer into molecules obtained from enzyme-based screening approaches. The recent development of novel biochemical and genomic technologies that facilitate identification and characterization of the mode of action of these agents has made this approach as attractive as the genomic target-based screening strategy.

Anti-Bacterial Agents↗
PubMed2004

Microbial serine carboxypeptidase inhibitors--comparative analysis of actions on homologous enzymes derived from man, yeast and wheat.

The actions of peptidase inhibitors derived from Streptomycete on human cathepsin A (hCath A), yeast carboxypeptidase Y (CPY), and wheat carboxypeptidase II (CPW) were analyzed comparatively. Lactacystin and omuralide (clasto-lactacystin beta-lactone), well-known cytoplasmic proteasome inhibitors, both had a potent and non-competitive inhibitory effect on these homologous serine carboxypeptidases, although they inhibited CPW and hCath A more effectively than CPY in vitro. Ebelactone B exhibited a mixed non-competitive inhibitory effect and selectivity for CPY. Piperastatin A showed competitive inhibition of CPY and hCath A but had little effect on CPW. In contrast, chymostatin inhibited CPW efficiently, while it had less effect on hCath A and CPY. In cell culture system, lactacystin was the most potent as to inactivation of the intralysosomal recombinant hCath A activity expressed in a genetically engineered fibroblastic cell line with galactosialidosis (hCath A deficiency). These results suggest that the specific inhibitory effects of lactacystin and its derivatives on hCath A might be applicable to elucidate the pathophysiological roles in the human deficinecy.

Carboxypeptidases↗
PubMed2026

A CRISPR-Cas9 Toolkit Enabling Tunable Integration and Transient Homologous Recombination Enhancement in Yarrowia lipolytica.

Although the oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory, its application remains constrained by inefficient homology-directed repair (HDR) and a lack of precise genomic integration tools. To address these limitations, we developed a comprehensive genetic toolkit featuring three synergistic advancements. First, we systematically identified 55 neutral integration sites with tunable expression profiles, enabling stable, position-independent gene integration with predictable transcriptional output across a 12.88-fold dynamic range. Second, we established a dual-readout high-throughput screening platform combining colony morphology analysis with hrGFP fluorescence. This approach accurately measures locus-specific homologous recombination (HR) efficiency while eliminating false positives by dominant non-homologous end joining (NHEJ). Third, we engineered a transient HR enhancement system by fusing the Sae2 exonuclease to Cas9 via a flexible (GGGGS)3 linker. This fusion significantly boosts HR efficiency and surpasses the cleavage activity of unmodified Cas9 without introducing permanent genomic modifications or compromising cellular fitness. Finally, HR efficiency for single-gene integration was increased from 46.5% to 77.5% while the dual-locus editing efficiency reached 64.1% when using 500-bp homology arms, and the engineered strains demonstrated improved genetic stability compared to those with constitutive HR enhancement.

Yarrowia↗
PubMed2003

Research and application of microbial enzymes--India's contribution.

Enzymes have attracted the attention of scientists world over due to their wide range of physiological, analytical and industrial applications. Although enzymes have been isolated, purified and studied from microbial, animal and plant sources, microorganisms represent the most common source of enzymes due to their broad biochemical diversity, feasibility of mass culture and ease of genetic manipulation. With the advent of molecular biology techniques, a number of genes of industrially important enzymes has been cloned and expressed in order to improve the production of enzymes, substrate utilization and other commercially useful properties. Special attention has been focused on enzymes isolated from thermophiles due to their inherent stability and industrial applications. In addition, a variety of methods have been employed to modify enzymes for their industrial usage including strain improvement, chemical modifications, modification of reaction environment, immobilization and protein engineering. A wide range of applications of enzymes in different bioprocess industries is discussed.

Archaea↗
PubMed2004

Prokaryotic diversity and its limits: microbial community structure in nature and implications for microbial ecology.

Recent advances in the estimation of prokaryotic diversity have brought us insight into two questions: what is the extent of prokaryotic diversity, and perhaps more importantly, why bother finding out. In this review, we highlight the insights about the extent of diversity that may be gained by considering patterns that occur, or are likely to occur, in the relative abundance of prokaryotic taxa. We posit that global reservoirs of diversity are an important driving force behind patterns in localised diversity seen in leaves, intestines and wastewater treatment reactors. Thus, where the reservoir community is very large and relatively even, chance alone will prevent physically identical communities from having the same, or sometimes even stable, communities. By contrast, communities that tend to be similar (even when not physically identical) and stable are observed where the source diversity is low. Thus the relationship between structure and function in a community can only be understood, predicted and engineered through an understanding of the source of diversity from which the community is drawn.

Ecology↗
PubMed2003

Directed evolution of a bacterial alpha-amylase: toward enhanced pH-performance and higher specific activity.

alpha-Amylases, in particular, microbial alpha-amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for alpha-amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the alpha-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild-type BAA and the mutants BAA S201N and BAA N297D were subjected to error-prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5-fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4- and 1.5-fold, respectively. Another mutant (BAA 29) possesses a wild-type-like pH profile but possesses a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial alpha-amylases.

Amino Acid Sequence↗
PubMed1988

Fate of genetically-engineered bacteria in activated sludge microcosms.

The conclusions that can be derived from this study regarding the fate of the GEMs and their ability to degrade added pollutants are as follows: 1. Both GEMs were able to survive in the microcoms. Because Pseudomonas sp. B13 has been cultured for a long time in the laboratory, it was not expected to survive well in the microcosm. Surprisingly, it and the derivative GEMs persisted at a high population level of approximately 10(5) bacteria/ml. Pure culture studies had demonstrated an ability of FR1(pFRC20P) to readily degrade simultaneous mixtures of 3CB and 4MB. In the microcosms, however, the GEM did not perform as well as expected, particularly when confronted with a shock load of a 3CB and 4MB mixture. Thus, the microcosm studies may be of potential help for making predictions concerning environmental applications of GEMs. 2. Pseudomonas sp. B13 derivative strains FR1 and FR1(pFRC20P) were able to degrade low concentrations of substituted benzoates within the complex ecosystem of the activated sludge microcosm. A good deal of information concerning the degradation pathway for aromatics by Pseudomonas sp. B13 was already known. This allowed for the construction of the stable, regulated pathways for the degradation of substituted aromatic compounds in both GEMs and indicates that the construction of similar GEMs for the degradation of environmental pollutants is a promising experimental strategy. 3. There was not any demonstrable, adverse effect of GEM addition to the microbial population level in the microcosm. The GEMs were even able to function in a protective manner for the indigenous populations by buffering them against the adverse effects of addition of substituted benzoates. In contrast, for microcosms lacking GEM addition, a wash-out of the bacterial population in the microcosm occurred (data not shown). 4. Lateral transfer of new genetic information (xylXYZLS) from the GEM chromosome to indigenous microorganisms was not detected, whereas transfer of the hybrid, mobilizable pFRC20P carrying the gene for lactone isomerase did apparently occur. In this particular case, transfer may have been beneficial for the community as a whole if it increased the ecosystem's ability to cope with the presence of toxic pollutants. As more GEMs are constructed for specific biotechnological applications the diversity and complexity of microcosms used to study their fate and function will increase. The ability of such studies to predict a priori the fate of these microorganisms will help to develop strategies both to decrease the risks associated with introducing GEMs into the environment and to increase and regulate the capacity of GEMs to degrade toxic pollutants.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteria↗
PubMed1993

[The Staphylococcus aureus carrier state among the workers of a machine-building plant].

120 workers of beet-harvesting combines plant and 120 students and teachers (control group) were examined for S. aureus carrying. In workers level of carrying on nasal and pharyngeal mucosa as well as on the skin of hands was 3-7 times as high as in members of control group. Number of carriers and quantity of microbial population on the skin of hands depended on the use of oils and emulsion in production process. Contact with oils favoured skin contamination while emulsions reduced bacterial colony. 36% of mechano-assembly shop workers and 10-12% of those employed at mechano-processing constitute the risk group which is likely to develop purulent skin diseases in microtraumas of hand and fingers.

Adolescent↗
PubMed1998

Polyhydroxyalkanoates, biopolyesters from renewable resources: physiological and engineering aspects.

Polyhdroxyalkanoates (PHAs), stored as bacterial reserve materials for carbon and energy, are biodegradable substitutes to fossil fuel plastics that can be produced from renewable raw materials. PHAs can be produced under controlled conditions by biotechnological processes. By varying the producing strains, substrates and cosubstrates, a number of polyesters can be synthesized which differ in monomer composition. By this means, PHAs with tailored interesting physical features can be produced. All of them are completely degradable to carbon dioxide and water through natural microbiological mineralization. Consequently, neither their production nor their use or degradation have a negative ecological impact. After a historical review, possibilities for the synthesis of novel PHAs applying different micro-organisms are discussed, and pathways of PHA synthesis and degradation are shown in detail for important PHA producers. This is followed by a discussion of the physiological role of the accumulation product in different micro-organisms. Detection, analysis, and extraction methods of PHAs from microbial biomass are shown, in addition to methods for polyester characterization. Strategies for PHA production under discontinuous and continuous regimes are discussed in detail in addition to the use of different cheap carbon sources from the point of view of different PHA producing strains. An outlook on PHA production by transgenic plants closes the review.

Bacteria↗
PubMed2007

Engineering of a sialic acid synthesis pathway in transgenic plants by expression of bacterial Neu5Ac-synthesizing enzymes.

Plants are a low-cost and contamination-free factory for the production of recombinant pharmaceutical proteins. However, plant-made pharmaceuticals differ from their mammalian homologues by the structure of their N-linked glycans. For instance, most mammalian glycoproteins harbour terminal sialic acids that control their half-life in the bloodstream. The absence of the whole sialylation machinery in plants is of major concern as non-sialylated plant-made pharmaceuticals may not perform at their full potential in humans, because of their removal from the circulation through the involvement of hepatic cell receptors. In this context, we have investigated the synthesis of N-acetylneuraminic acid (Neu5Ac) in the cytosol of plants by either the re-routing of the endogenous 3-deoxy-d-manno-2-octulosonic acid (Kdo) biosynthetic pathway or the expression of microbial Neu5Ac-synthesizing enzymes. In this paper, we demonstrate that the plant Kdo-8P synthase is not able to use N-acetyl d-mannosamine as a substrate, and thus re-routing of the Kdo pathway for the synthesis of Neu5Ac is not possible. Consequently, we expressed genes encoding Neu5Ac lyase from Escherichia coli and Neu5Ac synthase (neuB2) from Campylobacter jejuni in plants. These resulted in the production of functional enzymes in the cytosol, which in turn can catalyse the synthesis of Neu5Ac in vitro. Experiments were carried out on two models, Bright Yellow 2 (BY2) tobacco cells and Medicago sativa (alfalfa), the perennial legume crop.

Bacterial Proteins↗
PubMed2004

The surface-mosaic model in host-parasite relationships.

The dynamics of protein adsorption to a microbial surface could be of significance in host-parasite relationships because non-defense proteins might interfere with the binding of defense proteins. A surface mosaic of defense and non-defense proteins formed on the microbial surface could activate one of the tissue reactivity programs via a binary code (help or silence) generated by the adsorbed proteins. Understanding the mechanisms of the mosaic formation and its evolution might help to identify evasion mechanisms used by virulent microorganisms. This also provides a conceptual framework to design new strategies to control the infectious diseases they cause.

Adsorption↗
PubMed2002

Probing and overcoming a mould menace.

Thorough investigation of an environmental problem within Brockville General Hospital, Ontario, Canada, was vital. This special report is by William Roth, Chris Rahm and Bruce G. Fraser--all of Jacques Whitford Environment Limited. William Roth is the first point of contact for Jacques Whitford's hospital clients and is a project manager within the Environmental Engineering group of JWEL's Ottawa office. Chris Rahm is a member of the Hazardous Materials group in JWEL's Ottawa office and was the site supervisor for the mould abatement at BGH. Bruce Fraser is the corporate technical director of indoor air quality for JWEL and regularly provides technical assistance for mould related projects carried out by JWEL.

Air Microbiology↗
PubMed2006

Mass spectrometric identification of proteins in complex post-genomic projects. Soluble proteins of the metabolically versatile, denitrifying 'Aromatoleum' sp. strain EbN1.

The rapidly developing proteomics technologies help to advance the global understanding of physiological and cellular processes. The lifestyle of a study organism determines the type and complexity of a given proteomic project. The complexity of this study is characterized by a broad collection of pathway-specific subproteomes, reflecting the metabolic versatility as well as the regulatory potential of the aromatic-degrading, denitrifying bacterium 'Aromatoleum' sp. strain EbN1. Differences in protein profiles were determined using a gel-based approach. Protein identification was based on a progressive application of MALDI-TOF-MS, MALDI-TOF-MS/MS and LC-ESI-MS/MS. This progression was result-driven and automated by software control. The identification rate was increased by the assembly of a project-specific list of background signals that was used for internal calibration of the MS spectra, and by the combination of two search engines using a dedicated MetaScoring algorithm. In total, intelligent bioinformatics could increase the identification yield from 53 to 70% of the analyzed 5,050 gel spots; a total of 556 different proteins were identified. MS identification was highly reproducible: most proteins were identified more than twice from parallel 2DE gels with an average sequence coverage of >50% and rather restrictive score thresholds (Mascot >or=95, ProFound >or=2.2, MetaScore >or=97). The MS technologies and bioinformatics tools that were implemented and integrated to handle this complex proteomic project are presented. In addition, we describe the basic principles and current developments of the applied technologies and provide an overview over the current state of microbial proteome research.

Amino Acid Sequence↗
PubMed2000

Biochemical and genetic bases of microbial degradation of polychlorinated biphenyls (PCBs).

The microbial degradation of polychlorinated biphenyls (PCBs) has been extensively conducted by many workers, and the following general results have been obtained. (1) PCBs are degraded oxidatively by aerobic bacteria and other microorganisms such as white rot fungi. PCBs are also reductively dehalogenated by anaerobic microbial consortia. (2) The biodegradability of PCBs is highly dependent on chlorine substitution, i.e., number and position of chlorine. The degradation and dehalogenation capabilities are also highly strain dependent. (3) Biphenyl-utilizing bacteria can cometabolize many PCB congeners to chlorobenzoates by biphenl-catabolic enzymes. (4) Enzymes involved in the PCB degradation were purified and characterized. Biphenyl dioxygenase, ring-cleavage dioxygenase, and hydrolase are crystallized, and two ring-cleavage dioxygenases are being solved by x-ray crystallography. (5) The bph gene clusters responsible for PCB degradation are cloned from a variety of bacterial strains. The structure and function are analyzed with respect to the evolutionary relationship. (6) The molecular engineering of biphenyl dioxygenases is successfully performed by DNA shuffling, domain exchange, and subunit exchange. The evolved enzymes exhibit wide and enhanced degradation capacities for PCBs and other aromatic compounds.

Journal Article↗
PubMed2006

The effects of changing water flow velocity on the formation of biofilms and water quality in pilot distribution system consisting of copper or polyethylene pipes.

We studied the effects of flow velocity on the formation of biofilms and the concentration of bacteria in water in copper and plastic (polyethylene, PE) pipes. The formation of biofilms increased with the flow velocity of water. The increase in microbial numbers and contents of ATP was clearer in the PE pipes than in the copper pipes. This was also seen as increased consumption of microbial nutrients in the pipeline system. This indicates that the mass transfer of nutrients is in major role in the growth of biofilms. However, the increased biomass of biofilms did not affect microbial numbers in the water. Rapid changes in water flow rate resuspended biofilms and sediments which increased the concentrations of bacteria and copper in water. The disturbance caused by the changing water flow was also seen as an increase in the particle counts and water turbidity recorded with online instrumentation.

Biofilms↗
PubMed2004

Transgene expression in strawberries driven by a heterologous phloem-specific promoter.

Strawberry is susceptible to diseases caused by phytoplasmas, mycoplasma-like prokaryotes restricted to sieve elements in the phloem tissue of infected plants. One strategy to improve strawberry resistance to phytoplasmas involves transgenic expression of anti-microbial peptide genes in phloem. For targeted phloem-specific expression, we constructed a binary vector with an expression cassette bearing the beta-glucuronidase (GUS) reporter gene (uidA) under control of the Arabidopsis sucrose-H+ symporter gene (AtSUC2) promoter. Transgenic strawberry lines were generated with high efficiencies by a modified transformation protocol, which combines the adoption of a 3-day pre-selection period following transformation, and the addition of 10-microM thidiazuron to the regeneration medium. Histological GUS activity indicated that the reporter gene was expressed specifically in phloem of leaves, petioles, and roots of transgenic plants. The results suggest that the transformation protocol and the AtSUC2 promoter may be useful for engineering phytoplasma-resistant transgenic strawberries.

Arabidopsis↗
PubMed1996

Antibiotic activity of polyketide products derived from combinatorial biosynthesis: implications for directed evolution.

A library of over 100 polyketides, generated via combinatorial cloning of genes encoding subunits of aromatic polyketide synthases, was screened for molecules capable of inhibiting the growth of gram-positive bacteria. A total of 26 polyketides, with varying levels of antibiotic activity in filter-disk assays, were purified. Most bioactive polyketides were produced as relatively minor compounds (< 1 mg/l), although two major anthraquinones, with yields in the range of 10-100 mg/l, were also identified and structurally characterized. When tested against Bacillus subtilis 168 beta, they were found to cause a 50% reduction in colony-forming units at concentrations of 20 and 300 micrograms/ml, respectively. We speculate that many of the minor (and possibly more potent) bioactive polyketides are synthesized via nonspecific enzymatic modifications of shunt products derived from engineered polyketide synthase pathways. If so, then these 'fortuitous' pathways should be amenable to further rationally guided manipulation. Our results support the notion that combinatorial biosynthesis can be used to generate novel, biologically active molecules. They also point to the feasibility of designing mutagenesis selection experiments aimed at the directed evolution of organic molecules with desirable pharmaceutical properties.

Anti-Bacterial Agents↗