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PubMed1987

Role of motility, chemotaxis, and adhesion in microbial ecology.

This review emphasized the implications of recent data pertaining to the role that motility, chemotaxis, and adhesion play in microbial ecology. Some of these processes appear to promote colonization by allowing certain organisms to selectively "seek out" nutrients or sites of colonization. For example, chemotaxis to NO3- and NO2- may provide pseudomonads with such a strong competitive mechanism that it allows this group of bacteria to outcompete other members of the soil microbiota for these chemicals. Likewise, chemotaxis also allows other bacteria to enter and colonize the mucus gel lining the intestinal epithelium and thereby resist physical removal from the gut. On the other hand, the understanding of such mechanisms offers important new possibilities for the deliberate control of microorganisms for the benefit of man. To that end, much remains to be done before a thorough understanding of the ecology of the microflora of any ecosystem can be accomplished. It is hoped that this review will stimulate further work in this area, as well as to lead to collaboration between engineers and microbiologists, which should lead to fruitful and exciting research in the future.

Animals↗
PubMed2026

Limited efficacy of plazomicin against carbapenem resistant Enterobacterales in a tertiary care centre in Eastern India.

The global escalation of carbapenem resistant Enterobacterales (CRE) represents a critical threat to public health, necessitating the investigation of alternative therapeutic agents. Plazomicin, a newer aminoglycoside, was engineered to evade common aminoglycoside-modifying enzymes. This study evaluated the susceptibility of plazomicin among 100 CRE isolates. The findings reveal a strikingly low plazomicin susceptibility rate of only 15/100 (15%). Whole-genome sequencing identified a high prevalence of 16S rRNA methyltransferase (RMTase) genes, such as armA and rmtB1, which confer high-level resistance to plazomicin. These results suggest that plazomicin may have limited utility against CRE in regions where RMTase-mediated resistance is prevalent.

Sisomicin↗
PubMed1990

Biosafety considerations in industries with production methods based on the use of recombinant deoxyribonucleic acid.

Since no occupational accidents or diseases have been attributed specifically to the use of constructions containing recombinant deoxyribonucleic acid (rDNA), this paper evaluates the occupational health risks in industries utilizing genetically manipulated organisms mainly on the basis of theoretical considerations. Bacteria, filamentous fungi, yeasts, and mammalian cells in culture are in use. For each of these systems the possible hazards are considered. Concerning microbial production systems, infections are regarded as the main problem, but the risk of infection is considered extremely low. As for cells in culture, only dormant viruses are regarded as problematic, but well-defined production cell lines should not contain such undetected and dangerous viruses. Overall, the additional risks posed by rDNA-modified micro-organisms are minor. Only long-term observations can, however, confirm this assumption, and consequently the highest feasible containment measures should still be used in the years to come.

Accident Prevention↗
PubMed2005

Yeast surface display of a noncovalent MHC class II heterodimer complexed with antigenic peptide.

Microbial protein display technologies have enabled directed molecular evolution of binding and stability properties in numerous protein systems. In particular, dramatic improvements to antibody binding affinity and kinetics have been accomplished using these tools in recent years. Examples of successful application of display technologies to other immunological proteins have been limited to date. Herein, we describe the expression of human class II major histocompatibility complex allele (MHCII) HLA-DR4 on the surface of Saccharomyces cerevisiae as a noncovalently associated heterodimer. The yeast-displayed MHCII is fully native as assessed by binding of conformationally specific monoclonal antibodies; failure of antibodies specific for empty HLA-DR4 to bind yeast-displayed protein indicates antigenic peptide is bound. This report represents the first example of a noncovalent protein dimer displayed on yeast and of successful display of wild-type MHCII. Results further point to the potential for using yeast surface display for engineering and analyzing the antigen binding properties of MHCII.

Antibodies, Monoclonal↗
PubMed2004

Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts.

Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme. Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second. The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.

Bacteria↗
PubMed2006

A pilot study of an anti-MRSA bio-engineered lacteal complex (anti-MRSA BLC) in a murine septicemia model.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen of humans and other animals, causing septicemia, abcessation, toxemia, and other infectious diseases. Refined bioengineered lacteal complex (BLC), made specifically against MRSA, is a novel complex of low molecular weight immunogenic and antimicrobial molecules. It was evaluated in vivo using a mouse model of MRSA-induced peritonitis. Intraperitoneal dosing of anti-MRSA BLC demonstrated a therapeutic effect (83% survival) against an intraperitoneal MRSA challenge that caused 100% mortality in untreated animals. Anti-MRSA BLC is a promising therapeutic modality for MRSA infection.

Animals↗
PubMed2002

Novel Saccharomyces cerevisiae screen identifies WR99210 analogues that inhibit Mycobacterium tuberculosis dihydrofolate reductase.

The ongoing selection of multidrug-resistant strains of Mycobacterium tuberculosis has markedly reduced the effectiveness of the standard treatment regimens. Thus, there is an urgent need for new drugs that are potent inhibitors of M. tuberculosis, that exhibit favorable resistance profiles, and that are well tolerated by patients. One promising drug target for treatment of mycobacterial infections is dihydrofolate reductase (DHFR; EC 1.5.1.3), a key enzyme in folate utilization. DHFR is an important drug target in many pathogens, but it has not been exploited in the search for drugs effective against M. tuberculosis. The triazine DHFR inhibitor WR99210 has been shown to be effective against other mycobacteria. We show here that WR99210 is also a potent inhibitor of M. tuberculosis and Mycobacterium bovis BCG growth in vitro and that resistance to WR99210 occurred less frequently than resistance to either rifampin or isoniazid. Screening of drugs with M. tuberculosis cultures is slow and requires biosafety level 3 facilities and procedures. We have developed an alternative strategy: initial screening in an engineered strain of the budding yeast Saccharomyces cerevisiae that is dependent on the M. tuberculosis DHFR for its growth. Using this system, we have screened 19 compounds related to WR99210 and found that 7 of these related compounds are also potent inhibitors of the M. tuberculosis DHFR. These studies suggest that compounds of this class are excellent potential leads for further development of drugs effective against M. tuberculosis.

Antibiotics, Antitubercular↗
PubMedDate not supplied

Purification of bacterial exotoxins. The case of botulinum, tetanus, anthrax, pertussis and cholera toxins.

Bacterial protein toxins and their fragments have been isolated and purified for various reasons, including the development of efficient vaccines and for methods of identification of bacterial agents causing disease. This activity continues today but a new area of bacterial protein toxin research has recently emerged. Since it was shown that toxin molecules comprise several types of biological activity within their structural domains, it was suggested to use these domains (and their combinations) as biochemical tools for developing novel agents for disease imaging and and/or relieving. In this way eukaryotic cell-receptor specific fusion toxins have been developed to prevent malignancy in human. While human clinical trials of these preparations have only recently begun, the preliminary clinical findings are promising. Also fusion proteins which combine independent immunodominant epitopes from different antigens have also been developed thus opening a way for the generation of new vaccines for both human and veterinary use. Receptor binding fragments of microbial toxins when combined with other molecules may be useful in delivering these molecules into the cell. In this way novel agents may be developed with a potential for inducing specific changes at the molecular level for the correction of metabolic disorders causing human and animal diseases. Bacterial protein toxins such as anthrax, botulinum, cholera, pertussis and tetanus for which considerable progress has been achieved in structure-function analysis are promising candidates for such research. Particularly exciting appears the idea of extending this research to the cells of the nervous system, exploiting the unique specificity of the botulinum or tetanus toxin fragments which may bring long desired methods for treatment of various disorders of the nervous system. Data on functional domains of these toxins as well as methods of purification of the whole toxins and their fragments are considered in this review as they form a base for their further structure-function analysis and engineering applications.

Animals↗
PubMed1997

Construction and characterization of a recombinant ureolytic Streptococcus mutans and its use to demonstrate the relationship of urease activity to pH modulating capacity.

To begin to understand the contribution of oral microbial ureolysis to the inhibition of dental caries, we sought to construct a recombinant, ureolytic mutans streptococcus and correlate the ureolytic capacity of plaque bacteria with pH moderating ability. Streptococcus mutans GS-5 was transformed with a plasmid containing the urease genes from Streptococcus salivarius 57.I. The recombinant strain, S. mutans AC04, stably maintained the urease genes. High levels of urease activity were detected, with a maximum specific activity of 0.9 mumol of urea hydrolyzed/min/mg cell dry weight when the growth medium was supplemented with 50 microM exogenous NiCl2. Harboring the recombinant plasmid, or growth in NiCl2, did not markedly affect the glycolytic capacity of S. mutans. In vitro pH drop analysis of S. mutans AC04, metabolizing glucose and physiologically relevant concentrations of urea simultaneously, demonstrated that increasing the urease activity of plaque bacteria resulted in a corresponding reduction in the depth and the duration of the glycolytic pH fall. The results demonstrate the feasibility of engineering urease producing S. mutans and suggest that enhancing the ureolytic capacity of dental plaque, particularly cariogenic plaque, may help to offset the progression of the caries process.

Hydrogen-Ion Concentration↗
PubMed2002

Targets and assays for discovering novel antibacterial agents.

The increasing frequency of nosocomial infections due to multi-resistant pathogens exerts a significant toll and calls for novel and better antibiotics. Different approaches can be used in the search for novel antibiotics acting on drug-resistant bacterial pathogens. We present some considerations on valid bacterial targets to be used for searching new antibiotics, and how the information from bacterial genome sequences can assist in choosing the appropriate targets. Other factors to be considered in target selection are the chemical diversity available for screening and its uniqueness. We will conclude discussing our strategy for searching novel antibacterials. This is based on a large collection of microbial extracts as a source of chemical diversity and on the use of specific targets essential for the viability of bacterial pathogens. Two assay strategies have been implemented: a pathway-based assay, where a series of essential bacterial targets is screened in a single assay; and a binding assay, where many targets can be screened individually in the same format.

Anti-Bacterial Agents↗
PubMed1998

Substituted salicylanilides as inhibitors of two-component regulatory systems in bacteria.

A new class of inhibitors of the two-component regulatory systems (TCS) of bacteria was discovered based on the salicylanilide screening hits, closantel (1) and tetrachlorosalicylanilide (9). A systematic SAR study versus a model TCS, KinA/Spo0F, demonstrated the importance of electron-attracting substituents in the salicyloyl ring and hydrophobic groups in the anilide moiety for optimal activity. In addition, derivatives 8 and 16, containing the 2, 3-dihydroxybenzanilide structural motif, were potent inhibitors of the autophosphorylation of the KinA kinase, with IC50s of 2.8 and 6. 3 &microM, respectively. Compound 8 also inhibited the TCS mediating vancomycin resistance (VanS/VanR) in a genetically engineered Enterococcus faecalis cell line at concentrations subinhibitory for growth. Closantel (1), tetrachlorosalicylanilide (9), and several related derivatives (2, 7, 10, 11, 20) had antibacterial activity against the drug-resistant organisms, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF).

Anti-Bacterial Agents↗
PubMed2002

Construction of a fluorescent biosensor family.

Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand-mediated hinge-bending motion. Construction of cysteine mutations at these locations then permits site-specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three-dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.

Amino Acid Sequence↗
PubMed1999

Assessment of the application of baculoviruses for control of Lepidoptera.

Baculoviruses, among other insect viruses, are regarded as safe and selective bioinsecticides, restricted to invertebrates. They have been used worldwide against many insect pests, mainly Lepidoptera. Their application as microbial pesticides, however, has not met their potential to control pests in crops, forests, and pastures, with the exception of the nuclear polyhedrosis virus of the soybean caterpillar (Anticarsia gemmatalis), which is used on approximately 1 million ha annually in Brazil. Problems that have limited expansion of baculovirus use include narrow host range, slow killing speed, technical and economical difficulties for in vitro commercial production, timing of application based on frequent host population monitoring, variability of field efficacy due to climatic conditions, and farmers' attitudes toward pest control, which have been based on application of fast-killing chemical insecticides. Farmer education regarding use of biological insecticides and their characteristics is considered one of the major actions necessary for increased use of baculoviruses. Strategies to counteract some of the limitations of baculoviruses, especially their slow killing activity, have been investigated and are promising. These include the use of chemical or biological substances added to virus formulations and genetic engineering of the viruses themselves to express insect toxins or hormones. Such strategies can enhance viral activity and increase speed of kill as well as reduce larval feeding activity. The use of baculoviruses against Lepidoptera is reviewed, with the utilization of the nuclear polyhedrosis virus of A. gemmatalis in Brazil serving as a case-study.

Journal Article↗
PubMed2005

Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site.

Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.

Base Sequence↗
PubMed1991

Techniques for selection of industrially important microorganisms.

The screening of microorganisms for the production of useful products continues to be an important aspect of biotechnology. Although advances in instrumentation, genetics, and microbial physiology are having an impact, screening programs are still primarily based on so-called classical techniques of enrichment and mutagenesis. One area that needs strengthening is the advancement of knowledge in microbial physiology. Recent surveys indicate that industry leaders see trained microbial physiologists as being the limiting factor in development of biotechnology in the coming decade. The largest impediment to development of new screening techniques is the ironic lack of programs specifically directed at developing new techniques. Too much emphasis is placed on using available techniques and relying on sheer labor and screening of vast numbers of organisms to produce novel products. In this respect, the Japanese are the exception and have proven that the establishment of new programs is worth the cost and effort. They are undoubtedly the world leaders in development of screening techniques and consequently the discovery of novel products. The isolation of microbes from novel and extreme environments holds tremendous promise in two areas. First, as Omura (46) and others (9, 48, 65) state, novel organisms will yield novel products. Second, such organisms serve as models for the understanding of structure and function that will facilitate the genetic manipulation of organisms and advance our ability to engineer novel enzymes. Hopefully such advances will enable genetic and protein engineering to have a greater impact on screening programs and techniques in the future. The earth holds a vast amount of varied and unique environments, from natural extremes such as high-altitude deserts and thermal springs, to manmade environments such as industrial-waste-treatment facilities, from which, with the appropriate methods and techniques, we may isolate and evaluate new potential products.

Bacterial Physiological Phenomena↗
PubMedDate not supplied

Deletions in the beta3-beta4 hairpin loop of HIV-1 reverse transcriptase are observed in HIV-1 isolated from subjects during long-term antiretroviral therapy.

OBJECTIVES: To examine the effect of in-frame deletions in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) on plasma viremia and phenotypic resistance to antiretroviral drugs. STUDY DESIGN/METHODS: Plasma HIV-1 RNA was isolated from 168 antiretroviral therapy-experienced subjects for quantification of plasma viremia, RT sequence analysis, and phenotypic resistance assays. RESULTS: Four patients were found to harbor HIV-1 strains possessing in-frame, 3-nucleotide deletions at RT codons 67, 69, and 70. In these subjects, phenotypic resistance and high plasma viremia were observed only in a background of multiple resistance mutations. A recombinant virus engineered with an in-frame deletion of RT codon 67 did not have increased resistance to nucleoside reverse transcriptase inhibitors (NRTIs). CONCLUSIONS: Selection for deletions within the beta3-beta4 hairpin loop of the HIV-1 RT is an uncommon event most likely to occur in subjects with long-term antiretroviral experience. The codon 67 deletion does not appear to cause increased phenotypic resistance or increased viremia in the absence of concomitant RT mutations.

Adult↗
PubMed1995

Enzyme-catalyzed dehalogenation of pentachloroethane: why F87W-cytochrome P450cam is faster than wild type.

Under anaerobic conditions, cytochromes P450 can reductively dehalogenate heavily halogenated hydrocarbons, such as one- and two-carbon organic solvents. This catalytic capacity has drawn attention to the potential use of engineered forms of P450s in the remediation of contaminated deep subsurface ecosystems. Loida (1994, PhD Thesis, University of Illinois at Urbana-Champaign, IL) and S.G. Sligar (personal communication) have observed recently that an active-site variant of cytochrome P450cam (F87W) dechlorinates pentachloroethane approximately three times faster than the wild-type enzyme. Molecular dynamics simulations have revealed that the mutant enzyme binding pocket remains smaller, and that pentachloroethane assumes configurations closer to the heme-Fe in the F87W mutant twice as often as in the wild-type enzyme. This result is consistent with a collisional model of dehalogenation, which agrees with experimental observations [Li and Wackett (1993) Biochemistry, 32, 9355-9361] that solutions containing wild-type P450cam dehalogenate pentachloroethane 100 times faster than those containing free heme. The simulations suggest that it is unlikely that Trp87 significantly stabilizes the developing negative charge on the substrate during carbon-halogen bond reduction. The design of improved microbial enzymes that incorporate both steric and electronic effects continues for use in remediating halogenated contaminants in situ.

Binding Sites↗
PubMed1997

Precursor-directed biosynthesis of erythromycin analogs by an engineered polyketide synthase.

A genetic block was introduced in the first condensation step of the polyketide biosynthetic pathway that leads to the formation of 6-deoxyerythronolide B (6-dEB), the macrocyclic precursor of erythromycin. Exogenous addition of designed synthetic molecules to small-scale cultures of this null mutant resulted in highly selective multimilligram production of unnatural polyketides, including aromatic and ring-expanded variants of 6-dEB. Unexpected incorporation patterns were observed, illustrating the catalytic versatility of modular polyketide synthases. Further processing of some of these scaffolds by postpolyketide enzymes of the erythromycin pathway resulted in the generation of novel antibacterials with in vitro potency comparable to that of their natural counterparts.

Amino Acid Substitution↗