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PubMedDate not supplied

[Specific prophylaxis of shigellosis].

A high level of shigellosis morbidity requires new approaches to the control of bacterial dysentery. According to modern concepts, the outbreaks of Shigella infections are linked both with less intensive epidemic control measures and the objective cyclic character of the epidemic process. In this connection great importance is attached to the development of vaccines for the immunization of high risk groups in territories with unfavorable epidemic conditions and in zones of military conflicts, as well as children of school and pre-school age, who mostly determine seasonal rises of shigellosis morbidity. In this article the data describing new approaches to the creation of new live enteric vaccines on the basis of the knowledge of the genetic control of microbial pathogenicity and the regulation of its expression are presented. Attenuated Shigella mutants, created by different authors and having good prospects to be used for the development of vaccines, are characterized.

Dysentery, Bacillary↗
PubMed1999

Molecular and cellular mechanisms for microbial entry into the CNS.

A number of pathogenic microbes including neuroinvasive viruses, bacteria and parasites are capable of entry into the central nervous system (CNS) and cause a variety of clinical manifestations. The cellular and molecular mechanisms for the CNS invasion have been extensively studied in the last two decades. Viruses invade neurons and thereby cause encephalitis or peripheral neuritis, while bacteria enter the cerebrospinal fluid (CSF) and cause meningitis. In contrast, the mechanisms for parasitic neuroinvasion are much more complex and less clear. The capabilities that enable these elite subsets of pathogens to engineer uptake into the CNS will be the subject of this review.

Animals↗
PubMed1996

Lymphocytes utilize CD11b/CD18 for adhesion to Candida albicans.

Large granular lymphocytes require adherence to hyphae of Candida albicans to inhibit growth of this fungus. This study was undertaken to identify the lymphocyte surface structures that mediate this adhesion. Monoclonal antibodies specific for epitopes of the alpha subunit (CD11b) and the beta 2 subunit (CD18) of Mac-1 eliminated lymphocyte adhesion to C. albicans hyphae. Significant inhibition of lymphocyte adhesion to C. albicans was also achieved with known protein ligands of Mac-1. These proteins included the extracellular matrix proteins vitronectin, laminin, and fibrinogen as well as two engineered peptides containing RGD (arginine-glycine-aspartic acid) sequences. Carbohydrates including N-acetyl-D-glucosamine which have been demonstrated to inhibit Mac-l-mediated adhesion to whole yeast and yeast zymosan also blocked lymphocyte adhesion to hyphae. These results identify Mac-1 (CD11b/CD18) as the surface structure that mediates lymphocyte adhesion to C. albicans. A model is proposed for lymphocyte Mac-1 activation by microbial ligands.

Amino Acid Sequence↗
PubMed2005

Feasibility of acrylic acid production by fermentation.

Acrylic acid might become an important target for fermentative production from sugars on bulk industrial scale, as an alternative to its current production from petrochemicals. Metabolic engineering approaches will be required to develop a host microorganism that may enable such a fermentation process. Hypothetical metabolic pathways for insertion into a host organism are discussed. The pathway should have plausible mass and redox balances, plausible biochemistry, and plausible energetics, while giving the theoretically maximum yield of acrylate on glucose without the use of aeration or added electron acceptors. Candidate metabolic pathways that might lead to the theoretically maximum yield proceed via beta-alanine, methylcitrate, or methylmalonate-CoA. The energetics and enzymology of these pathways, including product excretion, should be studied in more detail to confirm this. Expression of the selected pathway in a host organism will require extensive genetic engineering. A 100,000-tons/year fermentation process for acrylic acid production, including product recovery, was conceptually designed based on the supposition that an efficient host organism for acrylic acid production can indeed be developed. The designed process is economically competitive when compared to the current petrochemical process for acrylic acid. Although the designed process is highly speculative, it provides a clear incentive for development of the required microbial host, especially considering the environmental sustainability of the designed process.

Acrylates↗
PubMed2002

The dynamic microbe: green fluorescent protein brings bacteria to light.

The demonstration that the green fluorescent protein (GFP) from the jellyfish Aequorea victoria required no jellyfish-specific cofactors and could be expressed as a fluorescent protein in heterologous hosts including both prokaryotes and eukaryotes sparked the development of GFP as one of the most common reporters in use today. Over the past several years, the utility of GFP as a reporter has been optimized through the isolation and engineering of variants with increased folding rates, different in vivo stabilities and colour variants with altered excitation and emission spectral properties. One of the great utilities of GFP is as a probe for characterizing spatial and temporal dynamics of gene expression, protein localization and protein-protein interactions in living cells. The innovative application of GFP as a reporter in bacteria has made a significant contribution to microbial cell biology. This review will highlight recent studies that demonstrate the potential of GFP for real-time analysis of gene expression, protein localization and the dynamics of signalling transduction pathways through protein-protein interactions.

Animals↗
PubMed2005

Microbial transformation of tannin-rich substrate to gallic acid through co-culture method.

Modified solid-state fermentation (MSSF) of tannin-rich substrate yielding tannase and gallic acid was carried out using a co-culture of the filamentous fungi, Rhizopus oryzae (RO IIT RB-13, NRRL 21498) and Aspergillus foetidus (GMRB013 MTCC 3557). Powdered fruits of Terminalia chebula and powdered pod cover of Caesalpinia digyna was used in the process and the different process parameters for maximum production of tannase and gallic acid by co-culture method were optimized through media engineering. MSSF was carried out at the optimum conditions of 30 degrees C and 80% relative humidity. The optimal pH and incubation period was 5.0 and 48 h respectively. Through the co-culture technique the maximum yield of tannase and gallic acid was found to be 41.3 U/ml and 94.8% respectively.

Bioreactors↗
PubMed2004

RC-101, a retrocyclin-1 analogue with enhanced activity against primary HIV type 1 isolates.

Rhesus macaques express three theta-defensins (RTDs 1-3), cyclic octadecapeptides with antiviral and lectin-like properties. Corresponding theta-defensin genes exist and are expressed in humans, but a signal sequence mutation prevents the formation of mature theta-defensin peptides. Retrocyclin-1 is a theta-defensin peptide whose precursor is encoded by human theta-defensin pseudogenes. It can protect human peripheral blood lymphocytes from infection by R5 and X4 strains of HIV-1, and provides a molecular template for designing novel antiviral agents. In this study, we used JC53-BL reporter cells to assess the activity of retrocyclin-1 (RC-100) and several analogues against primary HIV-1 isolates, including R5 and R5X4 strains of subtypes A-D, CRF-01_AE, and recombinants. Each analogue differed from retrocyclin-1 by a single amino acid substitution: Gly --> Tyr in RC-106, RC-115, and RC-116, and Arg --> Lys in RC-101. Although the modification in RC-101 was chemically conservative, this peptide was significantly more potent than retrocyclin-1 across the panel of primary isolates. We performed surface plasmon resonance binding studies, using recombinant gp120 and CD4 produced in insect cells. Although RC-100 and RC-101 bound gp120 LAV/IIIB with a K(d) of 30-35 nM, they bound gp120 from CRF-01_AE strains (CM 235 and 93TH975.15) with K(d) values of 200-750 nM. Overall, our findings suggest that clade-related differences in gp120 glycosylation impact the ability of retrocyclin-1 to bind this viral glycoprotein, and modulate the peptides' ability to prevent HIV-1 infection. The performance of RC-101 suggests that additional "engineering" could further enhance the antiviral properties of theta-defensins.

Cell Line↗
PubMed1981

Industrial microbiology.

Industrial microbiology has served humanity since prebiblical times, providing fermented beverages and foods to enhance the quality of life. The antibiotic era featured an explosion in the number of microbial products for medicine, nutrition, industry, and research. Revolutionary developments in molecular genetics are propelling the field into a new growth phase with promise of solutions to major world problems.

Animals↗
PubMed2001

Chryseobacterium (Flavobacterium) meningosepticum outbreak associated with colonization of water taps in a neonatal intensive care unit.

From September 1994 to May 1996, a strain of multi-resistant Chryseobacterium (Flavobacterium) meningosepticum was isolated from eight neonates on a neonatal intensive care unit. The strain was resistant to ampicillin, ceftazidime, imipenem, gentamicin, ciprofloxacin and trimethoprim-sulphamethoxazole, susceptible to piperacillin and amikacin, and had variable susceptibility to rifampicin and vancomycin. Two neonates were infected (one had pneumonia and one septicaemia and meningitis); the remaining six neonates were colonized in the respiratory secretions. Two cases occurred that could not be explained by cross-infection during the outbreak. Environmental screening recovered C. meningosepticum from sink taps. Pulsed-field gel electrophoresis of chromosomal macrorestriction digests of patient and environmental isolates showed them to be representatives of a single strain. The outbreak was controlled after staff were required to use an alcoholic handrub after washing hands, and toiletting of babies was done with sterile water instead of tap-water. Repair and chlorination of the water-tanks and changing the sink-taps resolves the outbreak.

Cross Infection↗
PubMed1993

Phenotypic suppression by incorporation of an alien amino acid.

Azaleucine is a naturally occurring amino acid antibiotic that can be incorporated into proteins by mimicking leucine. In spite of its highly toxic character, the contrast between the hydrophobic side-chain of leucine and the basic side-chain of azaleucine suggested a mechanism for rescuing certain mutants. We constructed a thymidine auxotrophic mutant of Escherichia coli by replacing an arginine residue with leucine in the catalytic centre of thymidylate synthase, and indeed showed that activity could be restored by incorporation of azaleucine. This result extends the current scope of phenotypic suppression to mischarging with amino acid analogues. Microbial strains with a clear-cut requirement for an additional amino acid, as reported here, should be instrumental for widening the genetic code experimentally.

Amino Acids↗
PubMed1995

Engineering liposomes for drug delivery: progress and problems.

Liposomes were first proposed and tested as a drug delivery system 25 years ago. Since then, advances in our understanding of the fate and behaviour of liposomes at the cellular and subcellular level in vivo have allowed the rational design of constructs for use in the treatment and prevention of disease, both in experimental animals and clinically. The involvement of several liposome-based biotechnology companies in the early 1980s, paralleled by great leaps in liposome technology, has culminated in the design and licencing of formulations for the treatment of certain microbial infections and cancers, and the first liposome-based vaccine for use in humans.

Drug Delivery Systems↗
PubMed1997

Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: a molecule for phylogenetic and enzymological investigation.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the key reaction of the Calvin reductive pentose phosphate cycle and as such is responsible for life as we know it. This enzyme has been intensively studied for decades. Evidence that RubisCO phylogenies are incongruent with those derived from other macromolecules has been accumulating and recent discoveries have driven home this point. Here we review findings regarding RubisCO phylogeny and discuss these in the context of the important biochemical and structural features of the enzyme. The implications for the engineering of improved RubisCO enzymes are considered.

Cyanobacteria↗
PubMed2006

Biofuel cells and their development.

This review considers the literature published since 1994 on microbial and enzymatic biofuel cells. Types of biofuel cell are classified according to the nature of the electrode reaction and the nature of the biochemical reactions. The performance of fuel cells is critically reviewed and a variety of possible applications is considered. The current direction of development of biofuel cells is carefully analysed. While considerable chemical development of enzyme electrodes has occurred, relatively little progress has been made towards the engineering development biofuel cells. The limit of performance of biofuel cells is highlighted and suggestions for future research directions are provided.

Bacteria↗
PubMed1997

Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases.

BACKGROUND: Microbial pectin and pectate lyases are virulence factors that degrade the pectic components of the plant cell wall. The homogalacturan backbone of pectin varies in its degree of methylation from the highly methylated and relatively hydrophobic form known as pectin, to the fully demethylated and highly charged form known as pectate. Methylated and demethylated regions of pectin are cleaved by pectin lyase and calcium-dependent pectate lyases, respectively. Protein engineering of lyases specific for particular patterns of methylation, will yield modified pectins of high value to the food and pharmaceutical industries. RESULTS: The crystal structures of pectin lyase A from two strains of Aspergillus niger, N400 and 4M-147, have been determined at pH 6.5 (2.4 A resolution) and pH 8.5 (1.93 A resolution), respectively. The structures were determined by a combination of molecular replacement, multiple isomorphous replacement and intercrystal averaging. Pectin lyase A folds into a parallel beta helix and shares many of the structural features of pectate lyases, despite no more than 17% sequence identity after pairwise structure-based alignment. These shared structural features include amino acid stacks and the asparagine ladder. However, the differences in the substrate-binding clefts of these two enzymes are striking. In pectin lyase A, the cleft is dominated by aromatic residues and is enveloped by negative electrostatic potential. In pectate lyases, this cleft is rich in charged residues and contains an elongated ribbon of positive potential when Ca2+ is bound. The major difference between the two pectin lyase A structures from the two strains is in the conformation of the loop formed by residues 182-187. These observed differences are due to the different pH values of crystallization. CONCLUSIONS: The substrate-binding clefts and catalytic machinery of pectin and pectate lyases have diverged significantly. Specificity is dictated by both the nature of the protein-carbohydrate interaction and long-range electrostatic forces. Three potential catalytic residues have been identified in pectin lyase, two of these are common to pectate lyases. Pectin lyase A does not bind Ca2+ but an arginine residue is found in an equivalent position to the Ca2+ ion in pectate lyase, suggesting a similar role in catalysis. The activity of pectin lyase A is pH -dependent with an optimum activity at pH 5.5. The activity drops above pH 7.0 due to a conformational change at the binding cleft, triggered by the proximity of two buried aspartate residues.

Aspergillus niger↗
PubMed2006

Rational design of macrolides by virtual screening of combinatorial libraries generated through in silico manipulation of polyketide synthases.

Bacterial secondary metabolites display diverse biological activities, thus having potential as pharmacological agents. Although most of these compounds are discovered by random screening, it is possible to predict and re-design their structures based on the information on their biosynthetic pathways. Biosynthesis of macrolides, governed by modular polyketide synthases (PKS), obeys certain rules, which can be simulated in silico. PKS mode of action theoretically allows for a huge number of macrolides to be produced upon combinatorial manipulation. Since engineering of all possible PKS variants is practically unfeasible, we created Biogenerator software, which simulates manipulation of PKS and generates virtual libraries of macrolides. These libraries can be screened by computer-aided prediction of biological activities, as exemplified by analysis of erythromycin and macrolactin libraries. This approach allows rational selection of macrolides with desired biological activities and provides instructions regarding the composition of the PKS gene clusters necessary for microbial production of such molecules.

Combinatorial Chemistry Techniques↗
PubMed2004

Comparison of the 1.85 A structure of CYP154A1 from Streptomyces coelicolor A3(2) with the closely related CYP154C1 and CYPs from antibiotic biosynthetic pathways.

The genus Streptomyces produces two-thirds of microbially derived antibiotics. Polyketides form the largest and most diverse group of these natural products. Antibiotic diversity of polyketides is generated during their biosynthesis by several means, including postpolyketide modification performed by oxidoreductases, a broad group of enzymes including cytochrome P450 monooxygenases (CYPs). CYPs catalyze site-specific oxidation of macrolide antibiotic precursors significantly affecting antibiotic activity. Efficient manipulation of Streptomyces CYPs in generating new antibiotics will require identification and/or engineering of monooxygenases with activities toward a diverse array of chemical substrates. To begin to link structure to function of CYPs involved in secondary metabolic pathways of industrially important species, we determined the X-ray structure of Streptomyces coelicolor A3(2) CYP154A1 at 1.85 A and analyzed it in the context of the closely related CYP154C1 and more distant CYPs from polyketide synthase (EryF) and nonribosomal peptide synthetase (OxyB) biosynthetic pathways. In contrast to CYP154C1, CYP154A1 reveals an active site inaccessible from the molecular surface, and an absence of catalytic activities observed for CYP154C1. Systematic variations in the amino acid patterns and length of the surface HI loop correlate with degree of rotation of the F and G helices relative to the active site in CYP154A1-related CYPs, presumably regulating the degree of active site accessibility and its dimensions. Heme in CYP154A1 is in a 180 degrees flipped orientation compared with most other structurally determined CYPs.

Amino Acid Sequence↗
PubMed1994

Three selectable markers for transformation of Ustilago maydis.

Although Ustilago maydis is readily amenable to molecular genetic experimentation, few antibiotic-resistance markers are available for DNA-mediated transformation. This poses constraints on experiments involving targeted gene disruption and complementation. To address this problem, we constructed vectors using one of three additional genes as dominant selectable markers for transformation. Two genes, sat-1 (encoding streptothricin acetyltransferase) and Sh-ble (encoding a phleomycin-resistance polypeptide), are of bacterial origin and have been engineered for expression in Ustilago sp. The third gene encodes an allele of U. maydis beta-tubulin that confers resistance to the fungicide benomyl.

Acetyltransferases↗
PubMed1999

[Anti-plague vaccination: past and future perspectives].

The impact of the three historic plague pandemics will remain engraved forever in the collective memory. During the first half of the XXth century, the development of vaccines inducing protection against bubonic plague, the first production of antibiotics, insecticides and raticides, could have lead some people to think that eradication was possible. But according to the data of epidemiological surveillance, far from disappearing, plague is remaining or so increasing that it is considered, in some places, as a reemerging disease. Yersinia pestis is highly variable, and a multidrug resistant strain has been isolated in 1995 in the Ambalavo district of Madagascar. This high-level of resistance includes the drugs recommended for plague prophylaxis and therapy, and this observation pointed the fact that Yersinia pestis is able to acquire the plasmid carrying the resistance genes, under natural conditions. Consequently, it is not unreasonable to think that clinically ominous events could occur again. Moreover, currently available vaccines do not induce protection against the pneumonic form of plague, and are reactogenic. Lastly, according to some accurate sources, one cannot turn down the assumption of a genetically engineered strain of Yersinia pestis used as a biological weapon by a terrorist organization. So, the surveillance of plague remains a topical activity, as the development of none reactogenic live and/or inactivated new vaccines, inducing protection against the pneumonic form of the disease.

Drug Resistance, Microbial↗