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PubMed1994

BIOESTIM: software for automatic design of estimators in bioprocess engineering.

This paper describes BIOESTIM, a software package devoted to on-line estimation in bioprocess engineering. BIOESTIM enables bioengineers automatically to design state and parameter estimators from a minimal knowledge of the process kinetics. Such estimators allow development of software sensors capable of coping with the lack of reliable instrumentation suited to real-time monitoring. The estimator building procedure through BIOESTIM starts up from a dynamical material balance model of the bioprocess. This model, supplied by the user, is next completed by other information with no requirement for numerical values: the user has only to specify available measurements, coupled reactions and the known yield coefficients. On the base of this knowledge, BIOESTIM proceeds to symbolic algebraic manipulations on the model in order to study estimation possibilities and check identifiability of yield coefficients. When the design of an estimator is possible, the corresponding equations are automatically generated. Moreover, these estimators are stored in a user-specified file which is automatically interfaced with a specialized simulation software including data treatment and numerical integration packages. Thus, the user can simulate the estimator performances under various operational conditions using available experimental measurements. A typical example dealing with microbial growth and biosynthesis reactions is given in order to illustrate the main functional capabilities of BIOESTIM. BIOESTIM has been designed and written in a modular fashion. The module dealing with estimators design makes use of symbolic computation; it is written in Mathematica and runs on every computer on which this language is available.

Algorithms↗
PubMed1989

Thymidine kinase-negative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate.

Herpes simplex virus infection of mammalian hosts involves lytic replication at a primary site, such as the cornea, translocation by axonal transport to sensory ganglia and replication, and latent infection at a secondary site, ganglionic neurons. The virus-encoded thymidine kinase, which is a target for antiviral drugs such as acyclovir, is not essential for lytic replication yet evidently is required at the secondary site for replication and some phase of latent infection. To determine the specific stage in viral pathogenesis at which this enzyme is required, we constructed virus deletion mutants that were acyclovir resistant and exhibited no detectable thymidine kinase activity. After corneal inoculation of mice, the mutants replicated to high titers in the eye but were severely impaired for acute replication in trigeminal ganglia and failed to reactivate from ganglia upon cocultivation with permissive cells. Nevertheless, latency-associated transcripts were expressed in neuronal nuclei of ganglia from mutant-infected mice and superinfection of the ganglia with a second virus rescued the latent mutant virus. Thus, contrary to a widely accepted hypothesis, the thymidine kinase-negative mutants established latent infections, implying that neither thymidine kinase activity nor ganglionic replication is necessary for establishment of latency. Rather, thymidine kinase appears to be necessary for reactivation from latency. These results suggest that acyclovir-resistant viruses could establish latent infections in clinical settings and have implications for the use of genetically engineered herpesviruses to deliver foreign genes to neurons.

Acyclovir↗
PubMed2003

A novel erythromycin, 6-desmethyl erythromycin D, made by substituting an acyltransferase domain of the erythromycin polyketide synthase.

The acyltransferase (AT) domain in module 4 of the erythromycin polyketide synthase (PKS) was substituted with an AT domain from the rapamycin PKS module 2 in order to alter the substrate specificity from methylmalonyl-CoA to malonyl-CoA. The resulting strain produced 6-desmethyl erythromycin D as the predominant product. This AT domain swap completes the library of malonyl-CoA AT swaps on the erythromycin PKS and reinforces PKS engineering as a robust and generic tool.

Acyltransferases↗
PubMed2004

Bioaugmentation of a sequencing batch biofilm reactor by horizontal gene transfer.

Bioaugmentation by introduction of catabolic genes residing on mobile genetic elements into the microbial community of a soil or wastewater environment might be an alternative to bioaugmentation by addition of bacterial cells with chromosomally encoded catabolic genes. This study investigates the possibility to enhance degradation of the xenobiotic model compound 2,4-dichlorophenoxyacetic acid in a sequencing batch biofilm reactor (SBBR) by using the conjugative plasmid pJP4 carrying genes for 2,4-D degradation. After introduction of a plasmid donor strain to a lab-scale SBBR operated without 2,4-D, the number of plasmid-carrying cells first dropped, and then increased after switching to 2,4-D as the sole carbon source. The donor cells were unable to grow in the applied synthetic wastewater with 2,4-D as the sole carbon source. Transconjugants could be detected both by culture-dependent and culture-independent methods in the 2,4-D degrading biofilm. In contrast to 90% 2,4-D degradation in the bioaugmented reactor within 40 h, a control reactor which had not received the plasmid still contained 60% of the initial 2,4-D concentration after 90 h. This experiment clearly demonstrates the introduction of 2,4-D degradative genes into a microbial biofilm and indicates that horizontal gene transfer is a promising tool for bioaugmentation of reactors treating wastewater.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1989

[Microbial contamination of drinking water by a polyamide feedpipe].

An incident is described with high colony counts in the drinking water from a well. The well was disinfected with sodium hypochlorite for several times, but without permanent success. After that the search for the reason of the high colony counts started. It turned out that the polyamide rising-pipe produced the microbial growth. Besides the colony increase in the water there was a microbial growth upon the surface of the polyamide pipe as well. When a high grade steel pipe was installed instead of the polyamide pipe there was no colony increase in the water any more.

Germany, West↗
PubMed2001

Technical evaluation of the potential for streamlining of equipment validation for fermentation applications.

The purpose of this article is to provide a few concrete examples of the potential to acceptably reduce the scope of validation and qualification testing based on scientific justification for the specific area of microbial fermentation. The key areas explored include: autoclave operational qualification (OQ) testing, autoclave load pattern testing, vessel sterilize-in-place testing, spore strip use and failure investigation, grouping of D-values for media and concentrated nutrients, influence of temperature on D-values, and equipment clean-in-place cleaning agent/recovery studies. Suggestions are offered based on technical data and engineering analysis of the procedures involved. Methodologies are described for how to evaluate the systems being tested relative to processing requirements to determine which testing might be minimized and which testing might warrant expansion. The ultimate risk to quality then must be evaluated by the designated quality control groups within the organization for the specific process and equipment in use.

Evaluation Studies as Topic↗
PubMed2003

Computational approach for prediction of domain organization and substrate specificity of modular polyketide synthases.

Modular polyketide synthases (PKSs) are large multi-enzymatic, multi-domain megasynthases, which are involved in the biosynthesis of a class of pharmaceutically important natural products, namely polyketides. These enzymes harbor a set of repetitive active sites termed modules and the domains present in each module dictate the chemical moiety that would add to a growing polyketide chain. This modular logic of biosynthesis has been exploited with reasonable success to produce several novel compounds by genetic manipulation. However, for harnessing their vast potential of combinatorial biosynthesis, it is essential to develop knowledge based in silico approaches for correlating the sequence and domain organization of PKSs to their polyketide products. In this work, we have carried out extensive sequence analysis of experimentally characterized PKS clusters to develop an automated computational protocol for unambiguous identification of various PKS domains in a polypeptide sequence. A structure based approach has been used to identify the putative active site residues of acyltransferase (AT) domains, which control the specificities for various starter and extender units during polyketide biosynthesis. On the basis of the analysis of the active site residues and molecular modelling of substrates in the active site of representative AT domains, we have identified a crucial residue that is likely to play a major role in discriminating between malonate and methylmalonate during selection of extender groups by this domain. Structural modelling has also explained the experimentally observed chiral preference of AT domain in substrate selection. This computational protocol has been used to predict the domain organization and substrate specificity for PKS clusters from various microbial genomes. The results of our analysis as well as the computational tools for prediction of domain organization and substrate specificity have been organized in the form of a searchable computerized database (PKSDB). PKSDB would serve as a valuable tool for identification of polyketide products biosynthesized by uncharacterized PKS clusters. This database can also provide guidelines for rational design of experiments to engineer novel polyketides.

Amino Acid Sequence↗
PubMed2001

Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation.

Pseudomonas spp. that can colonise the roots of crop plants and produce antifungal metabolites represent a real alternative to the application of chemical fungicides. Presently, much research is aimed at understanding, at the molecular level, the mechanisms that enable Pseudomonas strains to act as efficient biological control agents. This approach is facilitating the development of novel strains with modified traits for enhanced biocontrol efficacy. However, without solving some inherent problems associated with the effective delivery of microbial inoculants to seeds and without knowledge on the biosafety aspects of novel biocontrol agents, the commercial potential of Pseudomonas spp. for plant disease control will not be realised.

Fungi↗
PubMed1996

Genetic manipulations of microorganisms for the degradation of hexachlorocyclohexane.

Hexachlorocyclohexane (HCH) is an organochlorine insecticide which has been banned in technologically advanced countries. However, it is still in use in tropical countries for mosquito control and thus new areas continue to be contaminated. Anaerobic degradation of HCH isomers have been well documented but until recently there have been only a few reports on aerobic microbial degradation of HCH isomers. The isolation of these microbes made it possible to design experiments for the cloning of the catabolic genes responsible for degradation. We review the microbial degradation of HCH isomers coupled with the genetic manipulations of the catabolic genes. The first part discusses the persistence of residues in the environment and microbial degradation while the second part gives an account of the genetic manipulations of catabolic genes involved in the degradation.

Bacteria↗
PubMed2004

The induction of acute ileitis by a single microbial antigen of Toxoplasma gondii.

The role of specific microbial Ags in the induction of experimental inflammatory bowel disease is poorly understood. Oral infection of susceptible C57BL/6 mice with Toxoplasma gondii results in a lethal ileitis within 7-9 days postinfection. An immunodominant Ag of T. gondii (surface Ag 1 (SAG1)) that induces a robust B and T cell-specific response has been identified and a SAG1-deficient parasite (Deltasag1) engineered. We investigated the ability of Deltasag1 parasite to induce a lethal intestinal inflammatory response in susceptible mice. C57BL/6 mice orally infected with Deltasag1 parasites failed to develop ileitis. In vitro, the mutant parasites replicate in both enterocytes and dendritic cells. In vivo, infection with the mutant parasites was associated with a decrease in the chemokine and cytokine production within several compartments of the gut-associated cell population. RAG-deficient (RAG1(-/-)) mice are resistant to the development of the ileitis after T. gondii infection. Adoptive transfer of Ag-specific CD4(+) effector T lymphocytes isolated from C57BL/6-infected mice into RAG(-/-) mice conferred susceptibility to the development of the intestinal disease. In contrast, CD4(+) effector T lymphocytes from mice infected with the mutant Deltasag1 strain failed to transfer the pathology. In addition, resistant mice (BALB/c) that fail to develop ileitis following oral infection with T. gondii were rendered susceptible following intranasal presensitization with the SAG1 protein. This process was associated with a shift toward a Th1 response. These findings demonstrate that a single Ag (SAG1) of T. gondii can elicit a lethal inflammatory process in this experimental model of pathogen-driven ileitis.

Acute Disease↗
PubMed2006

Production of biopharmaceuticals and vaccines in plants via the chloroplast genome.

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation; heat stability; and absence of human pathogens. When therapeutic proteins are orally delivered, plant cells protect antigens in the stomach through bioencapsulation and eliminate the need for expensive purification and sterile injections, in addition to development of both systemic and mucosal immunity. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multi-gene expression in a single transformation event. Hyper-expression of vaccine antigens against cholera, tetanus, anthrax, plague or canine parvovirus (4-31% of total soluble protein, tsp) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato), as well as the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes, facilitate oral delivery. Hyper-expression of several therapeutic proteins, including human serum albumin (11.1% tsp), somatotropin (7% tsp), interferon-gamma (6% tsp), anti-microbial peptide (21.5% tsp), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitate assembly of complex multi-subunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLa cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

Biopharmaceutics↗
PubMed2006

A synthetic congener modeled on a microbicidal domain of thrombin- induced platelet microbicidal protein 1 recapitulates staphylocidal mechanisms of the native molecule.

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a staphylocidal peptide released by activated platelets. This peptide initiates its microbicidal activity by membrane permeabilization, with ensuing inhibition of intracellular macromolecular synthesis. RP-1 is a synthetic congener modeled on the C-terminal microbicidal alpha-helix of tPMP-1. This study compared the staphylocidal mechanisms of RP-1 with those of tPMP-1, focusing on isogenic tPMP-1-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains for the following quantitative evaluations: staphylocidal efficacy; comparative MIC; membrane permeabilization (MP) and depolarization; and DNA, RNA, and protein synthesis. Although the proteins had similar MICs, RP-1 caused significant killing of ISP479C (<50% survival), correlating with extensive MP (>95%) and inhibition of DNA and RNA synthesis (>90%), versus substantially reduced killing of ISP479R (>80% survival), with less MP (55%) and less inhibition of DNA or RNA synthesis (70 to 80%). Interestingly, RP-1-induced protein synthesis inhibition was equivalent in both strains. RP-1 did not depolarize the cell membrane and caused a relatively short postexposure growth inhibition. These data closely parallel those previously reported for tPMP-1 against this strain set and exemplify how synthetic molecules can be engineered to reflect structure-activity relationships of functional domains in native host defense effector molecules.

Anti-Bacterial Agents↗
PubMed1993

Zymomonas mobilis--science and industrial application.

Zymomonas mobilis is undoubtedly one of the most unique bacterium within the microbial world. Known since 1912 under the names Termobacterium mobilis, Pseudomonas linderi, and Zymomonas mobilis, reviews on its uniqueness have been published in 1977 and 1988. The bacterium Zymomonas mobilis not only exhibits an extraordinarily uniqueness in its biochemistry, but also in its growth behavior, energy production, and response to culture conditions, as well as cultivation techniques used. This uniqueness caused great interest in the scientific, biotechnological, and industrial worlds. Its ability to couple and uncouple energy production in favor of product formation, to respond to physical and chemical environment manipulation, as well as its restricted product formation, makes it an ideal microorganism for microbial process development. This review explores the advances made since 1987, together with new developments in the pure scientific and applied commercial areas.

Carbohydrate Metabolism↗
PubMed2004

A sensitivity study for the visualisation of bacterial weathering of concrete and stone with computerised X-ray microtomography.

Geologists and engineers recently have adopted computerised X-ray microtomography (microCT), a radiological imaging technique, for geological and petrophysical applications such as the assessment of sediment characteristics, CT-measurement of compressibility and compaction, multiphase flow studies, measurement of bulk density and moisture content, and of porosity and permeability. This study focuses on another application: the monitoring of biological weathering of natural building stones and concrete. Microbial activity as a determining factor in the deterioration process of building materials has a major economic impact. Because of its non-destructive character, microCT could be the ideal monitoring technique. With this technique, three-dimensional (3D) images of the entire inner structure of the material can be obtained, together with quantitative data. In depth changes of porosity of concrete and stone specimens due to bacterial weathering were assessed in this work. Also, porosity was visualised based on 3D data with homemade software. Scanning electron microscopy (SEM) images provided additional information and supported conclusions drawn from the X-ray microCT data. Resolution improvement will make the study of petrophysical aspects of physical weathering and/or biological deterioration processes of natural building stones and concrete a promising subject for further microCT-application.

Biodegradation, Environmental↗
PubMed1990

Beneficial interactions between micro-organisms and roots.

Microbial activity in the rhizosphere can have positive and negative effects on plants. Some of the beneficial processes act by minimizing the negative effects or by modifying the cropping environment to enhance productivity. Processes that are considered here include the provision of nitrogen, phosphorus and iron to the plant, the biocontrol of diseases and deleterious organisms and the stabilization of soil structures. The use of genetic engineering techniques in studying these processes and generating novel strains which may enhance them is discussed, along with the possible consequences of the release of organisms into the environment.

Journal Article↗
PubMed2000

Polymyxin permeabilization as a tool to investigate cytotoxicity of therapeutic aromatic alkylators in DNA repair-deficient Escherichia coli strains.

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) and its biologically active beta-oxidation product phenylacetic acid mustard (PAM; N,N-bis(2-chloroethyl)-p-aminophenylacetic acid) are bifunctional aromatic alkylators. CLB is in wide clinical use as an anticancer drug and also as an immunosuppressant. The chemical structures indicate that CLB and PAM are mutagenic, teratogenic and carcinogenic, but the mode of action has remained obscure. We have investigated the biological effects of CLB and PAM with DNA repair-deficient Escherichia coli strains. In contrast to MNNG (N-methyl-N'-nitro-N-nitrosoguanine), CLB and PAM were not toxic to E. coli, but permeabilization of the outer membrane of the cells through use of polymyxin B nonapeptide (PMBN) rendered them susceptible to these compounds. The importance of DNA repair, shown by reversal of damage and attenuation of the toxicity of CLB and PAM, was indicated by the susceptibility of cells lacking O(6)-methylguanine-DNA methyltransferase I and II (ada ogt). Similarly, the protective role of base excision repair (BER) was substantiated by demonstration of an even more increased susceptibility to CLB and PAM of cells lacking 3-methyladenine-DNA glycosylase I and II (alkA1 tag-1). Cells deficient in mismatch repair (mutS) appeared to be slightly more sensitive than normal cells to CLB and PAM, although no such sensitivity to MNNG was observed. This implicates the role of mismatches in CLB- and PAM-related cytotoxicity. It is generally believed that bifunctional alkylating agents, like CLB and PAM, exert their cytotoxic action via DNA cross-linking. Our results with O(6)-methyltransferase- and 3-methyladenine-DNA glycosylase-deficient cells indicate that removal of the adducts prior to the formation of cross-links is an important mechanism maintaining cell viability. We conclude that PMBN permeabilization provides a valuable tool to investigate genetically engineered E. coli cells, whose outer membrane is not naturally permeable to mutagens or other interesting compounds.

Alkylating Agents↗
PubMed1985

NIH Conference. Herpes simplex virus infection: biology, treatment, and prevention.

Herpes simplex viruses cause common mucocutaneous infections, but many aspects of their epidemiology and transmission are incompletely defined. Although the incidence of oral herpes remains relatively unchanged, the incidence of genital herpes is increasing significantly. Definitive diagnosis of herpes remains dependent on virus isolation, but techniques involving direct examination of clinical specimens are increasingly sensitive and may simplify and speed diagnosis. With the advent of acyclovir, effective therapy and suppression of infection are feasible for immunodeficient and selected normal patients. Unanswered questions remain regarding the long-term safety of acyclovir and the potential for emergence of clinically significant drug resistance. No effective vaccines are yet available for herpes virus infections. Promising strategies for vaccine development include preparation of immunogenic proteins, engineering of specially attenuated live virus strains, and incorporation of selected herpes genes into live vaccinia virus vectors.

Acyclovir↗
PubMed1975

Microbial contamination of mist therapy units on six pediatric wards.

A study was conducted in six pediatric wards to investigated factors that influence microbial contamination of mist therapy units. Most samples from nebulizer reservoirs were positive for gram-negative bacilli which multiplied rapidly in distilled water and reached levels as high as 3.0 X 10(8) viable microorganisms per ml. High prevalence rates of Pseudomonas aeruginosa and coliform bacteria as well as high levels of gram-negative bacilli were found to be associated with infrequent and inadequate cleaning and disinfection of the nebulizing equipment. In one of the wards exhibiting high rates, a move to new quarters permitted the implementation of improved cleaning and maintenance procedures for mist therapy units. The result was a significant reduction of microbial contamination. This study reemphasizes the role of microbiological surveillance in assuring proper care of nebulizing equipment in the hospital.

Aerosols↗