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Results for “strain engineering”

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PubMed1993

Survival differences among freeze-dried genetically engineered and wild-type bacteria.

Because the death mechanisms of freeze-dried and air-dried bacteria are thought to be similar, freeze-drying was used to investigate the survival differences between potentially airborne genetically engineered microorganisms and their wild types. To this end, engineered strains of Escherichia coli and Pseudomonas syringae were freeze-dried and exposed to air, visible light, or both. The death rates of all engineered strains were significantly higher than those of their parental strains. Light and air exposure were found to increase the death rates of all strains. Application of death rate models to freeze-dried engineered bacteria to be released into the environment is discussed.

Air↗

PubMed2002

Distribution of sympathetic preganglionic neurons innervating the kidney in the rat: PRV transneuronal tracing and serial reconstruction.

The organization of spinal motor circuitry to the kidney is not well-characterized and changes in renal innervation have been associated with disease states such as hypertension found in the spontaneously hypertensive rat or renal hypertension. Here, we describe the segmental and intra-segmental organization of the spinal motor circuitry that was resolved after neurotropic viral injection into the kidney and retrograde transneuronal transport to the spinal cord. In the first experiment, the serial reconstruction of infected neurons in the thoracolumbar spinal cord from T8-L1 was performed following injection of pseudorabies virus (PRV, Bartha strain) into either the cranial pole, the caudal pole or both the cranial and caudal poles of the left kidney in male rats. In the second experiment, rats received injections of two different PRV strains that were genetically engineered to express unique reporter molecules; one of the engineered strains was injected into the cranial pole and the other was injected into the caudal pole. Either 3- or 4-day post-infection, the animals were anesthetized and sacrificed by transcardial perfusion. PRV-infected neurons were located by immunocytochemistry against either PRV itself (experiment 1) or the unique marker proteins (experiment 2). After injection of both poles of the kidney, the majority of the infected neurons were found in the ipsilateral intermediolateral cell column (IML) from T10 to T12 with the mode at T11. Infected neurons were found in discrete neuron clusters in the intermediolateral cell column along the longitudinal axis in a repeating pattern of high and low density that has been called "beading". Three observations indicated a topographic distribution of renal sympathetic preganglionic neurons (SPN). First, after injection into either the cranial or caudal poles of the kidney, the mode of infected cells was located in segments T11 and T12, respectively. The one spinal segment shift in the mode suggested a topographic distribution. Second, in spinal segments T8-L1, comparison of the distributions of the neurons innervating each pole of the left kidney revealed an overlap in the distribution, except in the T11 segment. In the T11 segment, the neurons projecting to each pole tended to segregate into separate populations. Third, in rats that received injections of two PRV strains that were genetically engineered to express unique markers into opposite poles of the kidney, a segregation of neurons projecting to the cranial and caudal poles of the kidney was noted again in the T11 spinal segment and the segregation at adjacent spinal levels was obvious. The analysis of the distribution of infected neurons within each spinal cord segment (intra-segmental distribution) revealed three different patterns along the cranial-caudal dimension. In segments T8-T10, >60% of the infected neurons were located in the caudal half of the spinal segment. In segments T12-L1, >60% of the infected neurons were located in the cranial half of the spinal segment. In segment T11, the neurons were more evenly distributed throughout the segment. These intra-segmental distribution patterns were found after both 3- or 4-day survival periods post-infection and were found in most animals. The distribution of clusters of neurons revealed a similar intra-segmental pattern. Thus, as was described previously for the sympathetic postganglionic neurons that innervate the kidney, the present work indicates a topographic organization in the second-order neurons in the renal sympathetic efferent pathway. The physiological significance of this anatomical organization remains to be determined.

Animals↗

PubMedDate not supplied

Engineering the E. coli UDP-glucose synthesis pathway for oligosaccharide synthesis.

A metabolic engineering strategy was successfully applied to engineer the UDP-glucose synthesis pathway in E. coli. Two key enzymes of the pathway, phosphoglucomutase and UDP-glucose pyrophosphorylase, were overexpressed to increase the carbon flux toward UDP-glucose synthesis. When additional enzymes (a UDP-galactose epimerase and a galactosyltransferease) were introduced to the engineered strain, the increased flux to UDP-glucose synthesis led to an enhanced UDP-galactose derived disaccharide synthesis. Specifically, close to 20 mM UDP-galactose derived disaccharides were synthesized in the engineered strain, whereas in the control strain only 2.5 mM products were obtained, indicating that the metabolic engineering strategy was successful in channeling carbon flux (8-fold more) into the UDP-glucose synthesis pathway. UDP-sugar synthesis and oligosaccharide synthesis were shown to increase according to the enzyme expression levels when inducer concentration was between 0 and 0.5 mM. However, this dependence on the enzyme expression stopped when expression level was further increased (IPTG concentration was increased from 0.5 to 1 mM), indicating that other factors emerged as bottlenecks of the synthesis. Several likely bottlenecks and possible engineering strategies to further improve the synthesis are discussed.

Carbon↗

PubMed2006

[Expression of AIV subtype H5HA, H7HA and H9HA hemagglutinin gene in Pichia pastoris].

The expression of the hemagglutinins of Avian influenza virus H5 H7and H9 subtypes was studied in this article by Pichia pastoris, one of the eukaryotis expression systems. Three reconstructed expression plasmids and engineering strains, named pPIC9K-H5HA, pPIC9K-H7HA, pPIC9K-H9HA and GS115/pPIC9K-H5HA, GS115/pPIC9K-H7HA, GS115/pPIC9K-H9HA repectively, were obtained. The reconstructed yeast engineering strains were identified by MD and MM plate selecting and PCR. The induced interests proteins were examined by SDS-PAGE and Western-bloting,the results showed that the interest genes were expressed exactly. And this will be helpful in the future study of antigen detection and antibody detection kit, as well in the subunit vaccines developing.

Animals↗

PubMed2006

Expression of Vitreoscilla hemoglobin in Gordonia amarae enhances biosurfactant production.

The gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb) was electroporated into Gordonia amarae, where it was stably maintained, and expressed at about 4 nmol VHb g(-1) of cells. The maximum cell mass (OD(600)) of vgb-bearing G. amarae was greater than that of untransformed G. amarae for a variety of media and aeration conditions (2.8-fold under normal aeration and 3.4-fold under limited aeration in rich medium, and 3.5-fold under normal aeration and 3.2-fold under limited aeration in mineral salts medium). The maximum level of trehalose lipid from cultures grown in rich medium plus hexadecane was also increased for the recombinant strain, by 4.0-fold in broth and 1.8-fold in cells under normal aeration and 2.1-fold in broth and 1.4-fold in cells under limited aeration. Maximum overall biosurfactant production was also increased in the engineered strain, by 1.4-fold and 2.4-fold for limited and normal aeration, respectively. The engineered strain may be an improved source for producing purified biosurfactant or an aid to microorganisms bioremediating sparingly soluble contaminants in situ.

Biodegradation, Environmental↗

PubMed1998

The influence of strain rate on the passive and stimulated engineering stress--large strain behavior of the rabbit tibialis anterior muscle.

The passive and stimulated engineering stress-large strain mechanical properties of skeletal muscle were measured at the midbelly of the rabbit tibialis anterior. The purpose of these experiments was to provide previously unavailable constitutive information based on the true geometry of the muscle and to determine the effect of strain rate on these responses. An apparatus including an ultrasound imager, high-speed digital imager, and a servohydraulic linear actuator was used to apply constant velocity deformations to the tibialis anterior of an anesthetized neurovascularly intact rabbit. The average isometric tetanic stress prior to elongation was 0.44 +/- 0.15 MPa. During elongation the average stimulated modulus was 0.97 +/- 0.34 MPa and was insensitive to rate of loading. The passive stress-strain responses showed a nonlinear stiffening response typical of biologic soft tissue. Both the passive and stimulated stress-strain responses were sensitive to strain rate over the range of strain rates (1 to 25 s-1). Smaller changes in average strain rate (1 to 10, and 10 to 25 s-1) did not produce statistically significant changes in these responses, particularly in the stimulated responses, which were less sensitive to average strain rate than the passive responses. This relative insensitivity to strain rate suggests that pseudoelastic functions generated from an appropriate strain rate test may be suitable for the characterization of the responses of muscle over a narrow range of strain rates, particularly in stimulated muscle.

Animals↗

PubMed2000

Capacity of mercury volatilization by mer (from Escherichia coli) and glutathione S-transferase (from Schistosoma mansoni) genes cloned in Escherichia coli.

A study was carried out to evaluate the capacity for mercury volatilization by genetically engineered strains that express the mer and glutathione S-transferase genes from Escherichia coli and Schistosoma mansoni, respectively. This method enabled strains containing simultaneously mer and glutathione S-transferase genes to grow in high concentrations of mercuric chloride (30 microg/ml) and to volatilize part of the mercury (248 microg/g cell dry wt.) present in the culture medium, while strains bearing only a single gene, did not have the same behavior. Up to 70% of the total mercury of bacterial volatilization occurred in the first 4 h. Although the findings were preliminary, the genetically engineered strain containing simultaneously the mer and glutathione S-transferase genes show a great potential for bioremediation. It may be used in a closed system to remove by volatilization, and recover mercury (Hg0) from contaminated effluents, such as industrial effluent, for instance.

Animals↗

PubMed1997

Detection and Enumeration of a Tagged Pseudomonas fluorescens Strain by Using Soil with Markers Associated with an Engineered Catabolic Pathway.

Previously we described a novel gene tagging method, using the moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955, to identify microorganisms destined for release into the environment. Here, we used the engineered strain Pseudomonas fluorescens PF5MT12 carrying the moc region integrated into the bacterial chromosome to demonstrate the usefulness of the markers for detection and direct selection of marked organisms present in soil samples. Using this system, we routinely detected population levels as low as 10(sup2) CFU per g of soil sampled. In addition to direct selection, we developed an immunologically based assay using MOP cyclase, a unique enzyme associated with moc, as the epitope for detecting the tagged organism. The colony immunoblot assay proved to be highly specific and without any false-positive signals when used to identify organisms cultured from soil on nonselective medium. The numbers of colonies that were immunoreactive with the anti-MOP cyclase antibody were essentially equal to those that grew out on selection plates. This indicates that MOP cyclase can be used as a marker and that we can use nonselective medium to retrieve the marked genetically engineered microorganisms and then identify them by using colony immunoblot assays. These direct selection and colony immunoblot methods provide a sensitive and accurate strategy for identifying and enumerating marked organisms recovered from soil samples. We also developed a rapid assay for MOP cyclase that does not require cell permeabilization with toluene. This assay can be used to verify tagged organisms isolated by other methods or to screen large numbers of colonies for the tag following nonselective isolation.

Journal Article↗

PubMed1999

Hyperproduction of tryptophan by Corynebacterium glutamicum with the modified pentose phosphate pathway.

A classically derived tryptophan-producing Corynebacterium glutamicum strain was recently significantly improved both by plasmid-mediated amplification of the genes for the rate-limiting enzymes in the terminal pathways and by construction of a plasmid stabilization system so that it produced more tryptophan. This engineered strain, KY9218 carrying pKW9901, produced 50 g of tryptophan per liter from sucrose after 80 h in fed-batch cultivation without antibiotic pressure. Analysis of carbon balances showed that at the late stage of the fermentation, tryptophan yield decreased with a concomitant increase in CO2 yield, suggesting a transition in the distribution of carbon flow from aromatic biosynthesis toward the tricarboxylic acid cycle via glycolysis. To circumvent this transition by increasing the supply of erythrose 4-phosphate, a direct precursor of aromatic biosynthesis, the transketolase gene of C. glutamicum was coamplified in the engineered strain by using low- and high-copy-number plasmids which were compatible with the resident plasmid pKW9901. The presence of the gene in low copy numbers contributed to improvement of tryptophan yield, especially at the late stage, and led to accumulation of more tryptophan (57 g/liter) than did its absence, while high-copy-number amplification of the gene resulted in a tryptophan production level even lower than that resulting from the absence of the gene due to reduced growth and sugar consumption. In order to assemble all the cloned genes onto a low-copy-number plasmid, the high-copy-number origin of pKW9901 was replaced with the low-copy-number one, generating low-copy-number plasmid pSW9911, and the transketolase gene was inserted to yield pIK9960. The pSW9911-carrying producer showed almost the same fermentation profiles as the pKW9901 carrier in fed-batch cultivation without antibiotic pressure. Under the same culture conditions, however, the pIK9960 carrier achieved a final tryptophan titer of 58 g/liter, which represented a 15% enhancement over the titers achieved by the pKW9901 and pSW9911 carriers.

Biomass↗

PubMed1993

Protection of freeze-dried Escherichia coli by trehalose upon exposure to environmental conditions.

Freeze-dried cultures of wild-type and genetically engineered strains of Escherichia coli lost their colony-forming ability upon exposure to air, visible light, and certain relative humidity levels. Both strains could be maximally protected from these lethal effects with 100 mM trehalose, a concentration calculated to just saturate the interphospholipid spaces in the cell membrane, thus preserving the liquid-crystalline structure. The trehalose protection was observed for at least 96 h. Trehalose increased viability as much as 2000-4000% over nontreated populations. In all cases, exposure to environmental conditions was more damaging to the genetically engineered strain.

Cryoprotective Agents↗

PubMed1999

Gelatin-sealed polyester resists Staphylococcus epidermidis biofilm infection.

BACKGROUND: The purpose of this study was to show that gelatin-impregnated polyester grafts inhibit Staphylococcus epidermidis biofilm infection in a canine model of aortic graft interposition. A clinically native species and two engineered strains, which differed in slime and adhesin antigen components, were compared to determine differential gelatin and slime interactions. METHODS: In vitro bacterial graft colonization was validated by immersion of graft segments in inoculating solutions (10(6) colony forming units/ml) of a clinically native species RP62A and two genetically engineered S. epidermidis species, M187sn3 (SN3: slime and adhesin negative) or M187sp11 (SP11: slime and adhesin positive), for 18 h at 23 degrees C. The grafts were washed, sonicated, and cultured to assess in vitro bacterial graft adherence. Grafts similarly inoculated were placed as aortic interposition grafts in dogs. Three sterile grafts were implanted as controls. Grafts were excised after 6 weeks and cultured for bacterial growth as in the in vitro study. Infection was defined by a positive culture in the excised grafts. Data were analyzed with nonparametric statistical methods. RESULTS: In vitro bacterial graft adherence in colony forming units per milliliter was similar at 18 h postsonication for RP62A (8 x 10(4) +/- 1 x 10(4)), SN3 (7 x 10(4) +/- 2 x 10(4)), and SP11 (6 x 10(4) +/- 2 x 10(4)) (P = NS). Only one of five grafts inoculated with RP62A was culture positive after 6 weeks. No grafts inoculated with the engineered strains SN3 or SP11 were culture positive after explanation. CONCLUSION: In vitro bacterial inoculation of gelatin-impregnated polyester was similar among the species and not dependent upon the presence of slime and adhesin components. Gelatin-impregnated polyester grafts demonstrated in vivo resistance to coagulase-negative staphylococcal biofilm infection.

Animals↗

PubMed2026

A fluorescent reporter system for tracking Lactobacillus casei T1 in the murine gastrointestinal tract.

BACKGROUND: Fluorescent reporter systems are useful for studying probiotic colonization and host-microbe interactions. However, their use in lactic acid bacteria is still limited by relatively weak fluorescence signals, insufficient expression stability, and limited resolution during in vivo imaging. In particular, efficient strain-specific tracking systems remain scarce. METHODS: Here, we developed a red fluorescent reporter system for Lactobacillus casei T1 (L.c T1). Lactate dehydrogenase (LDH) promoters identified from the L.c T1 genome were compared with the constitutive P32 promoter to drive expression of the red fluorescent proteins mCherry and mKate. The different promoter-reporter combinations were evaluated in both Escherichia coli DH5α and L.c T1. Fluorescence expression was further examined under different environmental pH conditions. The optimized reporter strains were then evaluated by whole-body fluorescence imaging in living mice and ex vivo imaging of gastrointestinal tissues following oral administration. RESULTS: Among the constructs tested, P32-mKate produced the strongest and most stable fluorescence signal in L.c T1. Fluorescence intensity was influenced by environmental pH, with higher signals observed under mildly alkaline conditions. Whole-body fluorescence imaging showed that the engineered strain could be detected in living mice following oral administration. Ex vivo imaging of gastrointestinal tissues provided clearer localization of fluorescence, with signals mainly detected in the stomach and upper small intestine. CONCLUSION: We established a stable and efficient red fluorescent reporter system for L.c T1. The P32-mKate system enables detection of the engineered strain both in vitro and in vivo and provides a practical approach for tracking probiotic distribution and studying host-microbe interactions in preclinical animal models.

Lactobacillus casei T1↗

PubMed2006

Altered nucleotide sugar metabolism in Streptococcus thermophilus interferes with nitrogen metabolism.

Exopolysaccharide (EPS)-producing Streptococcus thermophilus strains have attracted interest recently, since the EPSs act as natural viscosifiers and texture enhancers of fermented foods. We have previously reported that the low level of EPS production by S. thermophilus LY03 could be improved by altering the activities of enzymes in the central carbon metabolism involved in the nucleotide sugar metabolism. In this study, we observed a reduced growth in milk for the strains with increased UDP-glucose pyrophosphorylase (GalU) activity together with either enhanced phosphoglucomutase activity, and/or enhanced activity of the Leloir enzymes. Rapid growth of these mutants in milk could be restored by the addition of four specific amino acids, i.e. Glu, His, Met, and Val. This amino acid requirement was confirmed in a defined medium. Furthermore, the 31P NMR spectra showed higher levels of the GalU reactants pyrophosphate (PPi) and UDP-glucose in the engineered strain, TMB 6013, compared to the parent strain, LY03. These products plus Glu and the GalU reactant UTP are known to be involved in the nitrogen regulatory system in many bacteria. Thus, these results suggest that the reaction catalyzed by GalU is connected to the nitrogen demand of these engineered strains.

Animals↗

PubMed2004

Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and alpha-amylase.

Direct and efficient production of ethanol by fermentation from raw corn starch was achieved by using the yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase by using the C-terminal-half region of alpha-agglutinin and the flocculation functional domain of Flo1p as the respective anchor proteins. In 72-h fermentation, this strain produced 61.8 g of ethanol/liter, with 86.5% of theoretical yield from raw corn starch.

Culture Media↗

PubMed2004

Compound 800, a natural product isolated from genetically engineered Pseudomonas: proposed structure, reactivity, and putative relation to heme d1.

Genetically engineered strains of Escherichia coli and Pseudomonas aeruginosa were prepared harboring the gene cluster nirFDLGH from Pseudomonas stutzeri substrain ZoBell on a high copy plasmid. These genes have been previously implicated as being essential for the biosynthesis of heme d(1), the prosthetic group of dissimilatory nitrite reductases in anaerobic, denitryfying bacteria. Tetrapyrroles detectable at steady-state levels were identified from both organisms, and cell-free extracts from each were also used to transform uroporphyrinogen in vitro. E. coli does not naturally produce d(1), and the engineered strain failed to produce d(1) or any tetrapyrrole foreign to E. coli. Therefore, while nirFDLGHmay be necessary for d(1) biosynthesis, it is not sufficient. In the denitrifier P. aeruginosa, the results were more positive. The presence of the plasmid led to increased levels of d(1). In addition, a previously unidentified tetrapyrrole was detected. This compound was characterized by visible absorption spectroscopy, infrared spectroscopy, X-ray photoelectron spectroscopy, mass spectrometry, and NMR, and a tentative structure was proposed for this compound. The tetrapyrrole has structural features similar to sirohydrochlorin (as precorrin-2 or sirotetrahydrochlorin, a known intermediate of d(1)) and d(1) itself. The most unusual substituents are epoxide and sulfoxide moieties. When this tetrapyrrole was treated with strong mineral acid and heat, it was converted into natural d(1).

Enzymes↗

PubMed1998

A Salmonella typhimurium strain genetically engineered to secrete effectively a bioactive human interleukin (hIL)-6 via the Escherichia coli hemolysin secretion apparatus.

Human interleukin-6 (hIL-6) cDNA was genetically fused with the Escherichia coli hemolysin secretorial signal (hlyA[S]) sequence in a plasmid vector. Recombinant E. coli XL-1 Blue and attenuated Salmonella typhimurium secreted a 30 kDa hIL-6-HlyA(S) fusion protein, with an additional form of higher apparent molecular mass produced by S. typhimurium. In S. typhimurium cultures hIL-6-HlyA(S) concentrations entered a plateau at 500 to 600 ng ml(-1) culture supernatant. In contrast to E. coli XL-1 Blue, in S. typhimurium culture supernatants hIL-6-HlyA(S) was accumulated faster reaching three-fold higher maximal concentrations. The cell proliferating activity of hIL-6-HlyA(S) fusion protein(s) was equivalent to that of mature recombinant hIL-6. Furthermore. hIL-6-secreting S. typhimurium were less invasive than the attenuated control strain. Therefore, the bulky hemolysin secretorial peptide at the C-terminus of the fusion protein does not markably affect hIL-6 activity, suggesting that the hemolysin secretion apparatus provides an excellent system to study immunomodulatory effects of in situ synthesized IL-6 in Salmonella vaccine strains.

Animals↗

PubMed2001

Collinone, a new recombinant angular polyketide antibiotic made by an engineered Streptomyces strain.

Large chromosomal DNA fragments containing different parts of the putative rubromycin polyketide synthase gene cluster were cloned and functionally expressed in S. coelicolor CH999. Expression of these clones yielded 5 approximately 10 metabolites that were not detected in S. collinus culture extracts. This paper focusses on one of the new metabolites, termed collinone, that was isolated in large quantities and purified for spectroscopic structure determination and biological screening assays. Collinone is a heavily oxidized angular hexacyclic compound containing an unusual 1,4,5,8(2H,3H)-anthracenetetrone moiety previously only reported to be present in antibiotics SF2446A1, A2, A3, B1 and B2 isolated from Streptomyces sp. SF2446. Structure analysis of collinone indicates a tridecaketide with a 26 carbon backbone. The basic benz[a]naphthacene ring system of collinone is angular, similar to the aglycones of the well-known angucycline and angucyclinone antibiotics. While collinone showed antibacterial activity against vancomycin-resistant enterococci, no antifungal or significant antiviral activities were detected. Collinone could be a good starting point to obtain new bioactive angucyclin(on)e-like compounds by further genetic engineering of its pathway.

Anthracenes↗

PubMed1994

Safety and immunogenicity of recombinants based on the genetically-engineered vaccinia strain, NYVAC.

NYVAC-based recombinants expressing pertinent immunogens from equine influenza virus (EIV), pseudorabies virus (PRV), Japanese encephalitis virus (JEV) and human immunodeficiency virus (HIV) were used to evaluate the safety and immunogenicity of this vector. Administration of either NYVAC recombinants or parenteral virus to mice, horses and swine was well tolerated with no notable local or systemic reactivities. Further, despite a highly attenuated phenotype, NYVAC was found to function effectively as an immunization vehicle capable of eliciting both humoral and cell-mediated immune responses.

AIDS Vaccines↗