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PubMed2006

Residual virulence and immunogenicity of CGV26 and CGV2631 B. melitensis Rev. 1 deletion mutant strains in sheep after subcutaneous or conjunctival vaccination.

The CGV26 and CGV2631 strains are novel engineered Brucella melitensis Rev.1 mutant strains deleted for the bp26 gene or for both bp26 and omp31 genes, respectively, coding for proteins of diagnostic significance. The residual virulence and immunogenicity of both mutants were compared to the parental Rev.1 strain in sheep after subcutaneous or conjunctival vaccination. The deletion of the bp26 gene or both bp26 and omp31 genes had no significant effect on the intracellular survival of the Rev.1 strain in ovine macrophage cultures. The kinetics of infection induced by both mutants in sheep was similar to the Rev.1 strain, and inoculation by the subcutaneous route produced wider and more generalized infections than the conjunctival route. All strains were cleared from lymph nodes and organs within 3 months after inoculation. The CGV26 and CGV2631 mutants induced both specific systemic antibody response and lymphoproliferation in sheep. The kinetics of the responses induced by the mutants was quite similar to that of the parental Rev.1 strain, except for the intensity of the lymphoproliferative response, which was attenuated for the CGV2631 mutant. In conclusion, the residual virulence of both CGV26 and CGV2631 mutants in sheep was similar to that of the parental Rev.1 vaccine strain. These mutants induced also significant specific antibody and cell-mediated immunity in sheep and are suitable to be evaluated as potential vaccine candidates against B. melitensis and B. ovis infections in sheep.

Animals↗
PubMed2006

Genetically engineered poly-gamma-glutamate producer from Bacillus subtilis ISW1214.

The pgsBCA-gene disruptant from Bacillus subtilis ISW1214, i.e., MA41, does not produce poly-gamma-glutamate (PGA). We newly constructed an MA41 recombinant bearing the plasmid-borne PGA synthetic system, in which PGA production was strictly controlled by the use of xylose. Unlike the parent strain, ISW1214, the genetically engineered strain produced abundant PGA in both L-glutamate-rich and D-glutamate-rich media.

Bacillus subtilis↗
PubMed2007

Failure properties of cervical spinal ligaments under fast strain rate deformations.

STUDY DESIGN: The failure responses of the anterior longitudinal ligament, posterior longitudinal ligament, and ligamentum flavum were examined in vitro under large strain-rate mechanical loading. OBJECTIVE: To quantify the failure properties for 3 cervical spinal ligaments at strain rates associated with traumatic events. SUMMARY OF BACKGROUND DATA: There exists little experimentation literature for fast-rate loading of the cervical spine ligaments. The small amount of available information is framed only in extensive experimental coordinates, and not in the context of strains. METHODS: Bone-ligament-bone complexes were strained at fast rates, in an incrementally increasing loading protocol using a servohydraulic mechanical test frame. Failure loads and displacements were converted to engineering and true stress and strain values, and compared for the different ligaments (anterior longitudinal ligament, posterior longitudinal ligament, and ligamentum flavum), spinal levels (C3-C4, C5-C6, and C7-T1), and for male versus female specimens. RESULTS: There were no significant differences in force or true stress for gender or spinal level. There was a significant difference in force and true stress for ligament type. A difference was found between the posterior longitudinal ligament and ligamentum flavum for failure force, and between the ligamentum flavum and both the anterior and posterior longitudinal ligaments for failure true stress. No significant differences were found in true strain for ligament, gender, or spinal level. The mean ligament failure true strain was 0.81. Failure true strains were approximately 57% of the failure engineering strains. CONCLUSIONS: Once the injury mechanisms of the cervical spine are fully understood, computational models can be employed to understand the potentially traumatic effects of clinical procedures, and mitigate injury in impact, falls, and other high-rate scenarios. The soft tissue failure properties in this study can be used to develop failure tolerances in fast-rate loading scenarios. Failure properties of the anterior and posterior longitudinal ligaments were similar, and the same properties can be used to model both ligaments.

Aged↗
PubMed2003

Bacteria engineered for fuel ethanol production: current status.

The lack of industrially suitable microorganisms for converting biomass into fuel ethanol has traditionally been cited as a major technical roadblock to developing a bioethanol industry. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. Lignocellulosic biomass contains complex carbohydrates that necessitate utilizing microorganisms capable of fermenting sugars not fermentable by brewers' yeast. The most significant of these is xylose. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars, and work has concentrated on engineering these strains to selectively produce ethanol. Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work on this organism has concentrated on introducing pathways for the fermentation of arabinose and xylose. The history of constructing these strains and current progress in refining them are detailed in this review.

Bacteria↗
PubMed2026

Bacillus thuringiensis pathogenicity islands encode regulatory circuits controlling insecticidal Cry toxin expression during vegetative growth.

Bacillus thuringiensis (Bt) produces insecticidal toxins, including Cry and Vip3 proteins, that are widely used for biological pest control. Cry proteins are classically expressed during sporulation under the control of sporulation-specific σ factors, whereas Vip3 is produced during vegetative growth, suggesting distinct regulatory pathways. Notably, many cry and vip3A genes are clustered within pathogenicity islands (PAIs), such as BtPAI-1. However, whether these PAIs also encode regulatory mechanisms coordinating toxin expression remains unclear. Here, we identify VipR, a BtPAI-1-encoded transcriptional regulator, as an activator of insecticidal gene expression during the vegetative phase in Bt strains HD-1 and CT-43. In these strains, VipR promotes the transcription of BtPAI-1 associated insecticidal genes, including vip3A and selected cry genes, resulting in premature Cry protein accumulation and increased insecticidal activity. In addition, VipR contributes to the vegetative-phase expression of the non-BtPAI-1 cry9Aa genes in strain BGSC 4AE1. Phylogenetic analysis revealed that vipR is widely distributed in one-third of Bt strains, and is strongly associated with PAIs. Futhermore, heterologous expression of vipR in BGSC 4J5 and HD-73 was sufficient to activate vegetative-phase transcription of some cry independently of sporulation-specific σ factor cascade. These results support a role for VipR in coordinating vegetative-phase expression of insecticidal genes in the Bt strains examined and suggest that BtPAI-1 can encode both insecticidal determinants and regulatory functions that influence their expression. These findings provide new insights into the regulatory architecture of Bt pathogenicity islands and may facilitate the engineering of strains with enhanced insecticidal activity.

Bacillus thuringiensis↗
PubMed2026

Transcriptomic shift in ethanol and amino acid metabolic genes regulated by Med15 during alcoholic fermentation.

Organisms that thrive in extreme environments provide natural experiments in evolution, revealing the genetic regulators that orchestrate complex phenotypic change. Wine yeast are specialized strains that are adapted to survive in the wine making environment while producing high concentrations of ethanol. In addition to large genomic changes that differentiate wine yeast from yeast used in other industries, single nucleotide and polyglutamine tract polymorphisms in the transcriptional regulator Med15 are associated with the fermentation efficiency and stress response phenotypes of wine yeast. In this study we investigated the transcriptional differences during wine fermentation in transgenic lab strain yeast having integrated wine yeast MED15 alleles. Compared to the unmodified lab strain (LAB or MED15 LAB ), the same strain in which the MED15 locus was replaced with a MED15 allele from yeast isolated from palm wine, the fermented sap of palm (oil, date, coconut) trees, (WY23, or MED15 WY23 ) exhibited enhanced expression of glycolytic, fermentation, and amino acid biosynthesis genes. Our experimental data confirms the importance of arginine biosynthetic genes during the fermentation process and suggests that the improvement in fermentation efficiency in strains with MED15 alleles from some wine yeast strains may be related to the role of Med15 in expression of the genes of the arginine biosynthetic pathway. The global benefit conferred by polymorphisms in a single transcriptional regulator, makes Med15 a prime target for engineering of strains devoted to various types of alcohol production.

Journal Article↗
PubMed2000

Use of pertussis toxin encoded by ptx genes from Bordetella bronchiseptica to model the effects of antigenic drift of pertussis toxin on antibody neutralization.

Recently, concern has been voiced about the potential effect that antigenic divergence of circulating strains of Bordetella pertussis might have on the efficacy of pertussis vaccines. In order to model antigenic drift of pertussis toxin, a critical component of many pertussis vaccines, and to examine the effects of such drift on antibody neutralization, we engineered a strain of B. pertussis to produce a variant pertussis toxin molecule that contains many of the amino acid changes found in the toxin encoded by Bordetella bronchiseptica ptx genes. This altered form of the toxin, which is efficiently secreted by B. pertussis and which displays significant biological activity, was found to be neutralized by antibodies induced by vaccination as readily as toxin produced by wild-type B. pertussis. These findings suggest that significant amino acid changes in the pertussis toxin sequence can occur without drastically altering the ability of antibodies to recognize and neutralize the toxin molecule.

Amino Acid Sequence↗
PubMed2004

Precursor-directed biosynthesis of epothilone in Escherichia coli.

Engineered biosynthetic pathways provide a powerful method for generating complex molecules. Precursor-directed biosynthesis, which combines chemical synthesis and enzymatic transformations, allows non-native starting materials to be incorporated into biosynthetic pathways. Using this approach, we achieved the production of the anticancer agent epothilone C in Escherichia coli. An E. coli strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosynthetic enzymes was obtained by chemical synthesis. Under high-density cell culture conditions, the E. coli strain processed exogenously fed synthetic substrate into epothilone C at levels comparable to the native host (1 mg/L) and at higher levels than other heterologous hosts. Importantly, this precursor-directed approach will allow chemical modifications to be introduced into the polyketide backbone and may ultimately provide access to epothilone analogues with improved pharmacological properties in quantities sufficient for clinical development.

Antibiotics, Antineoplastic↗
PubMed1990

Agrobacterium in plant disease, biological disease control and plant genetic engineering.

Plant pathogenic strains of Agrobacterium cause crown gall and hairy-root diseases. The abnormal cell proliferation of diseased tissues results from elevated plant hormonal levels within them. These levels are consequent upon the transfer of DNA portions (T-DNA) from Agrobacterium plasmids to host nuclei where they are integrated and where genes for hormone synthesis, borne on the T-DNA, are expressed in transformed host cells. This process has been exploited in plant genetic engineering by producing Agrobacterium mutants with deleterious genes removed from T-DNA, and with selected beneficial genes inserted in their place. Such mutants are able to infect and transform plants so that the beneficial genes are transferred to, and expressed in, the host plants, endowing them with desirable characteristics, including insect and disease resistance and herbicide tolerance. Biological control of Agrobacterium disease has been achieved using non-pathogenic Agrobacterium strains which produce an antibiotic specifically inhibitory to Agrobacterium pathogens. Use of one of these biocontrol strains is the first instance of a genetically engineered micro-organism being released commercially.

DNA Mutational Analysis↗
PubMed2005

Increased virulence and competitive advantage of a/alpha over a/a or alpha/alpha offspring conserves the mating system of Candida albicans.

The majority of Candida albicans strains in nature are a/alpha and must undergo homozygosis to a/a or alpha/alpha to mate. Here we have used a mouse model for systemic infection to test the hypothesis that a/alpha strains predominate in nature because they have a competitive advantage over a/a and alpha/alpha offspring in colonizing hosts. Single-strain injection experiments revealed that a/alpha strains were far more virulent than either their a/a or alpha/alpha offspring. When equal numbers of parent a/alpha and offspring a/a or alpha/alpha cells were co-injected, a/alpha always exhibited a competitive advantage at the time of extreme host morbidity or death. When equal numbers of an engineered a/a/alpha2 strain and its isogenic a/a parent strain were co-injected, the a/a/alpha2 strain exhibited a competitive advantage at the time of host morbidity or death, suggesting that the genotype of the mating-type (MTL) locus, not associated genes on chromosome 5, provides a competitive advantage. We therefore propose that heterozygosity at the MTL locus not only represses white-opaque switching and genes involved in the mating process, but also affects virulence, providing a competitive advantage to the a/alpha genotype that conserves the mating system of C. albicans in nature.

Animals↗
PubMed1998

Overexpression and purification of cytochrome c oxidase from Rhodobacter sphaeroides.

The aa3-type cytochrome c oxidase of Rhodobacter sphaeroides has been overexpressed up to seven fold over that in wild-type strains by engineering a multicopy plasmid with all the required oxidase genes and by establishing optimum growth conditions. The two operons containing the three structural genes and two assembly genes for cytochrome c oxidase were ligated into a pUC19 vector and reintroduced into several oxidase-deleted R. sphaeroides strains. Under conditions of relatively high pH and maximal aeration, high levels of expression were observed. A smaller expression vector, pBBR1MCS, and a fructose promoter (fruP)5 were found not to enhance cytochrome c oxidase expression in R. sphaeroides. An improved cytochrome c oxidase purification protocol is reported, which combines histidine elution from a nickel affinity column and anion-exchange chromatography, and results in a higher yield and purity than previously obtained.

Chromatography, Affinity↗
PubMed1996

Techniques for genetic engineering in mycobacteria. Alternative host strains, DNA-transfer systems and vectors.

The study of mycobacterial genetics has experienced quick technical developments in the past ten years, despite a relatively slow start, caused by difficulties in accessing these recalcitrant species. The study of mycobacterial pathogenesis is important in the development of new ways of treating tuberculosis and leprosy, now that the emergence of antibiotic-resistant strains has reduced the effectiveness of current therapies. The tuberculosis vaccine strain M. bovis BCG might be used as a vector for multivalent vaccination. Also, non-pathogenic mycobacterial strains have many possible biotechnological applications. After giving a historical overview of methods and techniques, we will discuss recent developments in the search for alternative host strains and DNA transfer systems. Special attention will be given to the development of vectors and techniques for stabilizing foreign DNA in mycobacteria.

Cloning, Molecular↗
PubMed1988

Genetic diversity in natural populations of a soil bacterium across a landscape gradient.

Genetic diversity in natural populations of the bacterium Pseudomonas cepacia was surveyed in 10 enzymes from 70 clones isolated along a landscape gradient. Estimates of genetic diversity, ranging from 0.54 to 0.70, were higher than any previously reported values of which we are aware and were positively correlated with habitat variability. Patterns of bacterial genetic diversity were correlated with habitat variability. Findings indicate that the source of strains used in genetic engineering will greatly affect the outcome of planned releases in variable environments. Selection of generalist strains may confer a large advantage to engineered populations, while selection of laboratory strains may result in quick elimination of the engineered strains.

Journal Article↗
PubMed1999

3D imaging of the 58 kDa cell binding subunit of the Helicobacter pylori cytotoxin.

Pathogenic strains of Helicobacter pylori produce a potent exotoxin, VacA, which intoxicates gastric epithelial cells and leads to peptic ulcer. The toxin is released from the bacteria as a high molecular mass homo-oligomer of a 95 kDa polypeptide which undergoes specific proteolytic cleavage to 37 kDa and 58 kDa subunits. We have engineered a strain of H. pylori to delete the gene sequence coding for the 37 kDa subunit. The remaining 58 kDa subunit is expressed efficiently and exported as a soluble dimer that is non-toxic but binds target cells in a manner similar to the holotoxin. A 3D reconstruction of the molecule from electron micrographs of quick-freeze, deep-etched preparations reveals the contribution of each building block to the structure and permits the reconstruction of the oligomeric holotoxin starting from individual subunits. In this model P58 subunits are assembled in a ring structure with P37 subunits laying on the top. The data indicate that the 58 kDa subunit is capable of folding autonomously into a discrete structure recognizable within the holotoxin and containing the cell binding domain.

Bacterial Proteins↗
PubMed2001

Engineering a homo-ethanol pathway in Escherichia coli: increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation.

Replacement of the native fermentation pathway in Escherichia coli B with a homo-ethanol pathway from Zymomonas mobilis (pdc and adhB genes) resulted in a 30 to 50% increase in growth rate and glycolytic flux during the anaerobic fermentation of xylose. Gene array analysis was used as a tool to investigate differences in expression levels for the 30 genes involved in xylose catabolism in the parent (strain B) and the engineered strain (KO11). Of the 4,290 total open reading frames, only 8% were expressed at a significantly higher level in KO11 (P < 0.05). In contrast, over half of the 30 genes involved in the catabolism of xylose to pyruvate were expressed at 1.5-fold- to 8-fold-higher levels in KO11. For 14 of the 30 genes, higher expression was statistically significant at the 95% confidence level (xylAB, xylE, xylFG, xylR, rpiA, rpiB, pfkA, fbaA, tpiA, gapA, pgk, and pykA) during active fermentation (6, 12, and 24 h). Values at single time points for only four of these genes (eno, fbaA, fbaB, and talA) were higher in strain B than in KO11. The relationship between changes in mRNA (cDNA) levels and changes in specific activities was verified for two genes (xylA and xylB) with good agreement. In KO11, expression levels and activities were threefold higher than in strain B for xylose isomerase (xylA) and twofold higher for xylulokinase (xylB). Increased expression of genes involved in xylose catabolism is proposed as the basis for the increase in growth rate and glycolytic flux in ethanologenic KO11.

Bacterial Proteins↗
PubMed1999

The development of bactericidal yeast strains by expressing the Pediococcus acidilactici pediocin gene (pedA) in Saccharomyces cerevisiae.

The excessive use of sulphur dioxide and other chemical preservatives in wine, beer and other fermented food and beverage products to prevent the growth of unwanted microbes holds various disadvantages for the quality of the end-products and is confronted by mounting consumer resistance. The objective of this study was to investigate the feasibility of controlling spoilage bacteria during yeast-based fermentations by engineering bactericidal strains of Saccharomyces cerevisiae. To test this novel concept, we have successfully expressed a bacteriocin gene in yeast. The pediocin operon of Pediococcus acidilactici PAC1.0 consists of four clustered genes, namely pedA (encoding a 62 amino acid precursor of the PA-1 pediocin), pedB (encoding an immunity factor), pedC (encoding a PA-1 transport protein) and pedD (encoding a protein involved in the transport and processing of PA-1). The pedA gene was inserted into a yeast expression/secretion cassette and introduced as a multicopy episomal plasmid into a laboratory strain (Y294) of S. cerevisiae. Northern blot analysis confirmed that the pedA structural gene in this construct (ADH1P-MFa1S-pedA-ADH1T, designated PED1), was efficiently expressed under the control of the yeast alcohol dehydrogenase I gene promoter (ADH1P) and terminator (ADH1T). Secretion of the PED1-encoded pediocin PA-1 was directed by the yeast mating pheromone alpha-factor's secretion signal (MFa1S). The presence of biologically active antimicrobial peptides produced by the yeast transformants was indicated by agar diffusion assays against sensitive indicator bacteria (e.g. Listeria monocytogenes B73). Protein analysis indicated the secreted heterologous peptide to be approximately 4.6 kDa, which conforms to the expected size. The heterologous peptide was present at relatively low levels in the yeast supernatant but pediocin activity was readily detected when intact yeast colonies were used in sensitive strain overlays. This study could lead to the development of bactericidal yeast strains where S. cerevisiae starter cultures not only conduct the fermentations in the wine, brewing and baking industries but also act as biological control agents to inhibit the growth of spoilage bacteria.

Bacteriocins↗
PubMed2006

In vivo characterization of brain morphometric and metabolic endophenotypes in three inbred strains of mice using magnetic resonance techniques.

C57BL6J, FVB/N and 129/SvJ mice are commonly used as background strains to engineer genetic models of brain pathologies and psychiatric disorders. Magnetic resonance imaging (MRI) and spectroscopy provide alternative approaches to neuroanatomy, histology and neurohistochemistry for investigating the correlation between genes and brain neuroanatomy and neurometabolism in vivo. We used these techniques to non-invasively characterize the cerebral morphologic and metabolic endophenotypes of inbred mouse strains commonly used in neurological and behavioral research. We observed a great variability in the volume of ventricles and of structures involved in cognitive function (cerebellum and hippocampus) among these strains. In addition, distinct metabolic profiles were evidenced with variable levels of N-acetylaspartate, a neuronal marker, and of choline, a compound found in membranes and myelin. Besides, significant differences in high-energy phosphates and phospholipids were detected. Our findings demonstrate the great morphologic and metabolic heterogeneity among C57BL/ 6J, FVB/N and 129/SvJ mice. They emphasize the importance of selecting the appropriate genetic background for over-expressing or silencing a gene and provide some directions for modeling symptoms that characterize psychiatric disorders such as autism, schizophrenia and depression.

Animals↗