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PubMed2006

Transition temperature of a magnetic semiconductor with angular momentum j.

We employ dynamical mean-field theory to identify the materials properties that optimize T(c) for a generalized double-exchange model. We reach the surprising conclusion that T(c) achieves a maximum when the band angular momentum j equals 3/2 and when the masses in the m(j) = +/- 1/2 and +/-3/2 and subbands are equal. However, we also find that T(c) is significantly reduced as the ratio of the masses decreases from one. Consequently, the search for dilute-magnetic semiconductor materials with high T(c) should proceed on two fronts. In semiconductors with p bands, such as the currently studied Mn-doped Ge and GaAs semiconductors, T(c) may be optimized by tuning the band masses through strain engineering or artificial nanostructures. On the other hand, semiconductors with s or d bands with nearly equal effective masses might prove to have higher T(c)'s than p-band materials with disparate effective masses.

Journal Article↗

PubMed2005

Novel and economical purification of recombinant proteins: intein-mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association.

This work combines two well-established technologies to generate a breakthrough in protein production and purification. The first is the production of polyhydroxybutyrate (PHB) granules in engineered strains of Escherichia coli. The second is a recently developed group of self-cleaving affinity tags based on protein splicing elements known as inteins. By combining these technologies with a PHB-specific binding protein, a self-contained protein expression and purification system has been developed. In this system, the PHB-binding protein effectively acts as an affinity tag for desired product proteins. The tagged product proteins are expressed in E. coli strains that also produce intracellular PHB granules, where they bind to the granules via the PHB-binding tag. The granules and attached proteins can then be easily recovered following cell lysis by simple mechanical means. Once purified, the product protein is self-cleaved from the granules and released into solution in a substantially purified form. This system has been successfully used at laboratory scale to purify several active test proteins at reasonable yield. By allowing the bacterial cells to effectively produce both the affinity resin and tagged target protein, the cost associated with the purification of recombinant proteins could be greatly reduced. It is expected that this combination of improved economics and simplicity will constitute a significant breakthrough in both large-scale production of purified proteins and enzymes and high-throughput proteomics studies of peptide libraries.

Bacterial Proteins↗

PubMed2006

Regulation of tylosin production: role of a TylP-interactive ligand.

Gamma-butyrolactones regulate secondary metabolism and, sometimes, sporulation in actinomycetes by binding to specific receptor proteins, causing their dissociation from DNA targets and releasing the latter from transcriptional repression. Previously, in engineered strains of Streptomyces lividans, we showed that TylP, a deduced gamma-butyrolactone receptor, downregulated reporter gene expression driven by tylP, tylQ or tylS promoter DNA. These genes all control tylosin production in Streptomyces fradiae. Thus, at early stages of fermentation, TylQ represses tylR whereas TylS is needed for transcriptional activation of tylR. Importantly, TylR is the key activator of tylosin-biosynthetic genes. Here, we show that HIS-tagged TylP binds to specific DNA sequences, similar to the targets for authentic gamma-butyrolactone receptors, in the promoters of tylP, tylQ and tylS. Moreover, such binding is disrupted by material produced in S. fradiae and extractable by organic solvent. That putative gamma-butyrolactone material was not produced when orf18 * was disrupted within the S. fradiae genome and only about 1% of that activity survived inactivation of orf16 *, suggesting roles for the respective gene products in gamma-butyrolactone synthesis. Continued synthesis of tylosin by the disrupted strains contrasts with other reports that loss of gamma-butyrolactones abolishes antibiotic production.

4-Butyrolactone↗

PubMed2004

Silicon device scaling to the sub-10-nm regime.

In the next decade, advances in complementary metal-oxide semiconductor fabrication will lead to devices with gate lengths (the region in the device that switches the current flow on and off) below 10 nanometers (nm), as compared with current gate lengths in chips that are now about 50 nm. However, conventional scaling will no longer be sufficient to continue device performance by creating smaller transistors. Alternatives that are being pursued include new device geometries such as ultrathin channel structures to control capacitive losses and multiple gates to better control leakage pathways. Improvement in device speed by enhancing the mobility of charge carriers may be obtained with strain engineering and the use of different crystal orientations. Here, we discuss challenges and possible solutions for continued silicon device performance trends down to the sub-10-nm gate regimes.

Journal Article↗

PubMed1990

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey.

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.

Cloning, Molecular↗

PubMed1996

Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis.

Continuous cultivation in a glucose-limited chemostat was used to determine the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coefficients of 0.45 and 0.66 mmol of glucose g-1 h-1 were determined for the wild-type and recombinant strains, respectively. However, the maximum molar growth yield of 82 to 85 g (cell dry weight)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin production rate and the dilution rate was observed, revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For molecular characterization of B. subtilis, the total amino acid composition of the wild type was experimentally determined and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approximately 9% of B. subtilis biomass, three- to fivefold more than in Escherichia coli. Estimation of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle.

Amino Acids↗

PubMed1996

Engineering Bacillus thuringiensis bioinsecticides with an indigenous site-specific recombination system.

The cry genes of Bacillus thuringiensis encode a diverse group of crystal-forming proteins that exhibit insecticidal activity, particularly against the larvae of lepidopteran, coleopteran, and dipteran insects. The efficacy of B. thuringiensis-based biopesticides may be improved through the genetic manipulation of these genes. A gene transfer system has been developed for the introduction and maintenance of cloned insecticidal cry genes on small plasmids in B. thuringiensis. This vector system combines a B. thuringiensis plasmid replicon and an indigenous site-specific recombination system that allows for the selective removal of ancillary or foreign DNA from the recombinant bacterium after introduction of the Cry-encoding plasmid. The site-specific recombination system is useful for engineering strains with unique combinations of cry genes, resulting in new active ingredients with improved insecticidal properties.

Animals↗

PubMed2003

Enhanced production of insulin-like growth factor I fusion protein in Escherichia coli by coexpression of the down-regulated genes identified by transcriptome profiling.

The transcriptome profiles of recombinant Escherichia coli producing human insulin-like growth factor I fusion protein (IGF-I(f)) during the high-cell-density fed-batch culture were analyzed using DNA microarrays. The expression levels of 529 genes were significantly altered after induction. About 200 genes were significantly down-regulated during the production of IGF-I(f) after induction. Among these down-regulated genes, we rationally selected and coexpressed in E. coli producing IGF-I(f) the prsA gene (encoding a phosphoribosyl pyrophosphate synthetase) and the glpF gene (encoding a glycerol transporter), which are involved in an early key step in the biosynthetic pathway of nucleotides and amino acids (Trp and His) and the first step in glycerol utilization, respectively. As a result, the production of IGF-I(f) could be increased from 1.8 +/- 0.13 (+/- standard deviation) to 4.3 +/- 0.24 g/liter. The volumetric productivity was also increased from 0.36 +/- 0.027 to 0.82 +/- 0.048 g/liter/h. These results demonstrate that transcriptome profiling can provide invaluable information in designing engineered strains showing enhanced performance.

Aquaporins↗

PubMed2005

Rhizobium leguminosarum biovar viciae symbiotic hydrogenase activity and processing are limited by the level of nickel in agricultural soils.

Analysis of levels of hydrogenase processing and activity in Rhizobium leguminosarum biovar viciae bacteroids from pea (Pisum sativum) plants showed that the oxidation of nitrogenase-evolved hydrogen is limited by the availability of nickel in agricultural soils. This limitation was overcome by using an inoculant strain engineered for higher hydrogenase expression.

Agriculture↗

PubMed2005

Production of the potent antibacterial polyketide erythromycin C in Escherichia coli.

An Escherichia coli strain capable of producing the potent antibiotic erythromycin C (Ery C) was developed by expressing 17 new heterologous genes in a 6-deoxyerythronolide B (6dEB) producer strain. The megalomicin gene cluster was used as the source for the construction of two artificial operons that contained the genes encoding the deoxysugar biosynthetic and tailoring enzymes necessary to convert 6dEB to Ery C. The reconstructed mycarose operon contained the seven genes coding for the enzymes that convert glucose-1-phosphate (G-1-P) to TDP-L-mycarose, a 6dEB mycarosyl transferase, and a 6dEB 6-hydroxylase. The activity of the pathway was confirmed by demonstrating conversion of exogenous 6dEB to 3-O-alpha-mycarosylerythronolide B (MEB). The reconstructed desosamine operon contained the six genes necessary to convert TDP-4-keto-6-deoxyglucose, an intermediate formed in the mycarose pathway, to TDP-D-desosamine, a desosamine transferase, a 6dEB 12-hydroxylase, and the rRNA methyltransferase ErmE; the last was required to confer resistance to the host cell upon production of mature macrolide antibiotics. The activity of this pathway was demonstrated by conversion of MEB to Ery C. When the mycarose and desosamine operons were expressed in an E. coli strain engineered to synthesize 6dEB, Ery C and Ery D were produced. The successful production of Ery C in E. coli shows the potentiality of this model microorganism to synthesize novel 6-deoxysugars and to produce bioactive glycosylated compounds and also establishes the basis for the future use of E. coli both in the production of new glycosylated polyketides and for the generation of novel bioactive compounds through combinatorial biosynthesis.

Aminoglycosides↗

PubMed2006

Cytosolic entry controls CD8+-T-cell potency during bacterial infection.

Interaction with host immunoreceptors during microbial infection directly impacts the magnitude of the ensuing innate immune response. How these signals affect the quality of the adaptive T-cell response remains poorly understood. Utilizing an engineered strain of the intracellular pathogen Listeria monocytogenes that infects cells but fails to escape from the phagosome, we demonstrate the induction of long-lived memory T cells that are capable of secondary expansion and effector function but are incapable of providing protective immunity. We demonstrate that microbial invasion of the cytosol is required for dendritic cell activation and integration of CD40 signaling, ultimately determining the ability of the elicited CD8+-T-cell pool to protect against lethal wild-type L. monocytogenes challenge. These results reveal a crucial role for phagosomal escape, not for delivery of antigen to the class I major histocompatibility complex pathway but for establishing the appropriate cellular context during CD8+-T-cell priming.

Animals↗

PubMed1999

Detection of live Trypanosoma cruzi in tissues of infected mice by using histochemical stain for beta-galactosidase.

The pathogenesis of tissue damage in chronic Trypanosoma cruzi infection has been a subject of long-standing debate. Conventional staining methods reveal a paucity of parasites in tissues from chronically infected individuals, which has led to the theory that the pathologic findings may be primarily autoimmune in origin. Immunostaining for T. cruzi antigens or in situ PCR methods show evidence for parasite components in chronic tissues; however, these methods do not address whether the stained material represents parasite debris or live organisms. An improved method for detecting intact T. cruzi in tissues was developed by making a genetically engineered strain that expresses Escherichia coli beta-galactosidase. The expression of this enzyme allows the detection of T. cruzi in tissues by using the histochemical stain 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal). The technique was used to monitor tissue parasitism and its relation to pathologic findings in the mouse model of Chagas' disease. Parasites were easily visible as bright blue structures in skeletal muscle, heart, bladder, peripheral nerve, liver, spleen, adrenal gland, brain, and adipose tissue in acutely infected mice. The number of viable parasites diminished >100-fold when tissues from 3-week-infected mice were compared with those from 10-month-infected mice. However, even at the lower level, parasites were clearly recognizable in sections of skeletal muscle and bladder at the 10-month time point. Inflammation remained robust in skeletal muscle, bladder, and sciatic nerve despite the near disappearance of parasites, suggesting three possibilities: exuberant host reactions to the few remaining parasites, autoimmune inflammation, or reactions to retained parasite antigens in the tissues.

3T3 Cells↗

PubMed1999

Genetic characterization of wild-type and mutant fur genes of Bordetella avium.

For most, if not all, organisms, iron (Fe) is an essential element. In response to the nutritional requirement for Fe, bacteria evolved complex systems to acquire the element from the environment. The genes encoding these systems are often coordinately regulated in response to the Fe concentration. Recent investigations revealed that Bordetella avium, a respiratory pathogen of birds, expressed a number of Fe-regulated genes (T. D. Connell, A. Dickenson, A. J. Martone, K. T. Militello, M. J. Filiatraut, M. L. Hayman, and J. Pitula, Infect. Immun. 66:3597-3605, 1998). By using manganese selection on an engineered strain of B. avium that carried an Fe-regulated alkaline phosphatase reporter gene, a mutant was obtained that was affected in expression of Fe-regulated genes. To determine if Fe-dependent regulation in B. avium was mediated by a fur-like gene, a fragment of the B. avium chromosome, corresponding to the fur locus of B. pertussis, was cloned by PCR. Sequencing revealed that the fragment from B. avium encoded a polypeptide with 92% identity to the Fur protein of B. pertussis. In vivo experiments showed that the cloned gene complemented H1780, a fur mutant of Escherichia coli. Southern hybridizations and PCRs demonstrated that the manganese mutant had a deletion of 2 to 3 kbp of nucleotide sequence in the region located immediately 5' of the fur open reading frame. A spontaneous PCR-derived mutant of the B. avium fur gene was isolated that encoded a Fur protein in which a histidine was substituted for an arginine at amino acid position 18 (R18H). Genetic analysis showed that the R18H mutant gene when cloned into a low-copy-number vector did not complement the fur mutation in H1780. However, the R18H mutant gene was able to complement the fur mutation when cloned into a high-copy-number vector. The cloned wild-type fur gene will be useful as a genetic tool to identify Fur-regulated genes in the B. avium chromosome.

Amino Acid Sequence↗

PubMed2000

Identification of a Porphyromonas gingivalis receptor for the Streptococcus gordonii SspB protein.

Colonization of the plaque biofilm by the oral pathogen Porphyromonas gingivalis is favored by the presence of antecedent organisms such as Streptococcus gordonii. Coadhesion between P. gingivalis and S. gordonii can be mediated by the SspB protein of S. gordonii; however, the P. gingivalis cognate receptor for this protein has not been identified. In this study, we identified a surface protein of P. gingivalis that interacts with the SspB protein. Coprecipitation between P. gingivalis outer membrane proteins and purified SspB protein demonstrated that a 100-kDa P. gingivalis protein bound to SspB. The 100-kDa protein also bound to an engineered strain of Enterococcus faecalis that expresses the SspB protein on the cell surface. Monospecific polyclonal antibodies to the 100-kDa protein inhibited the binding between P. gingivalis and S. gordonii in a dose-dependent manner up to 86%. Amino acid sequencing of the 100-kDa protein showed homology to a protein previously identified as the P. gingivalis minor fimbria. The minor fimbrial protein may exist as a complex with a hemagglutinin-like protein since the genes encoding these proteins are adjacent on the chromosome and are cotranscribed. Thus, the P. gingivalis receptor for S. gordonii SspB is a 100-kDa protein that structurally may be a minor fimbria-protein complex and functionally effectuates coadhesion.

Adhesins, Bacterial↗

PubMed2025

A novel regulation on the developmental checkpoint protein Sda that controls sporulation and biofilm formation in Bacillus subtilis.

UNLABELLED: Biofilm formation by Bacillus subtilis is triggered by an unusually simple environmental sensing mechanism. Certain serine codons, the four TCN codons (N for A, T, C, or G), in the gene for the biofilm repressor SinR caused lowered SinR translation and subsequent biofilm induction during transition from exponential to stationary growth. Global ribosome profiling showed that ribosomes pause when translating the four UCN (U for T on the mRNA) serine codons on mRNA, but not the two AGC/AGU serine codons. We proposed a serine codon hierarchy (AGC/AGT vs TCN) in that genes enriched in the TCN serine codons may experience reduced translation efficiency when serine is limited. In this study, we designed an algorithm to score all protein-coding genes in B. subtilis NCIB3610 based on the serine codon hierarchy. We generated a short list of 50 genes that could be subject to regulation by this novel mechanism. We further investigated one such gene from the list, sda, which encodes a developmental checkpoint protein regulating both sporulation and biofilm formation. We showed that synonymously switching the TCN serine codons to AGC in sda led to delayed biofilm formation and sporulation. This engineered strain also outgrew strains with other synonymously substituted sda alleles (TCN) in competition assays for biofilm formation and sporulation. Finally, we showed that the AGC serine codon substitutions in sda elevated the Sda protein levels. This serine codon hierarchy-based novel signaling mechanism could be exploited by bacteria in adapting to stationary phase and regulating important biological processes. IMPORTANCE: Genome-wide ribosome profiling in Bacillus subtilis shows that under serine limitation, ribosomes pause on the four TCN (N for A, C, G, and T), but not AGC/AGT serine codons, during translation at a global scale. This serine codon hierarchy (AGC/T vs TCN) differentially influences the translation efficiency of genes enriched in certain serine codons. In this study, we designed an algorithm to score all 4,000+ genes in the B. subtilis genome and generated a list of 50 genes that could be subject to this novel serine codon hierarchy-mediated regulation. We further investigated one such gene, sda, encoding a developmental checkpoint protein. We show that sda and cell developments controlled by Sda are also regulated by this novel mechanism.

Bacillus subtilis↗

PubMed1997

A feedback loop regulates the switch from one sigma factor to the next in the cascade controlling Bacillus subtilis mother cell gene expression.

Regulation of gene expression in the mother cell compartment of sporulating Bacillus subtilis involves sequential activation and inactivation of several transcription factors. Among them are two sigma factors, sigmaE and sigmaK, and a DNA-binding protein, SpoIIID. A decrease in the level of SpoIIID is thought to relieve its repressive effect on transcription by sigmaK RNA polymerase of certain spore coat genes. Previous studies showed that sigmaK negatively regulates the level of spoIIID mRNA. Here, it is shown that sigmaK does not affect the stability of spoIIID mRNA. Rather, sigmaK appears to negatively regulate the synthesis of spoIIID mRNA by accelerating the disappearance of sigmaE RNA polymerase, which transcribes spoIIID. As sigmaK begins to accumulate by 4 h into sporulation, the sigmaE level drops rapidly in wild-type cells but remains twofold to fivefold higher in sigK mutant cells during the subsequent 4 h. In a strain engineered to produce sigmaK 1 h earlier than normal, twofold less sigmaE than that in wild-type cells accumulates. SigmaK did not detectably alter the stability of sigmaE in pulse-chase experiments. However, beta-galactosidase expression from a sigE-lacZ transcriptional fusion showed a pattern similar to the level of sigmaE protein in sigK mutant cells and cells prematurely expressing sigmaK. These results suggest that the appearance of sigmaK initiates a negative feedback loop controlling not only transcription of spoIIID, but the entire sigmaE regulon, by directly or indirectly inhibiting the transcription of sigE.

Bacillus subtilis↗

PubMed1997

Acyltransferase domain substitutions in erythromycin polyketide synthase yield novel erythromycin derivatives.

The methylmalonyl coenzyme A (methylmalonyl-CoA)-specific acyltransferase (AT) domains of modules 1 and 2 of the 6-deoxyerythronolide B synthase (DEBS1) of Saccharopolyspora erythraea ER720 were replaced with three heterologous AT domains that are believed, based on sequence comparisons, to be specific for malonyl-CoA. The three substituted AT domains were "Hyg" AT2 from module 2 of a type I polyketide synthase (PKS)-like gene cluster isolated from the rapamycin producer Streptomyces hygroscopicus ATCC 29253, "Ven" AT isolated from a PKS-like gene cluster of the pikromycin producer Streptomyces venezuelae ATCC 15439, and RAPS AT14 from module 14 of the rapamycin PKS gene cluster of S. hygroscopicus ATCC 29253. These changes led to the production of novel erythromycin derivatives by the engineered strains of S. erythraea ER720. Specifically, 12-desmethyl-12-deoxyerythromycin A, which lacks the methyl group at C-12 of the macrolactone ring, was produced by the strains in which the resident AT1 domain was replaced, and 10-desmethylerythromycin A and 10-desmethyl-12-deoxyerythromycin A, both of which lack the methyl group at C-10 of the macrolactone ring, were produced by the recombinant strains in which the resident AT2 domain was replaced. All of the novel erythromycin derivatives exhibited antibiotic activity against Staphylococcus aureus. The production of the erythromycin derivatives through AT replacements confirms the computer predicted substrate specificities of "Hyg" AT2 and "Ven" AT and the substrate specificity of RAPS AT14 deduced from the structure of rapamycin. Moreover, these experiments demonstrate that at least some AT domains of the complete 6-deoxyerythronolide B synthase of S. erythraea can be replaced by functionally related domains from different organisms to make novel, bioactive compounds.

Acyl Coenzyme A↗

PubMed1999

A single-ring mitochondrial chaperonin (Hsp60-Hsp10) can substitute for GroEL-GroES in vivo.

Chaperonins participate in the facilitated folding of a variety of proteins in vivo. To see whether the same spectrum of target proteins can be productively folded by the double-ring prokaryotic chaperonin GroEL-GroES and its single-ring human mitochondrial homolog, Hsp60-Hsp10, we expressed the latter in an Escherichia coli strain engineered so that the groE operon is under strict regulatory control. We found that expression of Hsp60-Hsp10 restores viability to cells that no longer express GroEL-GroES, formally demonstrating that Hsp60-Hsp10 can carry out all essential in vivo functions of GroEL-GroES.

Chaperonin 10↗