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A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per μg of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (≥108 colonies per μg of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures. Molecular Sequence Numbers: GENBANK: AF291751, X53217; Chemicals/CAS: DNA Restriction-Modification Enzymes

Two Lactobacillus-Escherichia coli shuttle vectors, harbouring the levanase gene from Bacillus subtilis under the control of its own promoter (pLPEW1) or behind the E. coli tac promoter (pE-SIEW2), were constructed. Lactobacillus plantarum showed the same growth characteristics on selective plates and in liquid media containing inulin, after transformation with either pLPEW1 or pESIEW2. L. plantarum transformed with pLPEW1 could be selected on inulin plates, indicating that levanase expression can be used as a food-grade selection system for Lactobacillus. Lactobacillus casei grew faster in inulin-containing medium than L. plantarum after transformation with pE-SIEW2, but did not grow when harbouring pLPEW1. Inulin-degrading activities of 90 mU/ml were found in culture medium of L. plantarum containing pLPEW1 or pESIEW2, and of 500 mU/ml in medium of L. casei (pESIEW2). Addition of 1 mM isopropyl β-D-thiogalactoside to the culture medium had no effect on growth and levanase expression in L. plantarum (pESIEW2) and L. casei (pESIEW2) strains. Levanase produced by L. casei (pESIEW2) has a size of 75 kDa and 72 kDa, corresponding to that of unprocessed and mature B. subtilis levanase, respectively, suggesting that the protein produced is recognized and processed by a signal peptidase.

Chemicals/CAS: DNA Restriction Enzymes, EC 3.1.21; DNA, Recombinant; Plasmids; Tryptophan, 73-22-3

Bacterial strains differ in their ability to cause hospital outbreaks. Using comparative genomic hybridization, Enterobacter cloacae complex isolates were studied to identify genetic markers specific for Enterobacter cloacae complex outbreak strains. No outbreak-specific genes were found that were common in all investigated outbreak strains. Therefore, the aim of our study was to identify specific genetic markers for an Enterobacter hormaechei outbreak strain (EHOS) that caused a nationwide outbreak in The Netherlands. Most EHOS isolates carried a large conjugative plasmid (pQC) containing genes encoding heavy-metal resistance, mobile elements, pili-associated proteins and exported proteins as well as multiple-resistance genes. Furthermore, the chromosomally encoded high-pathogenicity island (HPI) was highly associated with the EHOS strain. In addition, other DNA fragments were identified that were associated with virulence: three DNA fragments known to be located on a virulence plasmid (pLVPK), as well as phage- and plasmid-related sequences. Also, four DNA fragments encoding putative pili with the most homology to pili of Salmonella enterica were associated with the EHOS. Finally, four DNA fragments encoding putative outer-membrane proteins were negatively associated with the EHOS. In conclusion, resistance and putative virulence genes were identified in the EHOS that may have contributed to increased epidemicity. The high number of genes detected in the EHOS that were related to transferable elements reflects the genomic plasticity of the E. cloacae complex and may explain the emergence of the EHOS in the hospital environment. © 2009 SGM.

The genes encoding the production of acidocin B, a bacteriocin produced by Lactobacillus acidophilus strain M46 which is active against Listeria monocytogenes, Clostridium sporogenes, Brochothrix thermosphacta, Lactobacillus fermentum and Lactobacillus delbrueckii subsp. bulgaricus, but inactive against most other Lactobacillus species, were previously localized on a 4 kb Xbal-HindIII fragment of plasmid pCV461. In the present work, DNA sequence analysis revealed the presence of three consecutive ORFs, which potentially code for hydrophobic peptides composed of 60, 91 and 114 amino acids, respectively, and a fourth ORF of opposite polarity which could potentially encode a peptide of 59 amino acids. The middle ORF (ORF-2; acdB) was identified as the gene encoding acidocin B by comparing the amino acid composition of highly purified acidocin B with the deduced amino acid sequence of ORF-2. Our results suggest that acidocin B is synthesized as a precursor which is processed at a site which conforms to the '-3, -1' rules of von Heijne to yield active acidocin B (59 amino acids). The presence of an immunity-protein-encoding gene on the 4 kb Xbal-BamHI fragment was deduced from the capacity of a plasmid vector harbouring this fragment to confer immunity upon transformation of L. fermentum NCK127. One of the three non-assigned ORFs may encode this immunity protein.

Synthesis of lactococcin 972 is plasmid-encoded. An operon composed of two genes that encode pre-bacteriocin and a putative immunity protein has been identified. The first gene encodes a 91-residue polypeptide that is exported via a sec-dependent system to give the mature 66-aa bacteriocin. The immunity protein is a 563-residue polypeptide with seven potential transmembrane domains. Two transcripts were observed from this region: one comprises the whole operon and is synthesized during the exponential phase of growth while the other, which corresponds just to the bacteriocin structural gene, presents a maximum in exponential cultures but is still present in late-stationary-phase cells.

We have identified and characterized the D-xylose transport system of Lactobacillus pentosus. Uptake of D-xylose was not driven by the proton motive force generated by malolactic fermentation and required D-xylose metabolism. The kinetics of D-xylose transport were indicative of a low- affinity facilitated-diffusion system with an apparent K(m) of 8.5 mM and a V(max) of 23 nmol min-1 mg of dry weight-1. In two mutants of L. pentosus defective in the phosphoenolpyruvate:mannose phosphotransferase system, growth on D-xylose was absent due to the lack of D-xylose transport. However, transport of the pentose was not totally abolished in a third mutant, which could be complemented after expression of the L. curvatus manB gene encoding the cytoplasmic EIIB(Man) component of the EII(Man) complex. The EII(Man) complex is also involved in D-xylose transport in L. casei ATCC 393 and L. plantarum 80. These two species could transport and metabolize D-xylose after transformation with plasmids which expressed the D-xylose-catabolizing genes of L. pentosus, xylAB. L. casei and L. plantarum mutants resistant to 2- deoxy-D-glucose were defective in EII(Man) activity and were unable to transport D-xylose when transformed with plasmids containing the xylAB genes. Finally, transport of D-xylose was found to be the rate-limiting step in the growth of L. pentosus and of L. plantarum and L. casei ATCC 393 containing plasmids coding for the D-xylose-catabolic enzymes, since the doubling time of these bacteria on D-xylose was proportional to the level of EII(Man) activity.

Several cutinase variants derived by molecular modelling and site- directed mutagenesis of a cutinase gene from Fusarium solani pisi are poorly secreted by Saccharomyces cerevisiae. The majority of these variants are successfully produced by the filamentous fungus Aspergillus awamori. However, the L51S and T179Y mutations caused reductions in the levels of extracellular production of two cutinase variants by A. awamori. Metabolic labelling studies were performed to analyze the bottleneck in enzyme production by the fungus in detail. These studies showed that because of the single L51S substitution, rapid extracellular degradation of cutinase occurred. The T179Y substitution did not result in enhanced sensitivity towards extracellular proteases. Presumably, the delay in the extracellular accumulation of this cutinase variant is caused by the enhanced hydrophobicity of the molecule. Overexpression of the A. awamori gene encoding the chaperone BiP in the cutinase-producing A. awamori strains had no significant effect on the secretion efficiency of the cutinases. A cutinase variant with the amino acid changes G28A, A85F, V184I, A185L, and L189F that was known to aggregate in the endoplasmic reticulum of S. cerevisiae, resulting in low extracellular protein levels, was successfully produced by A. awamori. An initial bottleneck in secretion occurred before or during translocation into the endoplasmic reticulum but was rapidly overcome by the fungus.

The xylP gene of Lactobacillus pentosus, the first gene of the xylPQR operon, was recently found to be involved in isoprimeverose metabolism. By expression of xylP on a multicopy plasmid in Lactobacillus plantarum 80, a strain which lacks active isoprimeverose and D-xylose transport activities, it was shown that xylP encodes a transporter. Functional expression of the XylP transporter was shown by uptake of isoprimeverose in L. plantarum 80 cells, and this transport was driven by the proton motive force generated by malolactic fermentation. XylP was unable to catalyze transport of D-xylose.

A protocol was developed for the introduction of foreign plasmid DNA into various Bifidobacterium strains. The method, which is applicable to all Bifidobacterium species tested so far, is based on electroporation of bacteria made competent by preincubation in electroporation buffer for several hours at 4°C. Transformation of Bifidobacterium could be achieved with a plasmid vector originating from Bifidobacterium and with plasmid vectors from Corynebacterium, but not with vectors carrying replicons from Lactococcus or Lactobacillus.

Bifidobacteria are among the most common bacteria in the human intestine and are thought to have a positive effect on human health. Therefore, there is an increasing interest in using these microorganisms as probiotics, either in fermented dairy products or formulated as tablets. However, convincing scientific data supporting their health claims are scarce. The study of the role of bifidobacteria in the colon is complicated by the fact that they are part of a complex ecosystem also interacting with the human host and by the fact that their in vivo study encounters many ethical constraints. Several tools have been developed at TNO with which the role of bifidobacteria can be studied. These include (i) an efficient transformation protocol for the introduction of foreign DNA into Bifidobacterium strains and (ii) in vitro models of the stomach/small intestine (TIM-1) and large intestine (TIM-2), creating an environment closely resembling that of the in vivo situation. With these tools, biomarkers from bifidobacteria quantifying their positive effect on gut health can be identified.

Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the p WCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and the number of fungi that can be transformed by Agrobacterium-mediated transformation (AMT) is still increasing. AMT has especially opened the field of molecular genetics for fungi that were difficult to transform with traditional methods or for which the traditional protocols failed to yield stable DNA integration. Because of the simplicity and efficiency of transformation via A. tumefaciens, it is relatively easy to generate a large number of stable transformants. In combination with the finding that the T-DNA integrates randomly and predominantly as a single copy, AMT is well suited to perform insertional mutagenesis in fungi. In addition, in various gene-targeting experiments, high homologous recombination frequencies were obtained, indicating that the T-DNA is also a useful substrate for targeted mutagenesis. In this review, we discuss the potential of the Agrobacterium DNA transfer system to be used as a tool for targeted and random mutagenesis in fungi. © Springer-Verlag 2005.

Recombinant lactobacilli are being developed which can be used as expression and delivery vectors of heterologous antigens in oral vaccination and other therapeutic applications. Because most Lactobacillus strains do not accept ligation mixtures, sufficiently pure plasmid DNA needs to be isolated from Lactobacillus casei to transform other Lactobacillus strains. The isolation of plasmid DNA from Gram-positive lactobacilli is complicated by the resilience of the peptidoglycan layer. Here a rapid, safe and efficient method is described that combines enzymatic breakdown of the cell wall and purification of the plasmid by commercially available DNA-binding columns. For the lysis-resistant L. casei strain, this method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency. Chemicals/CAS: Bacterial Proteins; Deoxyribonuclease BamHI, EC 3.1.21.-; Deoxyribonucleases, Type II Site-Specific, EC 3.1.21.4; DNA, Bacterial; endodeoxyribonuclease PvuI, EC 3.1.21.-; Immunodominant Epitopes

A method was developed to perform PCR directly on mycelial pellets or colonies treated with NOVOzym 234. The method allows rapid screening of large numbers of transformants of both sporulating and non-sporulating fungi for the presence of (co)transforming plasmid copies or for specific genetic modifications such as gene disruption and site specific integration. PCR fragments of at least 3.2 kb can be obtained. Using this method the identification of specific disruption mutants from Aspergillus niger and Beauveria bassiana was carried out.

Fibrinogen is a coagulation factor and an acute phase reactant up-regulated by inflammatory cytokines, such as interleukin 6 (IL-6). Elevated plasma fibrinogen levels are associated with coronary heart diseases. Fibrates are clinically used hypolipidemic drugs that act via the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). In addition, most fibrates also reduce plasma fibrinogen levels, but the molecular mechanism is unknown. In this study, we demonstrate that fibrates decrease basal and IL-6-stimulated expression of the human fibrinogen-β gene in human primary hepatocytes and hepatoma HepG2 cells. Fibrates diminish basal and IL-6-induced fibrinogen-β promoter activity, and this effect is enhanced in the presence of co-transfected PPARα. Site-directed mutagenesis experiments demonstrate that PPARα activators decrease human fibrinogen-β promoter activity via the CCAAT box/enhancer-binding protein (C/EBP) response element. Co-transfection of the transcriptional intermediary factor glucocorticoid receptor-interacting protein 1/transcriptional intermediary factor 2 (GRIP1/TIF2) enhances fibrinogen-β gene transcription and alleviates the repressive effect of PPARα. Co-immunoprecipitation experiments demonstrate that PPARα and GRIP1/TIF2 physically interact in vivo in human liver. These data demonstrate that PPARα agonists repress human fibrinogen gene expression by interference with the C/EBPβ pathway through titration of the coactivator GRIP1/TIF2. We observed that the anti-inflammatory action of PPARα is not restricted to fibrinogen but also applies to other acute phase genes containing a C/EBP response element; it also occurs under conditions in which the stimulating action of IL-6 is potentiated by dexamethasone. These findings identify a novel molecular mechanism of negative gene regulation by PPARα and reveal the direct implication of PPARα in the modulation of the inflammatory gene response in the liver. Chemicals/CAS: fibrinogen, 9001-32-5; peroxisome proliferator activated receptor alpha, 147258-70-6; CCAAT-Enhancer-Binding Protein-alpha; Fibrinogen, 9001-32-5; Interleukin-6; NCOA2 protein, human; Nuclear Receptor Coactivator 2; Peroxisome Proliferators; pirinixic acid, 50892-23-4; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Transcription Factors

The heterologous production of Arthromyces ramosus peroxidase (ARP) was analysed in the filamentous fungus Aspergillus awamori under control of the inducible endoxylanase promoter. Secretion of active ARP was achieved up to 800 mg l-1 in shake flask cultures. Western blot analysis showed that an rARP product of the correct molecular weight was produced. In contrast to several other studies about heterologous production of heme containing peroxidases, our results suggest that in A. awamori no heme limitation exists during overproduction of ARP. © 2003 Elsevier Science B.V. All rights reserved.

A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the subsequent secretion of the recombinant enzyme was achieved in Saccharomyces cerevisiae and Aspergillus awamori.

A cluster of three genes involved in D-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis D-xylose isomerase (68% and 77%, respectively), and to E. coli D-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment D-xylose. NMR analysis confirmed that 13C-xylose was converted into 13C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of D-xylose isomerases of different bacteria suggests that L. pentosus D-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli D-xylose isomerase and not to a second similarity group comprising D-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5'-xylR (1167 bp, repressor) - xylA (1350 bp, D-xylose isomerase) - xylB (1506 bp, D-xylulose kinase) - 3' is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB. Molecular Sequence Numbers: GENBANK: M57384, S55527, S55528, S55529, S55530, S55531, S55532, S63909, S69823, Z14057; Chemicals/CAS: Carbohydrate Epimerases, EC 5.1.3; DNA, Bacterial; Phosphotransferases, EC 2.7; Plasmids; Repressor Proteins; xylose isomerase, EC 5.3.1.5; Xylose; xylulokinase, EC 2.7.1.17

Site-directed mutagenesis has been used to investigate the role of glutamic acid 183 in chloroperoxidase catalysis. Based on the x-ray crystallographic structure of chloroperoxidase, Glu-183 is postulated to function on distal side of the heme prosthetic group as an acid-base catalyst in facilitating the reaction between the peroxidase and hydrogen peroxide with the formation of Compound I. In contrast, the other members of the heme peroxidase family use a histidine residue in this role. Plasmids have now been constructed in which the codon for Glu-183 is replaced with a histidine codon. The mutant recombinant gene has been expressed in Aspergillus niger. An analysis of the produced mutant gene shows that the substitution of Glu-183 with a His residue is detrimental to the chlorination and dismutation activity of chloroperoxidase. The activity is reduced by 85 and 50% of wild type activity, respectively. However, quite unexpectedly, the epoxidation activity of the mutant enzyme is significantly enhanced ∼2.5-fold. These results show that Glu-183 is important but not essential for the chlorination activity of chloroperoxidase. It is possible that the increased epoxidation of the mutant enzyme is based on an increase in the hydrophobicity of the active site.