Increased yield of a lysozyme after self-cloning of the gene in Streptomyces coelicolor “Müller”

Summary Streptomyces coelicolor Müller DSM3030 excretes a lysozyme comprising both -1,4-N-acetyl-and -1,4-N,6-O-diacetyl muramidase activities. The lysozyme is named Cellosyl. Gene libraries have been established using genomic DNA from the wild-type strain, S. coelicolor DSM3030, and from an overproducing mutant, S. coelicolor HP1, which exhibits about a twofold increase in lysozome production. The lysozyme-encoding genes (cel) from both strains were detected by oligodeoxynucleotide hybridization. The nucleotide sequence of the cel genes isolated from both strains was shown to be identical. The different levels of lysozyme production could not be correlated with any mutations at the cel gene locus. The cel gene isolated from the wild-type strain could not be expressed in some other species of Streptomyces. However, self-cloning of the cel gene into S. coelicolor DSM3030 and HP1 resulted in a 2.5-fold increase in lysozyme production.     

Nucleotide sequence of the celA gene encoding a cellodextrinase of Ruminococcus flavefaciens FD-1

Summary The nucleotide sequence of a 3.6 kb DNA fragment containing a cellodextrinase gene (celA) fromRuminococcus flavefaciens FD-1 was determined. The gene was expressed from its own regulatory region inEscherichia coli and a putative consensus promoter sequence was identified upstream of a ribosome binding site and a TTG start codon. The complete amino acid sequence of the CeIA enzyme (352 residues) was deduced and showed no significant homology to cellulases from other oganisms. Two lysozymetype active sites were found in the amino-terminal third of the enzyme. InE. coli the cloned CeIA protein was translocated into the periplasm. The lack of a typical signal sequence, and the results of transposonphoA mutagenesis experiments indicated that CeIA is secreted by a mechanism other than a leader peptide.Abbreviations CMCase carboxymethylcellulase - celA gene coding for CeIA - CelA cellodextrinase - ORF open reading frame - phoA gene encoding alkaline phosphatase - pNPC p-nitrophenyl--d-cellobioside     

Role of lipids in theNeurospora crassa membrane. I. Influence of fatty acid composition on membrane lipid phase transitions

Summary The relationship between lipid composition and phase transition was investigated by differential scanning calorimetry for intact and membrane phospholipid extracts of wild-type (w/t) and thecel ^– (Tw 40) mutant ofNeurospora crassa. Thecel ^– (Tw 40) mutant (grown on minimal, sucrose medium supplemented with Tween 40 at 34 °C) had approximately twice the saturated fatty acid content ofw/t organisms grown at 22 °C. The gel-liquid crystal phase transitions of ergosterol-free extracts derived fromw/t andcel ^– (Tw 40) occur at –31 and –11 °C, respectively. The heats of transition (H) of these extracts were 1 and 13 cal/g, respectively. The addition of ergosterol (the predominant sterol inNeurospora) to the phospholipid extracts decreased the observed heats of transition, but did not alter the transition temperature. IntactNeurospora, whetherw/t orcel ^– (Tw 40) did not manifest similar gel-liquid crystal phase transitions in the differential scanning calorimeter. However, an endothermic peak at approximately 30 °C was observed in intact cells and extracted phospholipids of bothw/t andcel ^– (Tw 40) organisms. This peak was insensitive to the addition of ergosterol, had a low heat content (H1 cal/g), and was reversible.     

Role of lipids in theNeurospora crassa membrane: IV. Biochemical and electrophysiological changes caused by growth on phytanic acid

Summary Neurospora crassa straincel, which is deficient in fatty acid synthesis, was grown with phytanic acid supplementation. The temperature dependence of membrane potential is increased by growth on phytanic acid. A temperature change of 40°C produces a change of 184 mV in phytanic acid-grown cells as compared to a 50 mV change forcel grown on palmitic acid or wild-type. Membrane resistance (measured as DC input resistance) of phytanic acid-grown cells did not differ fromcel grown on palmitic acid or wild-type. Lipid analysis ofcel grown on phytanic acid revealed 7 mole percent phytanic acid incorporation into phospholipids, no change in phospholipid base composition, a reduction of ergosterol content from 80 to 30 percent, and the induction of sitosterol, a sterol not usually present inNeurospora. sitosterol accounted for 60 percent of the sterol present. Incorporation of 7 mole percent phytamic acid into phospholipids lowers the phase transition temperature by 5°C, and decreases the heat content of the phase transition (H) slightly. Results are discussed in relation to Refsum's disease, a human neurological disorder associated with high plasma levels of phytanic acid. It is proposed that high intracellular phytanic acid concentration induces novel sterol synthesis and that the incorporation of the novel sterol into the membrane is responsible for the increased temperature sensitivity of membrane potential. The excitable membrane deficits observed in patients with Refsum's disease may also be explained by such a mechanism.     

Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between adhered enzyme system and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the V_max without significant K _m variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.     

Cloning of a gene involved in cellulose biosynthesis in Acetobacter xylinum: Complementation of cellulose-negative mutants by the UDPG pyrophosphorylase structural gene

Summary Three cellulose-negative (Cel^-) mutants of Acetobacter xylinum strain ATCC 23768 were complemented by a cloned 2.8 kb DNA fragment from the wild type. Biochemical analysis of the mutants showed that they were deficient in the enzyme uridine 5-diphosphoglucose (UDPG) pyrophosphorylase. The analysis also showed that the mutants could synthesize (1-4)-glucan in vitro from UDPG, but not in vivo from glucose. This result was expected, since UDPG is known to be the precursor for cellulose synthesis in A. xylinum. In order to analyze the function of the cloned gene in more detail, its biological activity in Escherichia coli was studied. These experiments showed that the cloned fragment could be used to complement an E. coli mutant deficient in the structural gene for UDPG pyrophosphorylase. It is therefore clear that the cloned fragment must contain this gene from A. xylinum. This is to our knowledge the first example of the cloning of a gene with a known function in cellulose biosynthesis from any organism, and we suggest the gene be designated celA.     

Nucleotide sequence of an endo-β-1,4-glucanase gene fromBacillus subtilis CK-2

The gene encoding endo--1,4-glucanase inBacillus subtilis CK-2 was cloned intoEscherichia coli DH5, and the nucleotide sequence determined. The 1500 bp gene encodes a protein of 499 amino-acid residues with a calculated molecular mass of 55 261, and is equipped with a typicalB. subtilis signal peptide. Nucleotide sequence comparison revealed only 2 basepairs deviation between this gene and the endo--1,4-glucanase gene ofB. subtilis PAP115, and 93% to 95% homology was found between the amino acid sequences of these enzymes and otherB. subtilis endo--1,4-glucanases. Regions of similarity were also observed between the carboxy-terminal part of these enzymes and the part of theB. lautus PL236celA enzyme constituting the cellulose-binding domain.     

Growth enhancement of transgenic poplar plants by overexpression of Arabidopsis thaliana endo-1,4–β-glucanase (cel1)

Poplar (Populus tremula) plants which had been transformed with Arabidopsis thaliana cel1 cDNA and successfully over-expressed the gene, exhibited significant phenotypic alterations which included taller plants, larger leaves, increased stem diameter, wood volume index, dry weight and a higher percentage of cellulose and hemicellulose, compared to the wild-type plants. Transgenic A. thaliana plants over-expressing A. thaliana cel1 exhibited similar levels of cel1 mRNA in the elongation zone of the flowering stem and higher levels in mature leaves when compared with wild-type plants. CEL1 protein levels in the elongation zone of the flowering stem of transgenic plants were similar or slightly higher compared to that of the wild-type plants, whereas mature leaves of transgenic plants contained a higher level of CEL1. These data indicate that in elongating zone of Arabidopsis, CEL1 level is tightly regulated. In contrast to transgenic poplar over-expressing the A. thaliana cel1, no phenotypic difference was found between A. thaliana transgenic and wild-type plants.     

Vector-free cloning of a bacterial endo-1,4-β-glucanase in Lactobacillus plantarum and its effect on the acidifying activity in silage: Use of recombinant cellulolytic Lactobacillus plantarum as silage inoculant

In this research, the advantage of use of cellulolytic recombinant Lactobacillus plantarum as microbial inoculants for alfalfa silage fermentation was evaluated. To such purpose, two L. plantarum strains, one (L. plantarum Lp80) currently commercialised and the other (L. plantarum B41) suitable as silage microbial additive, were genetically modified by integration of celA gene, encoding an alkaline endo-1,4--glucanase from Bacillus sp., in the chromosome, by means of a vector-free cloning technique. The heterologous gene was cloned in two fashions: preceded by two promoters (AC1 modification) or in translational coupling with a partial upstream ORF (AC2 modification). Therefore two different genetically modified organisms (GMOs) per each wild-type (WT), producing 43–59 U/l cellulase in 16 h, were examined. Thirty-five micro-ensiling experiments were carried out by inoculating the WT or the derived GMOs. L. plantarum B41AC1 cellulolytic clone exhibited significantly increased acidification capacity in silage samples incubated at 37°C. No advantage of use was evident for the other GMOs.     

Circadian period lengths of lipid synthesis mutants (cel,chol-1) of Neurospora show defective temperature,but intact pH-compensation

The influence of extracellular pH on the circadian sporulation rhythm of Neurospora crassa has been investigated for the mutants chol-1 and cel. Both mutants have a defect in the lipid synthesis pathway and require either choline or palmitate, respectively, as supplements for normal growth. The chol-1 and cel mutants also show an impaired temperature-compensation when growing on minimal medium. We investigated the possible correlation between loss of temperature- and pH-compensation in cel and chol-1 similar to the correlation found earlier for the frq⁷ mutant. Our results show that the cel and the chol-1 mutants, although defective in temperature-compensation have an intact pH-compensation of their circadian rhythms. At present, the products of the frq-locus are the only components of the clock that affect the sporulation rhythm of Neurospora both through pH- and temperature-compensation.     

Mapping and regulation of the cel genes in Erwinia chrysanthemi

Summary Chromosomal mutations of the celZ and celY genes which encode two different endoglucanases in Erwinia chrysanthemi 3937 were obtained by a three-step procedure: (i) in Escherichia coli, insertions of lacZ fusion-forming mini-Mu bacteriophages in the cel genes cloned on plasmids and screening of cel-lac fusions, (ii) Mu-mediated transduction in E. chrysanthemi of the plasmids carrying the fusions, (iii) recombinational exchange between the plasmidic mutated and the wild-type chromosomal alleles. These mutations allowed mapping of celZ between ura and pan and celY between xyl and met on the linkage map of E. chrysanthemi. The -galactosidase activity of these strains indicated that celZ is expressed in the late exponential and stationary growth phases, while celY expression is almost undetectable.     

Identification of the C. coli dnaK (groPC756) gene product.

Summary The E. coli dnaK (groPC756) gene product is essential for bacteriophage DNA replication. Bacterial DNA segments carrying this gene have been cloned onto a bacteriophage vector. The product of the dnaK gene has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells. The dnaK gene codes for a polypeptide with an apparent molecular weight of 93,000-Mr. Transducing phages carrying amber mutations in the dnaK gene fail to induce the synthesis of the 93,000-Mr polypeptide chain upon infection of sup ⁺ bacteria, but do so upon infection of supF bacteria. E. coli carrying the dnaK756 mutation are, in addition, temperature sensitive for growth at 43° C. It is shown that the dnaK756 mutation results in an overproduction of the dnaK gene product at that temperature.     

Identification of the protein products of the rrnC, ilv, rho region of the Escherichia coli K-12 chromosome

Summary Two methods have been used to identify the protein products of the Escherichia coli K-12 ilv region at 84 min and the flanking rrnC (counterclockwise) and rho (clockwise) loci. First, a set of dilv specialized transducing phages, including some phages that carry rho and others that carry part of rrnC, was used to infect UV irradiated cells. The proteins produced by the infecting dilv phage were selectively labelled with radioactive amino acids and identified by SDS gel electrophoresis and autoradiography. Second, restriction enzyme fragments were cloned from the dilv phage into pBR322 and the plasmid specific gene products produced in maxicells were similarly identified by SDS gel electrophoresis and autoradiography. The proteins produced were correlated with specific genes and restriction enzyme fragments present in the dilv phage and the pBR322 derivatives. Several ilv gene products that have previously been refractory to protein purification attempts have been identified for the first time by this technique. The presence of mutations at the ilvO site is shown to activate the cryptic ilvG gene and to result in the production of a 62,000 dalton protein. A 15,000 dalton protein of unknown function is synthesized from a DNA segment between ilv and rrnC. The rho gene was cloned from dilv phage into pBR322 and shown to be dominant to a rho mutation on the host cromosome. The rho gene product and four additional proteins coded by genes near or between rho and ilv have been detected.     

Construction of the bifunctional enzyme cellulase-β-glucosidase from the hyperthermophilic bacterium <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

An artificial bifunctional enzyme, cellulase-β-glucosidase, was prepared by gene fusion from the hyperthermophilic bacterium Thermotoga maritima MSB8. The fusion protein exhibited both cellulase (Cel5C) and β-glucosidase (BglB) activity when the bglB gene was fused to downstream of cel5C, but not when cel5C was fused to downstream of bglB. The specific activity of the bifunctional enzyme was 70% lower than that of cellulase or β-glucosidase. The fusion enzyme was purified, and the MW was estimated as 114 kDa. The fusion enzyme displayed optimum cellulase activity at pH 8.0 and 70°C over 30 min, and optimal β-glucosidase activity at pH 7.0 and 80°C over 30 min.     

In vivo detection of regulatory factor binding sites of Arabidopsis thaliana Adh

In vivo footprinting experiments have been used to analyze the binding of trans-acting regulatory factors in the 5 flanking region upstream of the alcohol dehydrogenase (Adh) gene from Arabidopsis thaliana. Protein-DNA interactions were detected by dimethyl sulfate footprinting and genomic sequencing, using an A. thaliana cell suspension culture that constitutively expressed the Adh gene. Several distinct footprinting domains have been characterized, and the potential effects of the corresponding trans-acting factors have been inferred from a comparison with data from the maize alcohol dehydrogenase-1 (Adh1) gene. One binding site is similar in sequence to one of the anaerobic response elements (ARE) of the maize gene, which has also been shown to bind to a trans-acting factor. Several of the remaining binding sites apparently represent a class of elements sharing the sequence 5-GTGG-3 within their footprint.Comparisons with maize Adh1 in vivo protein interactions reveal that the elements of Adh promoter structure are highly conserved, but the relative and absolute positions of the elements are variable.     

Identification of the E. coli groNB(nusB) gene product

Summary The E. coli groNB(nusB) gene product has been previously shown to be necessary for bacteriophage N protein function. The product of the groNB gene has been identified on SDS polyacrylamide gels after infection of UV-irradiated E. coli cells with various groNB ⁺ transducing phage derivatives. It is a polypeptide with an apparent molecular weight of 14,000 daltons. Transducing phage carrying either a deletion or an amber mutation in the groNB gene fail to synthesize the 14,000-M_r polypeptide chain upon infection of a sup ⁺ host. However, am ⁺ revertants of the groNBam phage do induce the synthesis of the polypeptide.     

Sequence of the mouse Q4 class I gene and characterization of the gene product

The Q4 class I gene has been shown to participate in gene conversion events within the mouse major histocompatibility complex. Its complete genomic nucleotide sequence has been determined. The 5 half of Q4 resembles H-2 genes more strongly than other Q genes. Its 3 end, in contrast, is Q-like and contains a translational stop signal in exon 5 which predicts a polypeptide with an incomplete membrane spanning segment. The presence of two inverted B1 repeats suggests that part of the Q4 gene may be mobile within the genome. Gene transfer experiments have shown that the Q4 gene encodes a ²-microglobulin associated polypeptide of M_r 41 000. A similar protein was found in activated mouse spleen cells. The Q4 polypeptide was found to be secreted both by spleen cells and by transfected fibroblasts and was not detectable on the cell surface. Antibody binding and two-dimensional gel electrophoresis indicate that the Q4 molecule is identical to a mouse class I polypeptide, Qb-1, which has been previously described.     

Fusion of the lac genes to the promotor for the cytidine deaminase gene of Escherichia coli K-12

Summary Phage Mu has been inserted into the structural gene for cytidine deaminase (cdd). By the use of phage (lac, Mu) the promoter for the cdd gene has been fused to lacZ. In these strains lacZ expression is regulated by the cytR repressor protein and is therefore induced by cytidine. The fusion strains were used for the isolation of cddo mutants. Plaque forming phages carrying the different cdd-lacZ fusions were isolated. Studies of the cdd-Mu strains showed that the cdd gene is transcribed clockwise with respect to the Escherichia coli map.     

Posttranscriptional repression of the cel gene of the ColE7 operon by the RNA-binding protein CsrA of Escherichia coli

Carbon storage regulator (CsrA) is a eubacterial RNA-binding protein that acts as a global regulator of many functionally diverse chromosomal genes. Here, we reveal that CsrA represses expression from an extrachromosomal element of Escherichia coli, the lysis gene (cel) of the ColE7 operon (cea-cei-cel). This operon and colicin expression are activated upon SOS response. Disruption of csrA caused ∼5-fold increase of the lysis protein. Gel mobility shift assays established that both the single-stranded loop of the T1 stem–loop distal to cei, and the putative CsrA binding site overlapping the Shine–Dalgarno sequence (SD) of the cel gene are important for CsrA binding. Substitution mutations at SD relieved CsrA-dependent repression of the cel gene in vivo. Steady-state levels and half-life of the cel mRNA were not affected by CsrA, implying that regulation is mediated at the translational level. Levels of CsrB and CsrC sRNAs, which bind to and antagonize CsrA, were drastically reduced upon induction of the SOS response, while the CsrA protein itself remained unaffected. Thus, CsrA is a trans-acting modulator that downregulates the expression of lysis protein, which may confer a survival advantage on colicinogenic E. coli under environment stress conditions.