H. Schrempf - Academia.edu (original) (raw)

Papers by H. Schrempf

transport. MsiK assists in cellobiose and maltose The Streptomyces ATP-binding component

Journal of Bacteriology, 1978

A number of plasmids have been isolated as covalently closed circular DNAs from strains of Bacill... more A number of plasmids have been isolated as covalently closed circular DNAs from strains of Bacillus cereus and B. subtilis. From 12 out of 15 strains of B. cereus, plasmids could be isolated. Most of the B. cereus strains contained two or more plasmids. Their molecular weights ranged from 1.6 X 10(6) to 105 X 10(6). Bacteriocin production could be attributed to a 45 X 10(6)-dalton plasmid (pBC7) from B. cereus DSM 336, and tetracycline resistance to a 2.8 X 10(6) plasmid (pBC16) from B. cereus GP7. Two streptomycin-resistant strains of B. subtilis harbored plasmids of 5.2 X 10(6) and 9 X 10(6), respectively, which were, however, not correlated with the antibiotic resistance. The plasmid carrying resistance to tetracycline, pBC16, which was originally isolated from B. cereus, could be subsequently transformed in B. subtilis, where it is stably maintained.

Journal of Bacteriology, 1982

The sporulating wild-type strain of Streptomyces reticuli, which produces a melanin pigment and t... more The sporulating wild-type strain of Streptomyces reticuli, which produces a melanin pigment and the macrolide leucomycin, contains plasmid DNA of 48 to 49 megadaltons. Plasmidless variants had an altered secondary metabolism and a changed antibiotic resistance pattern. By using a new colony hybridization technique developed for streptomycetes, it could be shown that plasmidless variants could be transformed with the wild-type plasmid DNA, which, however, is quickly lost from regenerated mycelium. In contrast to the wild-type strain, the plasmidless variants contain amplified nucleotide sequences within the chromosomal DNA. The number and size of these sequences vary with the strain tested. Hybridization studies revealed that the reiterated sequences are neither amplified ribosomal nor plasmid genes, but are present in small concentrations within the wild-type chromosome. Some of them share extensive homologies with each other and are located at different positions within the chromos...

Applied and environmental microbiology, 1995

Various Streptomyces strains [Streptomyces lividans 66, Streptomyces vinaceus, and Streptomyces c... more Various Streptomyces strains [Streptomyces lividans 66, Streptomyces vinaceus, and Streptomyces coelicolor A3 (2)] acquired the ability to utilize crystalline cellulose (Avicel) after transformation with a multicopy vector containing the cel-1 gene from Streptomyces reticuli. The expression level in these hosts was two to three times lower than in S. reticuli, indicating the absence of positive regulatory elements. Like S. reticuli, they processed the Avicelase to its catalytic domain and to an enzymatically inactive part. The cel-1 gene with its original upstream region was not expressed within Escherichia coli. When cel-1 had been fused in phase with the lacZ gene, large quantities of the fusion protein were produced in E. coli. However, this protein was enzymatically inactive and proteolytically degraded to a series of truncated forms. As the cellulase (Avicelase) synthesized by S. reticuli is not cleaved by the E. coli proteases, its posttranslational modification is proposed. With Bacillus subtilis as host, the cel-1 gene was expressed neither under its own promoter nor under the control of a strong Bacillus promoter.

Applied and Environmental Microbiology, 1995

Usually plasmid DNA is introduced into Streptomyces strains by polyethylene glycol-mediated trans... more Usually plasmid DNA is introduced into Streptomyces strains by polyethylene glycol-mediated transformation of protoplasts. However, many Streptomyces strains are only poorly or not at all transformable via protoplasts. Therefore, we have optimized the parameters critical for the application of electrotransformation of plasmid DNA into Streptomyces species. The most critical parameters evaluated for electrotransformation of the model strain Streptomyces rimosus R6 were the pretreatment of mycelia, buffer composition, and electric field strength. The electrocompetent mycelia were prepared from 24-h-old cultures, treated mildly with lysozyme, resuspended in sucrose-glycerol-polyethylene glycol buffer, and stored in aliquots at -70 deg C. The electric field strength of 10 kV/cm at 400 (Omega) and a capacitance of 25 (mu)F was applied. The method is simple and rapid, yielding transformant colonies in 48 to 72 h. Efficiencies of 10(sup5) to 10(sup6) transformants per (mu)g of plasmid DNA ...

Applied and Environmental Microbiology, 1998

The Streptomyces strains CHR3 and CHR28, isolated from the Baltimore Inner Harbor, contained two ... more The Streptomyces strains CHR3 and CHR28, isolated from the Baltimore Inner Harbor, contained two and one, respectively, giant linear plasmids which carry terminally bound proteins. The plasmids pRJ3L (322 kb), from CHR3, and pRJ28 (330 kb), from CHR28, carry genes homologous to the previously characterized chromosomal Streptomyces lividans 66 operon encoding resistance against mercuric compounds. Both plasmids are transmissible (without any detectable rearrangement) to the chloramphenicol-resistant S. lividans TK24 strain lacking plasmids and carrying a chromosomal deletion of the mer operon. S. lividans TK24 conjugants harboring pRJ3L or pRJ28 exhibited profiles of mercury resistance to mercuric compounds similar to those of Streptomyces strains CHR3 and CHR28.

Molecular Genetics and Genomics, 2003

Streptomyces reticuli produces a myceliumassociated enzyme, CpeB, whose N-terminal and C-terminal... more Streptomyces reticuli produces a myceliumassociated enzyme, CpeB, whose N-terminal and C-terminal portions mediate heme-dependent catalaseperoxidase and heme-independent manganese-peroxidase activities, respectively. The regulator FurS governs transcription of the furS-cpeB operon. The thiol form of FurS contains one zinc ion per monomer and binds in this state to its cognate operator. Oxidation of SH groups within FurS induces the release of the zinc ion. Substitution of the codons for the amino acids cysteine 96, histidine 92 and 93, and tyrosine 59 in furS disrupts the in vivo repressor activity of FurS and results in enhanced synthesis of CpeB in corresponding S. lividans transformants. Biochemical and footprinting studies with FurS and its mutant derivatives revealed that the cysteine residues 96 and 99 are involved in reversible S-S bond formation, while cysteine 96 and the histidine residues 92 and 93 are required for zinc coordination, and tyrosine 59 is necessary for the binding of FurS to DNA. On the basis of these data, functional predictions can be made for the mycobacterial regulator FurA, a close homologue of FurS.

The EMBO Journal, 1995

We report the identification, functional expression, purification, reconstitution and electrophys... more We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of an up to now unique prokaryotic potassium ion channel (KcsA). It has a rectifying current-voltage relationship and displays subconductance states, the largest of which amounts to A _ 90 pS. The channel is blocked by Cs' ions and gating requires the presence of Mg2+ ions. The kcsA gene has been identified in the gram-positive soil bacterium Streptomyces lividans. It encodes a predicted 17.6 kDa protein with two potential membrane-spanning helices linked by a central domain which shares a high degree of similarity with the H5 segment conserved among eukaryotic ion channels. Multiple alignments of deduced amino acids suggest that the novel channel has the closest kinship to the S5, H5 and S6 regions of voltage-gated K+ channel families, mainly to the subfamily represented by the Shaker protein from Drosophila melanogaster. Moreover, KcsA is most distantly related to eukaryotic inwardly rectifying channels with two putative predicted transmembrane segments.

Journal of Bacteriology, 1995

pBL2 was identified genetically but not physically in Streptomyces lividans after its mating with... more pBL2 was identified genetically but not physically in Streptomyces lividans after its mating with S. bambergiensis. During conjugation, pBL2 was transferred at high frequency to S. lividans and S. coelicolor. pBL2.1 DNA isolated from S. coelicolor exconjugants as a circular plasmid was shown to derive from the genome of S. bambergiensis. S. lividans carrying pBL2 or pBL2.1 acquired a methyl-specific restriction (MsrA+) phenotype. The corresponding enzyme was partially purified and shown to resemble a class II endonuclease which cleaves Dam-methylated DNA preferentially.

Journal of Bacteriology, 1987

Streptomyces lividans 66 exhibits genetic instability, involving sequential loss of resistance to... more Streptomyces lividans 66 exhibits genetic instability, involving sequential loss of resistance to chloramphenicol (Cams) and subsequent mutation of argG. Associated with this instability is the amplification of a 5.7-kilobase (kb) amplified DNA sequence (ADS). We have characterized a second, independent pathway of genetic instability, involving sequential loss of resistance to tetracycline (Tets) followed by mutation in nitrogen assimilation (Ntr). We detected DNA amplification in many of these mutant strains, as well as other reiterations coresident with the 5.7-kb ADS in Cams Arg mutants. However, in contrast to the 5.7-kb ADS, none of the novel elements were observed to amplify at high frequency. The mutation of argG is due to a deletion, one endpoint of which is defined by the 5.7-kb ADS. This amplification derives from a structure, the tandemly duplicated amplifiable unit of DNA (AUD), present in the wild-type genome. We found that progenitor strains containing just a single-co...

Fermentation Products, 1981

ABSTRACT Extrachromosomal elements have been identified in several strains of Streptomycetes prod... more ABSTRACT Extrachromosomal elements have been identified in several strains of Streptomycetes producing different macrolide antibiotics. Streptomyces reticuli which produces melanin and the macrolide antibiotic leucomycin has been studied in most detail. This strain contains one large plasmid with a molecular weight of 48–49 Mdal. The strain is genetically unstable. Several different variants have been characterized which lack the ability to produce melanin and (or) aerial mycelium. The variants synthesize normal, no or reduced amounts of the macrolide. Most of the variants still contain plasmid DNA and dependinq on the variant tested, the plasmid DNA consists of a homoqeneous or a very heteroaeneous population of covalently closed circular (CCC) DNA molecules. Genetical and biochemical studies have revealed that genetic instability of this species results from chanqes within both the extrachromosomal and chromosomal DNA. KEYWORDS Function of plasmids; qenetic instability; deletions; amplifications; plasmid loss.

Chitin is the second most abundant renewable polysaccharide, as it is a component of the exoskele... more Chitin is the second most abundant renewable polysaccharide, as it is a component of the exoskeleton of many organisms and of the cell walls of numerous fungi. Most streptomycetes secrete a number of chitinases, hydrolyzing chitin to oligomers, chitobiose or N-acetylglucosamine which can be utilized as carbon or nitrogen source. The chitinases of several streptomycetes have been shown to have a modular arrangement comprising catalytic, substrate binding as well as linker domains. Moreover, during growth in the presence of chitin-containing substrates, many Streptomyces strains have been shown to secrete formerly unknown, small (about 200 aa) chitin binding proteins (CHBs) which lack enzymatic activity and specifically target and invade chitin. Several motifs, including the relative location and spacing of four tryptophan residues, are conserved in the investigated CHB types, CHB1 and CHB2. The affinity of CHB1 to crab shell chitin is two times higher than that of CHB2. Comparative studies of various generated mutant CHB1 proteins led to the conclusion that it is one of the exposed tryptophan residues that directly contributes to the interaction with chitin. On the basis of immunological, biochemical and physiological studies, it can be concluded that the CHBs act like a glue with which streptomycetes target chitin-containing samples or organisms. The ecological implications of these findings are discussed.

European Journal of Biochemistry, 1995

European Journal of Biochemistry, 1993

Streptomyces olivaceoviridis efficiently degrades chitin. Shotgun cloning of partially Sau3Acleav... more Streptomyces olivaceoviridis efficiently degrades chitin. Shotgun cloning of partially Sau3Acleaved DNA using the multicopy vector pIJ702 and Streptomyces lividans 66 as host resulted in the identification of the plasmid pCHI 01 which harbours an insert of 4.6 kb. In the presence of chitin as sole carbon source, transformants of S. Zividans 66 carrying pCHI 01 or its derivatives with smaller inserts overproduced an exochitinase which was purified to homogeneity. The chitininducible enzyme with an isoelectric point of 4.0 shows optimal activity at pH 7.3 and S°C , has an apparent molecular mass of 47 kDa and is competitively inhibited by the pseudosugar allosamidin. The enzyme was identified as an exochitinase since it generates exclusively chitobiose from chitotetraose, chitohexaose, and colloidal high-molecular mass chitin. Sequence analysis of a reading frame of 1794 base pairs and comparison of the deduced amino-acid sequence allowed the identification of the putative catalytic domain, one region with significant similarity to the type-I11 module of fibronectin and one domain of unknown function. Multiple sequence alignment and hydrophobiccluster analysis of 25 chitinolytic enzymes from bacteria, fungi and plants allowed the identification of their characteristic domains. The exochitinase from S. olivaceoviridis shares highest similarity with the chitinase D from Bacillus circulans. Chitin, a polymer of N-acetyl-D-glucosamine, is highly abundant in nature, since it is present in the exoskeleton of insects, in mollusca, coelenterata, nematodes, protozoa and the cell walls of certain fungi. Naturally occurring chitin varies in the length of its chains which are stabilized by hydrogen bridges to a highly ordered crystalline structure. It is frequently associated with proteins and may be stabilized by additional inorganic compounds. The natural annual production is judged to amount to 10' tons. Thus, apart from cellulose, it constitutes the second most abundant polysaccharide in nature (Muzarelli, 1977). Chitin can be hydrolyzed by enzymes produced by plants, fungi and bacteria. Mainly a few species of the bacterial genera Aeromonas (

European Journal of Biochemistry, 2000

Streptomyces reticuli produces a heme-containing homodimeric enzyme (160 kDa), the catalase-perox... more Streptomyces reticuli produces a heme-containing homodimeric enzyme (160 kDa), the catalase-peroxidase CpeB, which is processed to the enzyme CpeC during prolonged growth. CpeC contains four subunits of 60 kDa each that do not include the C-terminal portion of the progenitor subunits. A genetically engineered cpeB gene encodes a truncated subunit lacking 195 of the C-terminal amino acids; four of these subunits assemble to form the enzyme CpeD. Heme binds most strongly in CpeB, least in CpeD. The catalaseperoxidase CpeB and its apo-form (obtained after extraction of heme) catalyze the peroxidation of Mn(II) to Mn(III), independent of the presence or absence of the heme inhibitor KCN. CpeC and CpeD, in contrast, do not exhibit manganese-peroxidase activity. The data show for the first time that a bacterial catalase-peroxidase has a heme-independent manganeseperoxidase activity, which depends on the presence of the C-terminal domain.

Microbiology, 1997

Resistance to fusidic acid in Streptomyces lividans is due to secretion of an extracellular enzym... more Resistance to fusidic acid in Streptomyces lividans is due to secretion of an extracellular enzyme (FusH) that converts the steroid antibiotic into an inactive derivative. NH2-terminal and several internal amino acid sequences were prepared from the purified enzyme. Using one of the deduced oligonucleotides to probe a subgenomic DNA library, the fusH gene was cloned and sequenced. Sequence analysis located an ORF which, owing to the presence of two putative start codons, indicates a predicted protein with 520 or 509 amino acids. A signal peptide was identified by aligning the deduced amino acids with the N-terminal sequence determined for the mature enzyme. The C-terminal part of the deduced FusH contains a region of three tandemly repeated stretches of 50 amino acids, which is preceded and followed by amino acids showing high homology with the repeats. FusH was found to share a GDS motif with some deduced esterases. S. lividans transformants carrying fusH on a multicopy vector synt...

Microbiological Research, 2013

Candida guilliermondii is an ascomycetous yeast widely studied due to its clinical importance, bi... more Candida guilliermondii is an ascomycetous yeast widely studied due to its clinical importance, biotechnological interest, and biological control potential. During a series of preliminary experiments aiming at optimizing the electroporation procedure of C. guilliermondii cells, we observed that the efficiency of transformation of an ura5 recipient strain with the corresponding dominant marker URA5 was more than a thousand fold higher as compared with the transformation of an ura3 strain with the URA3 wild type allele. This result allowed the identification of an autonomously replicating sequence (ARS) within an A/T rich region located upstream of the URA5 open reading frame (ORF). Interestingly, linear double strand DNAs (dsDNAs) containing this putative ARS are circularized and then autonomously replicated in C. guilliermondii transformed cells. We demonstrated that the C. guilliermondii Lig4p ligase, involved in the canonical non-homologous end-joining (NHEJ) pathway, was responsible for this phenomenon since a lig4 mutant was unable to circularize and to autonomously maintain transforming dsDNAs containing the putative ARS. Finally, a functional dissection of the C. guilliermondii A/T rich region located upstream of the URA5 ORF revealed the presence of a 60 bp-length sequence essential and sufficient to confer ARS properties to shuttle plasmid and linear dsDNAs.