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T Kieser

Publications and source records attributed to T Kieser.

At least 19 recordsLinked to original sources
PubMed2004

SCP1, a 356,023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2).

The sequencing of the entire genetic complement of Streptomyces coelicolor A3(2) has been completed with the determination of the 365,023 bp sequence of the linear plasmid SCP1. Remarkably, the functional distribution of SCP1 genes somewhat resembles that of the chromosome: predicted gene products/functions include ECF sigma factors, antibiotic biosynthesis, a gamma-butyrolactone signalling system, members of the actinomycete-specific Wbl class of regulatory proteins and 14 secreted proteins. Some of these genes are among the 18 that contain a TTA codon, making them targets for the developmentally important tRNA encoded by the bldA gene. RNA analysis and gene fusions showed that one of the TTA-containing genes is part of a large bldA-dependent operon, the gene products of which include three proteins isolated from the spore surface by detergent washing (SapC, D and E), and several probable metabolic enzymes. SCP1 shows much evidence of recombinational interactions with other replicons and transposable elements during its history. For example, it has two sets of partitioning genes (which may explain why an integrated copy of SCP1 partially suppressed the defective partitioning of a parAB-deleted chromosome during sporulation). SCP1 carries a cluster of probable transfer determinants and genes encoding likely DNA polymerase III subunits, but it lacks an obvious candidate gene for the terminal protein associated with its ends. This may be related to atypical features of its end sequences.

Amino Acid Sequence↗
PubMed2002

Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2).

Streptomyces coelicolor is a representative of the group of soil-dwelling, filamentous bacteria responsible for producing most natural antibiotics used in human and veterinary medicine. Here we report the 8,667,507 base pair linear chromosome of this organism, containing the largest number of genes so far discovered in a bacterium. The 7,825 predicted genes include more than 20 clusters coding for known or predicted secondary metabolites. The genome contains an unprecedented proportion of regulatory genes, predominantly those likely to be involved in responses to external stimuli and stresses, and many duplicated gene sets that may represent 'tissue-specific' isoforms operating in different phases of colonial development, a unique situation for a bacterium. An ancient synteny was revealed between the central 'core' of the chromosome and the whole chromosome of pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae. The genome sequence will greatly increase our understanding of microbial life in the soil as well as aiding the generation of new drug candidates by genetic engineering.

Bacterial Proteins↗
PubMed2000

The ram-dependence of Streptomyces lividans differentiation is bypassed by copper.

The onset of morphological differentiation in Streptomyces lividans is intrinsically delayed in comparison to Streptomyces coelicolor, but can be advanced by adding extra copper to the medium. Copper-specific chelators block aerial hyphae formation in both strains illustrating the crucial role of copper in morphogenesis. The S. coelicolor ram cluster was isolated as a clone that complements the copper-dependent differentiation of S. lividans. The S. lividans ram cluster was cloned and shown to be 99.6% identical to the S. coelicolor clone. The difference in development between S. lividans and S. coelicolor could neither be related to functional differences between the two ram clusters nor to differences in the transcription level. In both strains the low level of ramAB transcription correlated with aerial mycelium formation and was coupled to the upstream ORF ramS. An increased ramAB expression level in S. lividans by the introduction of an extra copy of ram stimulated the development. In S. lividans disruption of ramABR resulted in the inability to produce aerial hyphae. Conversely, the identical mutant of S. coelicolor retained its developmental capacities, indicating the presence of a ram-independent developmental route that is not present or not activated in S. lividans. Aerial hyphae and spore formation in the S. lividans ramABR mutant was restored when grown near wild-type strains, suggesting that the ram gene products are involved in transport of a factor essential for normal development. In addition, an elevated copper concentration in the medium also relieved the developmental block of these mutants. These findings suggest that higher copper concentrations render this ram-associated factor obsolete.

ATP-Binding Cassette Transporters↗
PubMed1999

[Over-expression of a polyketide synthase (PKS) module of a giant polyene antibiotic gene cluster in E. coli by double induction].

A 2,671 bp DNA carrying a type I PKS module with KS and AT domains from Streptomyces sp. FR-008 was cloned in-frame into the BamHI site immediately downstream of the PT7 promoter of the E. coli expression vector pET-15b, no considerable expression under IPTG induction was detected. The same PKS gene cloned downstream of the tandem PRPL promoters of pBV220 also yielded no over-expression under 42 degrees C induction. This gene was, however, over-expressed when it was cloned downstream of the tandem PRPT7 or PRPLPT7 promoters. In the case of the tandem PRPLPT7 promoters, the over-expression was dependent on the 42 degrees C plus IPTG double induction. While in the case of the tandem PRPT7 promoters, over-expression could be achieved when the gene was induced by IPTG or 42 degrees C individually or by IPTG and 42 degrees C double induction. Based on these experiences an expression vector pHZ330 containing the tandem PRPT7 promoters was constructed. In addition, the PKS protein expressed in E. coli was injected into rabbits to generate PKS-specific antibodies. Western blotting experiment indicated that these antibodies were PKS-specific which could be used either for the study of the PKS gene cluster or for the detection of the heterologous expression of Streptomyces sp. FR-008 PKS genes.

Animals↗
PubMed1998

Effect of dual-chamber pacing with automatic rate-drop sensing on recurrent neurally mediated syncope.

We tested the hypotheses that a dual-chamber pacemaker that paces when intrinsic rate drops abruptly would reduce the number of syncopal spells and improve the quality of life in patients with highly recurrent neurally mediated syncope. Twelve patients with highly frequent neurally mediated syncope and at least 1 syncopal spell after tilt testing received dual-chamber pacemakers with automatic rate-drop sensing. The pacemakers were implanted 17+/-26 months after tilt testing, and the patients then were followed for 12+/-2 months. We compared the time to the first recurrence of syncope, syncope frequency, and quality of life for the 2 periods between tilt testing and pacemaker implantation, and between implantation and last follow-up. Only 6 of 12 patients fainted after pacemaker insertion. The median time to syncope recurrence before and after pacing was 7 days and 5.3 months, respectively. The geometric mean frequency of faints before and after pacing was 5.0 spells/month (95% confidence interval 2.7 to 9.2) and 0.30 spells/month (95% confidence interval 0.2 to 0.4), p <0.001. After 6 months the mean perception of health on the 100-point EuroQol scale rose from 55 to 82 (p = 0.003), and the general health perception on the SF-36 scale rose from 51 to 72 (p = 0.005). Permanent dual-chamber pacing with automatic rate-drop sensing in patients with highly frequent syncope is associated with a marked reduction in the likelihood of syncope and a marked improvement in quality of life.

Adolescent↗
PubMed1994

Transposition of IS117, the 2.5 kb Streptomyces coelicolor A3(2) 'minicircle': roles of open reading frames and origin of tandem insertions.

IS117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of ORF1 of IS117, presumed to encode a transposase, abolished transposition. Deletion or mutation of ORF2 and ORF3, which overlap each other on opposite strands of IS117, caused a c. 20-fold reduction in integration frequency of the circular form of IS117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. ORF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.

Base Sequence↗
PubMed1994

Streptomyces lividans 66 contains a gene for phage resistance which is similar to the phage lambda ea59 endonuclease gene.

The DNA of wild-type Streptomyces lividans 66 is degraded during electrophoresis in buffers containing traces of ferrous iron. S. lividans ZX1, a mutant selected for resistance to DNA degradation, simultaneously became sensitive to phi HAU3, a wide-host-range temperate bacteriophage. A DNA fragment conferring phi HAU3 resistance was cloned; it contains a phage resistance gene whose deduced amino acid sequence is similar to the phage lambda Ea59 endonuclease. The S. lividans phi HAU3 resistance does not seem to be a classical restriction-modification system, because no host-modified phages able to propagate on the wild-type strain could be isolated. The cloned fragment did not make the host DNA prone to degradation during electrophoresis, indicating that the two phenotypes are controlled by different genes which were deleted together from the chromosome of ZX1.

Amino Acid Sequence↗
PubMed1994

Repeated polyketide synthase modules involved in the biosynthesis of a heptaene macrolide by Streptomyces sp. FR-008.

Genes for biosynthesis of a Streptomyces sp. FR-008 heptaene macrolide antibiotic with antifungal and mosquito larvicidal activity were cloned in Escherichia coli using heterologous DNA probes. The cloned genes were implicated in heptaene biosynthesis by gene replacement. The FR-008 antibiotic contains a 38-membered, polyketide-derived macrolide ring. Southern hybridization using probes encoding domains of the type I modular erythromycin polyketide synthase (PKS) showed that the Streptomyces sp. FR-008 PKS gene cluster contains repeated sequences spanning c. 105kb of contiguous DNA; assuming c. 5 kb for each PKS module, this is in striking agreement with the expectation for the 21-step condensation process required for synthesis of the FR-008 carbon chain. The methods developed for transformation and gene replacement in Streptomyces sp. FR-008 make it possible to genetically manipulate polyene macrolide production, and may later lead to the biosynthesis of novel polyene macrolides.

Anti-Bacterial Agents↗
PubMed1994

High-frequency homologous plasmid-plasmid recombination coupled with conjugation of plasmid SCP2* in Streptomyces.

Non-transmissible derivatives of the Streptomyces multi-copy plasmid plJ101 were mobilized, by cointegrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low-copy-number Streptomyces coelicolor A3(2) plasmid SCP2*. Efficient co-integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co-integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.

Conjugation, Genetic↗
PubMed1994

Development of a gene cloning system for Streptomyces hygroscopicus subsp. yingchengensis, a producer of three useful antifungal compounds, by elimination of three barriers to DNA transfer.

Streptomyces hygroscopicus 10-22 could not be transformed with any of the commonly used Streptomyces plasmid vectors and was resistant to plaque formation by the Streptomyces phages phi C31 and R4. Repeated selection resulted in the isolation of derivatives of S. hygroscopicus 10-22 that could be transformed with pIJ101- and pJV1-derived cloning vectors and of restriction-deficient derivatives that could accept DNA propagated in Streptomyces lividans 66. These new strains, which include three that still produce the original antibiotics, can be used as hosts for gene cloning. Insertion of nonreplicating vectors by homologous recombination and transposition of Tn4560 were demonstrated in S. hygroscopicus 10-22.

Antifungal Agents↗
PubMed1993

Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages.

Effective chemotherapy of tuberculosis requires rapid assessment of drug sensitivity because of the emergence of multidrug-resistant Mycobacterium tuberculosis. Drug susceptibility was assessed by a simple method based on the efficient production of photons by viable mycobacteria infected with specific reporter phages expressing the firefly luciferase gene. Light production was dependent on phage infection, expression of the luciferase gene, and the level of cellular adenosine triphosphate. Signals could be detected within minutes after infection of virulent M. tuberculosis with reporter phages. Culture of conventional strains with antituberculosis drugs, including isoniazid or rifampicin, resulted in extinction of light production. In contrast, light signals after luciferase reporter phage infection of drug-resistant strains continued to be produced. Luciferase reporter phages may help to reduce the time required for establishing antibiotic sensitivity of M. tuberculosis strains from weeks to days and to accelerate screening for new antituberculosis drugs.

Adenosine Triphosphate↗
PubMed1993

Triggered propagated contractions in human atrial trabeculae.

OBJECTIVE: Mechanical stretch and rapid release of locally damaged regions of rat cardiac trabeculae due to contraction of undamaged cells during the regular twitch have been shown to trigger local aftercontractions in the damaged region. These aftercontractions appear to propagate along the length of the trabeculae and to precede triggered arrhythmias. The aim of this study was to determine whether triggered propagated contractions can also be elicited in human cardiac tissue. METHODS: Trabeculae were dissected from a biopsy of the right atrial appendage which was obtained during cardiac surgery. They were mounted horizontally, superfused with a modified Krebs-Henseleit solution, and monitored with a video system. Force was measured with a silicon strain gauge; sarcomere length was measured with laser diffraction techniques. Triggered contractions were elicited in 10 trabeculae following trains of 15 stimuli at a rate of 2 Hz, separated by 15 s rest intervals, at 21 degrees C and a [Ca2+]o above 5.0 mM. RESULTS: Both video analysis and sarcomere length recordings showed that the contractions started at one end of the trabeculae and travelled from there as a localised contraction along the muscle. Increasing [Ca2+]o or the number of conditioning stimuli increased force of the stimulated twitches up to a maximum; further increase of [Ca2+]o or the number of stimuli was accompanied by a decrease of force. Force of the triggered propagated contractions increased monotonically with increasing [Ca2+]o and number of stimuli, while the interval between last stimulus and the peak of the force transient (or latency) decreased progressively, and propagation velocity increased monotonically. Propagation velocity was calculated from the interval between peak sarcomere shortening at two sites and the distance between these sites, as well as from the ratio between the length of the muscle and the duration of the force transient in the remainder of the trabeculae. With increasing temperature at constant [Ca2+]o twitch force increased while latency, force, and duration of the force transient decreased. At 37 degrees C, localised contractions travelling along the muscle could still be induced at sufficiently increased [Ca2+]o. For all interventions, propagation velocity varied from 0.8 to 3.9 mm.s-1. CONCLUSIONS: Contractions can be elicited in human atrial cardiac trabeculae. These contractions have the same basic characteristics as the triggered propagated contractions that have been described previously in rat ventricular trabeculae.

Arrhythmias, Cardiac↗
PubMed1993

Analysis of the transfer region of the Streptomyces plasmid SCP2.

pIJ903, a bifunctional derivative of the 31.4 kb low-copy-number, conjugative Streptomyces plasmid SCP2*, was mutagenized in Streptomyces lividans using Tn4560. Mutant plasmids differing in their transfer frequencies, chromosome mobilization abilities, pock formation, and complementation properties were isolated. The mutations defined five transfer-related genes, traA, traB, traC, traD and spd, clustered in a region of 9 kb. The deduced sequences of the putative TraA and TraB proteins showed no overall similarity to known protein sequences, but the phenotype of traA mutant plasmids and sequence motifs in the putative TraA protein suggested that it might be a DNA helicase.

Amino Acid Sequence↗
PubMed1992

A combined genetic and physical map of the Streptomyces coelicolor A3(2) chromosome.

The restriction enzymes AseI (ATTAAT), DraI (TTTAAA), and SspI (AATATT) cut the Streptomyces coelicolor A3(2) chromosome into 17, 8, and 25 fragments separable by pulsed-field gel electrophoresis (PFGE). The sums of their lengths indicated that the chromosome consists of about 8 Mb of DNA, some 75% more than that of Escherichia coli K-12. A physical map of the chromosome was constructed for AseI and DraI, using single and double digests, linking clones, cross-hybridization of restriction fragments, and locations of genetically mapped genes, insertion sequences, prophages, and the integrated SCP1 and SLP1 plasmids on the physical map. The physical map was aligned with the previously established genetic map, revealing that the two long opposite quadrants of the genetic map that are almost devoid of markers (the silent regions at 3 o'clock and 9 o'clock) are indeed physically long rather than being hot spots for genetic exchange. They must therefore contain long stretches of DNA different in function from the remainder of the genome. Consistent with this conclusion are the locations of significant deletions in both of the silent regions. Of these, a 40-kb deletion in the 9 o'clock region accompanied or followed integration of the SCP1 linear plasmid to produce the NF fertility state. PFGE analysis of Streptomyces lividans 66, a close relative of S. coelicolor A3(2), was hampered by the previously described susceptibility of its DNA to degradation during electrophoresis. However, ZX7, a mutant derivative of S. lividans lacking the DNA modification responsible for this degradation, yielded good PFGE preparations. Not more than 7 of the 17 S. coelicolor AseI fragments could be shared by the S. lividans strain.

Blotting, Southern↗
PubMed1990

Transposition of IS117 (the Streptomyces coelicolor A 3 (2) mini-circle) to and from a cloned target site and into secondary chromosomal sites.

IS117, previously known as the 2.6 kb mini-circle, is a transposable element found in Streptomyces coelicolor A 3(2). It integrates predominantly into one preferred site when introduced into the closely related Streptomyces lividans 66, which lacks IS117. This preferred integration site was deleted from the S. lividans chromosome by replacement with an erythromycin resistance gene delivered by a phi C31 phage vector. When IS117 was introduced into the resulting strain it integrated into many other sites, with some indication of site preference. By cloning a 200 bp fragment centred on the preferred integration site onto a low copy number, self-transmissible Streptomyces plasmid derived from SCP2* it was shown that this sequence is sufficient to define the preferred site: IS117 integrates efficiently into this sequence from its preferred site in the host chromosome and at a lower frequency from the plasmid into the preferred site on the S. lividans chromosome.

Base Sequence↗
PubMed1990

Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis.

Recent development of vectors and methodologies to introduce recombinant DNA into members of the genus Mycobacterium has provided new approaches for investigating these important bacteria. While most pathogenic mycobacteria are slow-growing, Mycobacterium smegmatis is a fast-growing, non-pathogenic species that has been used for many years as a host for mycobacteriophage propagation and, recently, as a host for the introduction of recombinant DNA. Its use as a cloning host for the analysis of mycobacterial genes has been limited by its inability to be efficiently transformed with plasmid vectors. This work describes the isolation and characterization of mutants of M. smegmatis that can be transformed, using electroporation, at efficiencies 10(4) to 10(5) times greater than those of the parent strain, yielding more than 10(5) transformants per microgram of plasmid DNA. The mutations conferring this efficient plasmid transformation (Ept) phenotype do not affect phage transfection or the integration of DNA into the M. smegmatis chromosome, but seem to be specific for plasmid transformation. Such Ept mutants have been used to characterize plasmid DNA sequences essential for replication of the Mycobacterium fortuitum plasmid pAL5000 in mycobacteria by permitting the transformation of a library of hybrid plasmid constructs. Efficient plasmid transformation of M. smegmatis will facilitate the analysis of mycobacterial gene function, expression and replication and thus aid in the development of BCG as a multivalent recombinant vaccine vector and in the genetic analysis of the virulence determinants of pathogenic mycobacteria.

DNA, Bacterial↗