Vol. 8 No 1, 1994

Published by the Society for Actinomycetes, Japan

ABSTRACTS OF PAPERS

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E. Takano and M. J. Bibb

Department of Genetics, John Innes Centre, Colney, Norwich, NR4 7UH, UK

Actinomycetologica, 8: 1-16, 1994

The correlation between ppGpp synthesis and the onset of antibiotic production in Streptomyces coelicolor A3(2) is reviewed. A3(2) produces at least four antibiotics. Two of these, actinorhodin and undecylprodigiosin (whose synthesis is determined by the act and red genes respectively), are pigmented compounds. Conditions were established that gave dispersed and rapid exponential growth in liquid culture. Nitrogen limitation resulted in a short transition phase, during which a peak of ppGpp was observed, followed by a stationary phase. Undecylprodigiosin production commenced in transition phase, while actinorhodin synthesis was detected 5-6 hr later. Whereas the level of act and red biosynthetic transcripts increased markedly during transition phase, the level of the primary bldA transcript, which encodes the only tRNA of S. coelicolor A3(2) that can efficiently translate the rare leucine codon UUA and which is required for both actinorhodin and undecylprodigiosin production, decreased during early exponential phase; this decrease corresponded to an increased signal for the 5' end of the mature tRNA, which then remained constant throughout growth. Nutritional shiftdown of exponentially growing cultures evoked immediate synthesis of ppGpp and a severe reduction in growth rate; transcription of act genes occurred shortly after, and was followed by actinorhodin production. However, there was no immediate stimulatory effect on red gene transcription, and undecylprodigiosin production did not occur. Addition of serine hydroxamate, a seryl tRNA synthetase inhibitor, to exponentially growing cultures led to ppGpp accumulation, but to lower levels than after nutritional shiftdown, and did not elicit antibiotic production. Thus only a partial correlation was observed between ppGpp synthesis and the transcription of antibiotic biosynthetic genes. S. coelicolor A3(2) DNA that hybridised to the Escherichia coli (p)ppGpp synthetase gene (relA) was cloned, but it did not encode a relA homologue, and polyclonal antibodies against RelA failed to reveal a homologue in S. coelicolor A3(2) cell extracts. A relA homologue of Serratia marcescens was isolated using the same approach, but failed to hybridise to S. coelicolor A3(2) DNA.

Authors' Abstract

PLASMID-MEDIATED GENE DISRUPTION IN STREPTOMYCES GRISEUS

N. Kudo, K. Ueda, H. Ikeda*, S. Omura*, T. Beppu and S. Horinouchi

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo , *School of

Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo

Actinomycetologica, 8: 16-20, 1994

An SCP2-derived plasmid, pKU206, was found to be unstable in Streptomyces griseus and used as a plasmid vector for disruption of the chromosomal amfC gene. The plasmid containing a mutated amfC gene, obtained by insertion of the neomycin resistance (aph) gene in its coding region, was successfully used for chromosomal gene disruption via homologous recombination in S. griseus. Cultivation of S. griseus harboring the recombinant plasmid in a drug-free medium at 28øC for 4 days resulted in the loss of the plasmid at a very high frequency. About one disruptant of 2x10^4 colonies formed on drug-free medium was obtained. The plasmid pKU206 system turned out to be useful for gene disruption and replacement in S. griseus .

Authors' Abstract

COSMID VECTOR FOR CLONING AND ANALYSIS OF STREPTOMYCES DNA

C.-H. Pang, M. Shiiyama, H. Ikeda, H. Tanaka and S. Omura*

School of Pharmaceutical Sciences, Kitasato University, *The Kitasato Institute, 5-9-1 Shirokane, Minato-Ku Tokyo 108

Actinomycetologica, 8: 21-25, 1994

We have constructed a new type of cosmid vector pKU402. Since it contains a long palindromic sequence, the construction of a genomic library was easier and all transductants contain only the recombinant molecule. The restriction enzyme analysis of large cloned DNA fragments was also performed more easily by using the vector and rare cutters. DraI and AseI were detected in Streptomyces DNA, while SwaI seems extremely rare. RNA probes, generated by transcription with T3 and T7 RNA polymerases, were used for mapping large DNA fragments. The restriction mapping was carried out using labelled oligonucleotide probes.

The new cosmid vector appears useful in the construction of genomic libraries, the establishment of gene linkage and the analysis of large gene clusters or of genomic regions in Streptomyces.

Abridged Authors' Abstract

Abstracts of the 9th and 10th SAJ Colloquia

9th SAJ Colloquium

Takeda Chem. Ind., Ltd., Osaka

Dec.3, 1993

CLONING AND SEQUENCE ANALYSIS OF THE GENE ENCODING A THERMOSTABLE CHITINASE FROM STREPTOMYCES THERMOVIOLACEUS OPC-520

H. Tsujibo, H. Endo, K. Minoura, KMiyamoto and Y.Inamori

Osaka University of Pharmaceutical Sciences

Actinomycetologica, 8: S5, 1994

A genomic library of S. thermoviolaceus OPC-520 was constructed in E.coli JM109 using SphI partially digested chromosomal DNA ligated into the SphI site of pUC18. Among the approximately 2000 transformants tested, only one clone (pST292) showed clear halo formation. The plasmid isolated from the clone contained a 4.2-kb SphI fragment. To determine the location of the chitinase gene (chi40) in the 4.2kb DNA insert, we prepared various subclones. The results of subcloning showed that the 2,4kb BamHI-KpnI fragment was the region necessary for chitinase activity. The nucleoside sequence of both strands of the BamHIKpnI fragment of pST292 was determined. An open reading frame (ORF) starts at base 233 and ends at base 1474, and is followed by palindromic sequence and repeated DNA sequences. The ORF of 1239-bp would encode a protein of 414 amino acids with an Mr of 43,838. The putative -10 and -35 regions, which showed relatively weak homology with the consensus sequence in E.coli, were detected upstream of the SD sequence. Interestingly, the nucleotide sequence of the chi40 promoter region, which contains an 11-bp direct repeated sequence and l0-bp inverted repeated sequence, is very similar to those of the chi63 and chi35 genes of S. plicatus (Delic, I. et al., Proc.Natl.Acad.Sci. USA, 89: 1885-1889, 1992). These authors suggested that chitinase regulation occurs at the level of transcription and that this regulation is mediated by sequences within the promoter region. The protein sequence of Chi40 showed striking homology (74%) with Chi63, but the type-III homology unit of fibronectin found in Chi63 does not exist in the amino acid sequence of Chi40. Chi40 is more stable than Chi63 despite the striking homology. Thus, Chi40 seems to be a good candidate for studying the relationship between structure and thermostability. Chi40 contains the two conserved regions common to microbial and plant chitinases. One of the regions an Asp and Glu present in the active site of Iysozyme. These two portions of Chi40 may therefore constitute the catalytic site of chitinases.

Authors' Abstract

FUNCTIONAL ANALYSIS OF STREPTOMYCES PLASMID pSN22

M. Kataoka, T. Seki and T. Yoshida

Intl. Center of Coop. Res. in Biotechnol., Fac. Engineering, Osaka University, Osaka

Actinomycetologica, 8: S5-6, 1994

A 11 kbp (10,922 bp) multi-copy plasmid, pSN22, was isolated from Streptomyces nigrifaciens SN22. pSN22 is self-transmissible (conjugative), maintained stably in S. lividans, and forms pocks in a wide range of Streptomyces strains. Mutational analyses showed that a fragment of pSN22 contained five genes involved in plasmid transfer and pockformation. traB was essential for plasmid transfer. traA was required for pock-formation, but not for plasmid transfer. Mutation of spdA or spdB decreased the pock size. The fifth gene, traR, could be deleted together with other genes to give non-transmissible plasmids, but plasmids with insertions/deletions only within traR became non-viable. The plasmid transfer of pSN22 promoted the co-factor of nontransmissible plasmids and enhanced the chromosome recombination between the host and recipient strains, suggesting that plasmid transfer accompanied cytoplasmic mixing.

traR contributed as a repressor to the lethal effect-causing gene like the kor gene in pIJ101. In uiuo promoter-probing experiments identified a 550 bp BgIII-SmaI DNA fragment with promoter activity in both orientations. The traR gene product repressed its own transcription and also the transcription of the traA-traB-spdB operon. Plasmids containing a functional traB gene could not "survive" without traR being present in the same cell either in cis or in trans, presumably because unregulated expression of traB is lethal to the host. Plasmids with a functional traA gene but without traR gave a low transformation efficiency and inhibited the growth of host cells.

DNA replication of pSN22 was also analyzed. The minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a trans-acting element functioning as replication protein and a cis-acting sequence (probably a replication origin). Southern hybridization showed that minimal replication plasmids accumulated much more specific single-stranded plasmid molecules than wild type pSN22. A 500 bp fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids. This 500 bp DNA sequence may be an origin of second strand synthesis. The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism .

The DNA sequence analysis showed 10 ORFs, nine of which agreed well with the biological functions. The deduced rep proteins of pSN22 and pIJ101 are very similar, suggesting that both are derived from a recent common ancestor. The transfer regions of the two plasmids are, however, very different, indicating that both regions are of separate origins.

Authors' Abstract

BIOLOGY OF A STREPTOMYCES INTEGRATING ELEMENT, pSAM2

M. Guerineau, J. Hagege, G. Sezonov, A. Friedmann and J.-L. Pernodet

Institut de Genetique et Microbiologie, Laboratoire de Biologie et Genetique Moleculaire France

Actinomycetologica, 8: S6-7, 1994

pSAM2 is an 11 kb-element originally isolated from Streptomyces ambofaciens, the producer of the macrolide antibiotic spiramycin. It can be maintained in two different forms: in the wild type strain, S. ambofaciens ATCC 23877 (strain B1), pSAM2 is found integrated in the chromosome. In a survivor of U.V. treatment, S. ambofaciens JI3212 (strain B3), pSAM2 is found simultaneously in the integrated state and as an

autonomous plasmid at about 10 copies per chromosome. The pSAM2 element can replicate, is self-transmissible, elicits the lethal zygosis reaction (pock formation) and mobilizes chromosomal markers. Both the free form of pSAM2 and the integrated sequences of strains B1 and B2 can be transferred very efficiently. pSAM also has a site-specific recombination system very similar to that of temperate phages. It consists of an attachment site, attP, an int gene, whose product promotes site-specific integration of pSAM by recombination with the chromosomal attachment site (attB), and a xis gene, whose product could be involved in excision of pSAM2.

We constructed integrative vectors derived from pSAM2 such as pTS33. It can replicate in E.coli and allows the stable integration of cloned DNA in Streptomyces. It has the same host range as pSAM. After introduction in Streptomyces , pTS33 integrates site-specifically, as pSAM from which it derived. Neither non-specific integration nor free form were detected by Southern hybridizations. Moreover DNA fragments indicating excision by a reversal of the integration event could not be detected even on overexposed films. In addition, clones containing these vectors could be propagated without any selective pressure and without any loss of the vector.

pSAM2 regions involved in transfer and pock formation have been localized on a functional map and sequenced.

pSAM2 replicates through a rolling circle replication mechanism (RCR), but unlike most ss DNA elements, it does not possess a unique region involved in replication, but two regions, one of which contains ori and the other does rep SA that encodes a protein, RepSA, presenting similarities to the replication initiator proteins (Rep) of elements that replicate by an RCR mechanism. repSA might be transcribed with the genes involved in integration and excision of pSAM2. In conclusion, pSAM2 has a complex organisation as it integrates as a temperate phage and transfer as replicative plasmids. As it has a very wide host range, it should be a very good tool for building integrative vectors and studying plasmid mediated transfer.

Authors' Abstract

PLANTS, ANIMALS AND THEIR CIRCUMSTANCE IN KENYA

E. LIigashide

Okayama University

Actinomycetologica, 8: S7-8, 1994

Kenya is located in the middle east of Africa under the equator and is about 1.5 times larger in area than Japan. It has a population of about 18.7 million whose 81% lived in the urban territories (as of 1989). This country was founded thirty years ago (1963) and the successive presidents were eager in education for development of the country. I had a chance to visit this country in 1993 by request from Japan International Cooperation Agency (JICA). I provided a lecture on Food Toxicology and a training course in applied microbiological experiments for two months at Jomo Kenyatta University, College of Agriculture and Technology (JKUCAT) that is located about 50 km from Nairobi, the capital. Because of its altitude of about 1800 m, Nairobi has a mild and warm climate (annual average temperature and precipitation are 18.3øC and 830 mm, respectively). One can enjoy many beautiful flowers and birds there throughout the year.

Kenya is called "the Kingdom of Wild Animals and Plants in the World". I

had an opportunity to visit Nairobi National Park, Masai Mara National Reserve, Lake Nakuru National Park and Mt. Kenya National Park for collecting soil samples as well as watching wild animals and plants. Nairobi National Park (117 km2 in width) is the first and smallest national park of the country. This park is so close to Nairobi that high buildings in the city can be seen from the center of the park. I enjoyed watching many buffaloes, gazelles, waterbucks there, and few others. It seems that a relatively small number of animals are currently living in the park. There is a monument which was built five years ago and stands for the prohibition of elephant poaching. It is said that many tusks which had been collected from poached elephants were burned down to ash on that spot.

Masai Mara National Reserve contains the largest number of wild animals in an area of 1,812 km2 which almost equals the area of Osaka Prefecture. The reserve is located about 300 km west from Nairobi and boasts of the largest population of wild lions. I watched many topis, buffaloes, impalas, gazelles, antelopes, western whitebeaded wildbeasts, waterbucks, zebras, giraffes, lions, warthogs, and elephants, but few cheetahs, hippopotamuses and black rhinoceroses. As to birds, there are many marabou storks and vultures, but few ostriches.

In and around Lake Nakuru National Park, there are baboons, savannah monkeys and buffaloes. In addition, a huge number of flamingos are there in the lake, a so-called soda lake which is surrounded by the white shore soil containing salt and is polluted with flamingos' faeces.

As to plants, I often saw high aroes and traveller's trees. In a vast plain, there are a large number of acacias which are also called table trees or umbrella trees. In addition, there inhabit beautifully flowered trees such as jacaranda, nandi flame, pachystach lutea, bottlebrush and flame trees.

I could collect many soil samples from a variety of uncommon places in Kenya and so hope to be successful in isolating many novel actinomycetes from those samples in the near future.

Author's Abstract

10th SA.J Colloquium

Kitasato Institute, Tokyo

Feb. 18, 1994

CONSTRUCTION OF GENE PHYLOGENY: THE FIRST STEP TOWARD THE PERSPECTIVE VIEW OF LIFE FROM EVOLUTION

N. Saitou

Lab. of Evolutionary Genetics, National Institute of Genetics

Actinomycetologica, 8: S8, 1994

How will the currently diversified areas of biology be integrated into one coherent view of life in the next century? I'm quite certain that "evolution" will be the key word for this unifying process. As Charles Darwin pointed out, evolution is "descent with modification", or self-replication of nucleotides with mutations in modern terms. This aspect is ubiquitously applicable to any gene of any organism including viruses. Therefore, I think it natural that construction of gene phylogeny is fundamental for description of evolution.

Gene phylogeny and species phylogeny (or phylogenetic tree of organisms in the ordinary sense) are different from each other in many aspects. Gene

phylogeny has clear-cut branching (nucleotide replicating) points, and can be clearly defined for a certain gene region with no recombination. Species phylogeny, in contrast, always becomes blurred, if we look at it in detail. This is because many genes and individuals are involved in speciation.

There are three levels in gene phylogeny; expected phylogeny, realized phylogeny, and estimated phylogeny. This complicates the estimation process of expected gene phylogeny from the real data. We should be careful about distinguishing these three levels of phylogeny.

Author's Abstract

SCREENING OF ANTIOXIDATIVE AGENTS OF MICROBIAL ORIGIN AND THEIR APPLICATION

K. Shin-ya

Inst.Molecular and Cellular Biosciences, Univ. Tokyo

Actinomycetologica, 8: S8-9, 1994

Free radicals including active oxygen are of highly reactive species. They attack cellular membranes, proteins and genes, and result to cause several pathological states such as atherosclerosis, inflammation and ischemia-reperfusion injury. In addition, senility and cancer initiation are also believed to be triggered by the active oxygen species. With the hope to obtain compounds which are effective to prevent such pathological states, we have screened antioxidants of microbial origin by employing rat liver microsomes as an assay system. As a result, we have succeeded in isolation of new metabolites, benthocyanins A, B and C, and benthophoenin, from Streptomyces prunicolor.

Another pathological state caused by oxygen-derived free radicals is the ischemia-reperfusion injury of the central nervous system (CNS) such as stroke. LGlutamate, which acts as exciting amino acid in a part of brain, has been known to induce delayed neuronal death following ischemic attack. Since the cerebral injury was reported to be prevented by free radical scavengers under some conditions, we have attempted to screen antioxidants which protect neuronal cells from the toxicity of L-glutamate, using neuronal hybridoma N18RE105 cells. As a result, we have isolated from Streptomyces exfoliatus a compound named carquinostatin A with a brain-protecting activity. In the evaluation system we employed, carquinostatin A completely inhibited the glutamate toxicity in N18-RE-105 cells at higher concentrations than 20.0nM. In addition, it showed an antioxidative activity in rat liver microsomes which was comparable to that of vitamin E. In the primarily cultured hippocampal neurone system, it also suppressed the glutamate toxicity.

Author's Abstract

NOVEL ANTIOXIDANTS FROM MICROORGANISMS AND THEIR PHARMACEUTICAL EFFECTS

S.Kato

Pharmaceutical Res.Lab., Kirin Brewery Co., Ltd.

Actinomycetologica, 8: S9, 1994

The last few years have seen an increasing amount of knowledge about the important role of free oxygen radicals in various diseases. The involvement of subsequent peroxidative disintegration of cell membranes has also been widely indicated in a variety of pathological processes. These pathological and clinical backgrounds have prompted us to investigate novel and potent antioxidant compounds from microorganisms which are ultimately of therapeutic use. In the program of microbial screening for radical scavenging antioxidants based on the inhibitory activity against lipid peroxidation in the rat brain homogenate, we have isolated several novel compounds such as carazostatin, pyrrolostatin and phenazoviridin. Further investigation has resulted in the finding that an antibacterial antibiotic, carbazomycin B, has the antioxidant activity. Both carazostatin and carbazomycin B, which contain a carbazole nucleus in their structures, showed much stronger antioxidant activity in vitro and ex vivo than aIpha-tocopherol. We also investigated the protective effect of these compounds on ischemic tissue damage in vivo. Carbazole compounds had no protective effect either on KCNinduced acute hypoxic death in mice or on delayed neuronal cell death induced by transient forebrain ischemia in rats. On the other hand, pyrrolostatin and phenazoviridin, which has weaker antioxidant activity than the said carbazoles, showed the protective effect on the hypoxic death. These results suggest that the novel antioxidants isolated in our study may be useful as a new class of therapeutic agents to alleviate the ischemic tissue injury. The antioxidant potency-in vivo effect relationship of these compounds, however, remains to be studied soon.

Author's Abstract

Copyright 1994 C. E. T A.