Department of Human Genetics, University Medical School of Debrecen, H-4012 Debrecen Nagyerdei körút 98, Hungary1
Department of Medicinal Chemistry, Albert Szent-Györgyi Medical University, H-6720 Szeged Dóm tér 8, Hungary2
Department of Biochemistry, Leiden University, Gorlaeus Laboratory, PO Box 9502, 2300 RA Leiden, The Netherlands3
Author for correspondence: Sándor Biró. Tel: +36 52 416 531. Fax: +36 52 416 531. e-mail: sbiro{at}jaguar.dote.hu
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ABSTRACT |
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Keywords: extracellular signalling, cell differentiation, sporulation, autoregulator, Streptomyces
Abbreviations: DIG, digoxigenin; ESI, electrospray ionization
This paper is dedicated to the memory of Professor Gábor Szabó.
The GenBank accession number for the sequence reported in this paper is AF103943.
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INTRODUCTION |
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Studies on morphological differentiation and its intimate connection to secondary metabolism in Streptomyces coelicolor, the prototype and most extensively studied Streptomyces strain, have recently been reviewed by Chater (1998 ). In this strain, continuously increasing collections of developmental mutants and the corresponding cloned genes are available.
In Streptomyces griseus the loss of aerial mycelium formation, streptomycin production and ability to produce the low-molecular-mass -butyrolactone A-factor are frequently associated. Aerial mycelium formation and antibiotic synthesis can be restored by the addition of A-factor at nanomolar concentrations (Khokhlov, 1991
). Studies of the genetics of A-factor biosynthesis have shown that A-factor-dependent initiation of sporulation involves phosphorylation of two regulatory proteins by cognate membrane-bound protein kinases (Horinouchi, 1996
) and have also led to an understanding of the regulatory cascades involved in antibiotic biosynthesis (Horinouchi & Beppu, 1992
).
Factor C was detected and later isolated as a protein from the culture fluid of S. griseus 45H (Szabó et al., 1962 ), a strain that readily sporulates in liquid medium (Szabó et al., 1961
). Factor C induced the formation of preconidia in liquid cultures of the susceptible S. griseus strain 52-1 (Vitális & Szabó, 1969
), at concentrations as low as 0·51 ng ml-1 (Szeszák et al., 1991
). This strain is otherwise blocked in submerged sporulation (Szabó et al., 1961). Factor C also stimulated sporulation on solid medium (Szabó et al., 1995
). The pattern of proteins present in S. griseus 52-1 mycelium showed a specific change after the administration of factor C (Vitális et al., 1988
). The majority or all of the factor-C-like antigen was released into the fermentation liquid of Streptomyces cultures (Szeszák et al., 1990
, 1991
).
Factor C has been purified to electrophoretic homogeneity by phosphocellulose and DNA-agarose chromatography. Its molecular mass was estimated to be about 34 kDa (Biró et al., 1980 ), and its isoelectric point is around 9·9 (Szeszák et al., 1991
). By using polyclonal and monoclonal antibodies raised against factor C in conjunction with ELISA and immunoblotting techniques, the factor-C-like antigen was detected in 23 Streptomyces strains, in Bacillus subtilis, Escherichia coli and an archaeon, as well as in mammalian cells. On this evidence, factor C was assumed to be evolutionarily conserved.
Preliminary experiments aimed at examining the possible mechanism of action of factor C detected an effect on transcription (Szeszák & Szabó, 1973 , Szabó et al., 1984
), on the release of potassium (Szeszák et al., 1989
) and on NAD glycohydrolase activity in washed mycelia (Szabó et al., 1988
). Factor C was also shown to bind to single-stranded and double-stranded heterologous DNA (Szabó et al., 1984
).
In the present paper the cloning, sequence analysis and expression of the cloned factor C gene, facC, are described.
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METHODS |
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N-terminal amino acid sequencing.
Factor C protein was blotted electrophoretically to PVDF membrane and sequenced by the Edman degradation method.
Molecular cloning and DNA manipulations.
Purification of Streptomyces chromosomal DNA and transformation of S. griseus 52-1 protoplasts were carried out by standard methods (Hopwood et al., 1985 ). Plasmid DNA was isolated from E. coli strains using the techniques described by Sambrook et al. (1989
), except if required for DNA sequencing, when Wizard miniprep columns (Promega) were used. Restriction enzymes were used according to the instructions of the suppliers. To construct the mini gene-library of S. griseus 45H, chromosomal DNA was digested with SacII. The DNA fragments were separated electrophoretically on a 1% agarose gel in TAE buffer and transferred to a Hybond-N membrane (Amersham). The membrane was hybridized with the 5'-digoxigenin (DIG)-labelled 39-mer oligonucleotide (TIB MOLBIOL Syntheselabor; Table 1
) in 5x SSC, 2% (w/v) blocking reagent, 0·1% (w/v) N-lauroylsarcosine, 0·02% (w/v) SDS, and 50% (v/v) formamide at 42 °C, overnight. At highest stringency the membrane was washed in 0·5x SSC, 0·1% (w/v) SDS at 55 °C for 30 min. Hybridizing bands were detected by immunostaining using a DIG nucleic acid detection Kit (Boehringer Mannheim). On the basis of the hybridization signal, the SacII fragments in the 2·8 kb region from a parallel gel were purified using a DNA recovery purification kit (Hybaid). The purified DNA fragments were cloned in SacII-digested pBluescript II KS+ (Stratagene) and the recombinant vector was used to transform E. coli XL-1 Blue. Ampicillin-resistant white colonies were screened by colony hybridization with the 39-mer DIG-labelled probe as described above.
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DNA sequencing.
Plasmids were sequenced either by the dideoxy chain-termination method (Sanger et al., 1977 ) using the T7 Sequencing Kit (Pharmacia Biotech) and [
-35S]dCTP
S as the labelled nucleotide, or with an ABI 373 automated sequencer (Applied Biosystems) using a dye terminator cycle sequencing kit with Amplitaq DNA polymerase FS (Applied Biosystems). In all sequencing reactions, double-stranded plasmids were used with universal or specific oligonucleotides (Table 1
) as primers. Specific oligonucleotide primers were synthesized by Pharmacia Biotech. All sequence information reported was obtained from the independent analysis of both strands.
Computer analysis of nucleotide and amino acid sequence.
Sequence data were analysed with the pc/gene nucleic acid and protein sequence analysis software system (IntelliGenetics). For coding region analysis, FramePlot Version 2.2.1 (based on Bibb et al., 1984 , developed by Ishikawa Jun) was also used. Amino acid sequence homology searches made use of the blast network service and non-redundant protein sequence databases (Altschul et al., 1997
).
Molecular mass and sequence determination of tryptic fragments.
Gel bands containing about 13 µg protein from SDS-PAGE were cut into 1 mm2 pieces and soaked in 400 µl 100 mM NH4HCO3 (pH 8) with stirring for 10 min. The supernatant was discarded and the gel slices were dehydrated in 800 µl acetonitrile. The protein within the gel slices was reduced with 300 µl 10 mM dithiothreitol at 56 °C for 1 h and repeatedly washed with NH4HCO3 and acetonitrile. For alkylation, 300 µl 55 mM iodoacetamide was added at room temperature for 45 min. The washing procedure was repeated three times and the gel slices were dried under vacuum. For tryptic digestion (18 h at 37 °C) the ratio of factor C to trypsin (w/w) was 10:1. The peptide fragments were then washed with 400 µl NH4HCO3 solution and extracted with a 1:1 (v/v) acetonitrile/water mixture containing 5% (v/v) formic acid, concentrated in a Speedvac Evaporator, and dissolved for HPLC-MS analysis in 1% (v/v) acetic acid. The peptide fragments were separated using a 0·32x200 mm fused silica capillary column (Novotny, 1988 ) packed with Vydac C18. For mass spectrometric measurements a Finnigan TSQ 7000 tandem mass spectrometer equipped with a microelectrospray ion source (Kele et al., 1998
) was used. The amino acid sequence of the most intense peptide fragments was determined by HPLC-MS/MS measurements. Molecular masses obtained in HPLC-MS experiments were compared with theoretical tryptic digestion profiles of candidate proteins generated by a computer program (Mann et al., 1993
).
Southern hybridization.
Chromosomal DNA samples from 12 Streptomyces strains were digested to completion with SacII. The DNA fragments were size-fractionated by gel electrophoresis on a 1% (w/v) TAE agarose gel and transferred to a Hybond-N (Amersham) membrane using the manufacturers instructions. Hybridization was carried out at 42 °C in a mixture containing 50% (v/v) formamide, 2x SSC, 5x Denhardts solution, 0·2 mg denatured salmon sperm DNA ml-1 and 0·1% (w/v) SDS. The highest stringency washing was in 0·1x SSC, 0·1% (w/v) SDS at 65 °C for 30 min. The 860 bp EcoRVSalI fragment (nucleotide positions 9301790 in Fig. 1), was used as the probe. The DNA was radiolabelled by random priming with nonamers and the Klenow fragment of E. coli DNA polymerase I, using a Megaprime Kit from Amersham to incorporate [
-32P]dCTP.
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RESULTS AND DISCUSSION |
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Cloning of facC
Instead of screening a complete chromosomal gene library, we used a mini-genebank, which was constructed in pBluescript II KS+ by cloning the gel-purified SacII fragments of about 2·8 kb in size, corresponding to the signal obtained by Southern hybridization. The 39-mer oligonucleotide flanked by the two primers in the 76 bp amplified fragment was used as probe. Colony hybridization identified one clone (pBZ3) with a 2·9 kb insert that repeatedly gave a positive signal in high stringency hybridizations. The DNA sequence of this clone was determined.
Sequence analysis of pBZ3
Sequencing was started from primers complementary to the T3 and T7 promoters flanking the cloned DNA fragment in the vector. The 39-mer DIG-labelled oligonucleotide served as an internal primer. Gaps were filled by directed sequencing from walking primers. Both strands of the DNA were sequenced at least twice. FramePlot and coding region analysis by pc/gene programs identified a 975 bp ORF, typical of Streptomyces, with 96·9% GC in the third letter position and an overall G+C content of 70·7 mol% (Fig. 2). The coding sequence starts with a GTG codon at nt 1007 and extends to the TGA stop codon at nt 1978 (Fig. 1
). It encodes a protein of 324 amino acids with a predicted molecular mass of 34523 Da. Computer analysis of this protein located a sequence Ser-Ala-Ala-Ala/Ala/Val-Pro-Ala that contains two potential secretory signal cleavage sites at the positions indicated by slashes. Since the known N-terminal sequence began with the sequence Ala-Val-Pro it follows that the factor C propeptide contains a 38 amino acid secretion signal conforming to the consensus for prokaryotic signal sequences. It has the usual three domains: a positively charged N domain, a hydrophobic H domain required to initiate translocation across the cytoplasmic membrane, and a C domain preceding the cleavage site (Pugsley, 1993
). The signal peptidase cleavage site also conforms to the -3, -1 rules (von Heijne, 1986
). Since factor C was identified and isolated from the culture fluid of S. griseus 45H, and most or all of the factor-C-like antigen was found in the fermentation liquid, secretion of the protein was predictable. The mature protein contained 286 amino acids and its molecular mass was calculated to be 31038 Da, in good agreement with the 34500 Da estimated by SDS-PAGE (Biró et al., 1980
), especially if we consider that factor C is a strongly basic protein. Its calculated isoelectric point was 9·59, which is close to our previous estimate of 9·9 (Szeszák et al., 1991
).
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Molecular mass determination of factor C and amino acid sequencing of its tryptic fragments
Only a small amount of factor C is produced by S. griseus 45H. Typically a few micrograms of purified protein can be obtained from 10 litres of culture broth. Therefore we used capillary chromatography/microelectrospray mass spectrometry, a method that required only minute amounts of protein for molecular mass determination of factor C and sequence verification of tryptic fragments. The molecular mass obtained for the mature factor C was 31045±9 Da, very close to the value of 31038 Da calculated from the sequence.
After tryptic digestion the most intense peptide peaks were selected for sequence analysis in the HPLC-MS/MS measurements. The peptides identified by MS/MS are underlined in Fig. 1. Although the identified peptide fragments do not cover the whole sequence, the fragments are well distributed over the N-terminal, middle and C-terminal regions. Therefore, we concluded that the ORF identified by FramePlot analysis and the deduced amino acid sequence of the factor C protein are correct. One possible explanation for our previous, firmly established finding that factor C binds very specifically to zinc affinity columns, is that binding occurs in association with another protein. To study this possibility and detect any other protein that eluted with, or later than, factor C, we are examining proteins in S. griseus 45H cultures.
Factor C shows no significant homology to other proteins in the databases
Comparison of the amino acid sequence of the mature factor C protein with proteins in the databases using blast Search (Altschul et al., 1997 ) showed a low level of similarity to teichoic acid biosynthesis protein C (tagC) of B. subtilis 168 (29% amino acid identity with 6 gaps). This seemed at first to be an interesting homology, because teichoic acid is a cell wall component of Gram-positive bacteria and expression of the tag genes is sporulation specific (Mauël et al., 1991
, 1994
). However, recent reports have shown that tagC corresponds to dinC (Cheo et al., 1991
, 1993
), and thus belongs to the SOS regulon. Therefore, the relevance of the low similarity between factor C and the TagC protein, if any, is unclear.
facC is present in several Streptomyces strains that sporulate in submerged culture
To test for the presence of the factor C gene in other Streptomyces strains, Southern blots of chromosomal digests of 12 strains from 10 species were hybridized with a 32P-labelled DNA fragment spanning nt positions 9301790 (Fig. 1), covering approximately 75 bp of upstream region (Zs. Birkó and others, unpublished results) and about 80% of the coding region. As expected, a strong hybridization signal was observed in the lane with DNA from S. griseus 45H (Fig. 3.
) The lanes containing DNA from S. albus R-55, S. flavofungini and S. albus 391 each gave a strong signal, suggesting the presence of a gene with high homology to facC. No comparable band was observed with the DNA from any other strains tested, including our factor-C-sensitive test strain S. griseus 52-1. This contrasts with our previous results obtained using monoclonal antibody raised against factor C, which showed the presence of a factor-C-like antigen in all tested Streptomyces strains, including some of those that failed to hybridize in the present experiment (Szeszák et al., 1990
). This might be explained by the high stringency of washing. In other experiments when the DIG-labelled 39-mer oligonucleotide (Table 1
) was hybridized with chromosomal digests of the same strains, and a lower stringency washing was used, we could detect a hybridizing band or bands in all tested strains (S. Biró, unpublished results). The meaning and significance of these results await further clarification but our findings are reminiscent of those of Onaka et al. (1998
), who found two low homologues of the S. griseus A-factor receptor protein, which has a regulatory role in secondary metabolism and morphogenesis, in S. coelicolor. The four Streptomyces species shown to harbour a high homologue of facC are known to sporulate in submerged culture (Kendrick & Ensign, 1983
; Vitális et al., 1981
; Daza et al., 1989
; S. Biró, unpublished results). This points to a possible involvement of factor C in the onset of submerged sporulation.
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Expression of facC in S. griseus 52-1
To study the phenotypic effect of the cloned gene, an E. coliStreptomyces SCP2*-based shuttle vector containing the entire structural gene for factor C and approximately 560 bp of upstream region was constructed; it was designated pSGF4. It is present at approximately 10 copies per chromosome in Streptomyces. Preliminary transcriptional analysis revealed that the 560 bp upstream region contains the facC promoter (Zs. Birkó and others, unpublished results), allowing expression of facC from its own promoter. In submerged culture, under our standard test conditions, S. griseus 52-1 forms long, smooth, non-branching vegetative hyphae. It fails to sporulate and lacks the reproductive, frequently branching hyphae containing club-like thickening at their ends (Fig. 4b), characteristic of the factor C producer S. griseus 45H (Fig. 4a
; Vitális et al., 1963
). Addition of purified factor C to submerged cultures of S. griseus 52-1 resulted in a morphology very similar to that of S. griseus 45H (Fig. 4c
). Introducing pSGF4 into S. griseus 52-1 resulted in a phenotype (Fig. 4d
) almost identical to that of the facC+ strain S. griseus 45H. The transformant showed this typical cytomorphology irrespective whether it was grown in the presence or absence of thiostrepton.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bibb, M. J., Findlay, P. R. & Johnson, M. W. (1984). The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein coding sequences. Gene 30, 157-166.[Medline]
Biró, S., Békési, I., Vitális, S. & Szabó, G. (1980). A substance affecting differentiation in Streptomyces griseus. Eur J Biochem 103, 359-363.[Abstract]
Chater, K. F. (1998). Taking a genetic scalpel to the Streptomyces colony. Microbiology 144, 1465-1478.
Cheo, D. L., Bayles, K. W. & Yasbin, R. E. (1991). Cloning and characterization of DNA damage-inducible promoter regions from Bacillus subtilis. J Bacteriol 173, 1696-1703.[Medline]
Cheo, D. L., Bayles, K. W. & Yasbin, R. E. (1993). Elucidation of regulatory elements that control damage induction and competence induction of the Bacillus subtilis SOS system. J Bacteriol 175, 5907-5915.[Abstract]
Daza, A., Martin, J. F., Dominguez, A. & Gil, J. A. (1989). Sporulation of several species of Streptomyces in submerged culture after a nutritional downshift. J Gen Microbiol 135, 2483-2491.[Medline]
von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14, 4683-4690.[Abstract]
Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton, C. J., Lydiate, D. J., Smith, C. P., Ward, J. M. & Schrempf, H. (1985). Genetic Manipulation of Streptomyces. A Laboratory Manual. Norwich: John Innes Foundation.
Horinouchi, S. (1996). Streptomyces genes involved in aerial mycelium formation. FEMS Microbiol Lett 141, 1-9.
Horinouchi, S. & Beppu, T. (1992). Autoregulatory factors and communication in Actinomycetes. Annu Rev Microbiol 46, 377-398.[Medline]
Janssen, G. R. & Bibb, M. J. (1993). Derivatives of pUC18 that have BglII sites flanking a modified multiple cloning site and that retain the ability to identify recombinant clones by visual screening of Escherichia coli colonies. Gene 124, 133-134.[Medline]
Kawamoto, S. & Ensign, J. C. (1995). Cloning and characterization of a gene involved in regulation of sporulation and cell division in Streptomyces griseus. Actinomycetes 9, 136-151.
Kawamoto, S., Watanabe, H., Hesketh, A., Ensign, J. C. & Ochi, K. (1997). Expression of the ssgA gene product, associated with sporulation and cell division in Streptomyces griseus. Microbiology 143, 1077-1086.[Abstract]
Kele, Z., Janáky, T., Mészáros, T., Fehér, A., Dudits, D. & Szabó, P. T. (1998). Capillary chromatography/microelectrospray mass spectrometry used for the identification of putative cyclin-dependent kinase inhibitory protein in Medicago. Rapid Commun Mass Spectrom 12, 1564-1568.[Medline]
Kendrick, K. E. & Ensign, J. C. (1983). Sporulation of Streptomyces griseus in submerged culture. J Bacteriol 155, 357-366.[Medline]
Khokhlov, A. S. (1991). Microbial Autoregulators. Chur: Harwood Academic Publishers.
Larson, J. L. & Herschberger, C. L. (1986). The minimal replicon of a streptomycete plasmid produces an ultrahigh level of plasmid DNA. Plasmid 15, 199-209.[Medline]
Lydiate, D. J., Malpartida, F. & Hopwood, D. A. (1985). The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors. Gene 35, 223-235.[Medline]
Mann, M., Hojrup, P. & Roepstorff, P. (1993). Use of mass spectrometric molecular weight information to identify proteins in sequence database. Biol Mass Spectrom 22, 338-345.[Medline]
Mauël, C., Young, M. & Karamata, D. (1991). Genes concerned with synthesis of poly(glycerol phosphate), the essential teichoic acid in Bacillus subtilis strain 168, are organized in two divergent transcription units. J Gen Microbiol 137, 929-941.[Medline]
Mauël, C., Young, M., Monsutti-Grecescu, A., Marriott, S. A. & Karamata, D. (1994). Analysis of Bacillus subtilis tag gene expression using transcriptional fusions. Microbiology 140, 2279-2288.[Abstract]
Messing, J., Crea, R. & Seeburg, P. H. (1981). A system for shotgun DNA sequencing. Nucleic Acids Res 9, 309-321.[Abstract]
Novotny, M. (1988). Recent advances in microcolumn liquid chromatography. Anal Chem 60, 500A-510A.[Medline]
Onaka, H., Nakagawa, T. & Horinouchi, S. (1998). Involvement of two A-factor receptor homologues in Streptomyces coelicolor A3(2) in the regulation of secondary metabolism and morphogenesis. Mol Microbiol 28, 743-753.[Medline]
Pugsley, A. P. (1993). The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57, 50-108.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sanger, F., Nicklen, S. & Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[Abstract]
Szabó, G., Barabás, G. & Vályi-Nagy, T. (1961). Comparison of Streptomyces griseus strains which produce streptomycin and those which do not. Arch Mikrobiol 40, 261-274.
Szabó, G., Vályi-Nagy, T. & Vitális, S. (1962). A new factor regulating life cycle of Streptomyces griseus. In Genetics of Microorganisms, Proceedings of a Symposium on Heredity and Variability of Microorganisms, pp. 282-292. Edited by V. D. Timakova. Moscow: State Publishing House of Medical Literature.
Szabó, G., Biró, S., Trón, L., Valu, G. & Vitális, S. (1984). Mode of action of factor C upon the differentiation process of Streptomyces griseus. In Biochemical and Biomedical Aspects of Actinomycetes, pp. 197-214. Edited by L. Ortiz-Ortiz, L. F. Bojalil & V. Yakoleff. Orlando, FL: Academic Press.
Szabó, G., Szeszák, F., Vitális, S. & Tóth, F. (1988). New data on the formation and mode of action of factor C. In Biology of Actinomycetes 88, pp. 324-329. Edited by Y. Okami, T. Beppu & H. Ogawara. Tokyo: Japan Scientific Societies Press.
Szabó, G., Vitális, S., Szeszák, F. & Biró, S. (1995). Developmental heterogeneity of Sterptomyces griseus conidia. In Sekundärmetabolismus bei Mikroorganismen, pp. 109-122. Edited by W. Kuhn & H.-P. Fiedler. Tübingen: Attempto Verlag.
Szeszák, F. & Szabó, G. (1973). Alteration of RNA synthesis in vitro with an endogenous regulating factor of cytodifferentiation of Streptomyces griseus. Acta Biol Acad Sci Hung 24, 11-17.[Medline]
Szeszák, F., Vitális, S. & Szabó, G. (1989). Factor C, a regulatory protein of Streptomyces griseus, induces release of potassium from the mycelium. J Basic Microbiol 29, 233-240.
Szeszák, F., Vitális, S., Tóth, F., Valu, G., Fachet, J. & Szabó, G. (1990). Detection and determination of factor C a regulatory protein in Streptomyces strains by antiserum and monoclonal antibody. Arch Microbiol 154, 82-84.[Medline]
Szeszák, F., Vitális, S. & Szabó, G. (1991). Presence of factor C in streptomycetes and other bacteria. In Genetics and Product Formation in Streptomyces, pp. 11-18. Edited by S. Baumberg, H. Krügel & D. Noack. New York: Plenum.
Szeszák, F., Vitális, S., Biró, S. & Dalmi, L. (1997). Amino acid sequence homology of factor C produced by Streptomyces griseus with regulatory proteins of zinc finger type. Acta Biol Acad Sci Hung 48, 265-273.
Vitális, S. & Szabó, G. (1969). Cytomorphological effect of factor C in submerged cultures on the hyphae of Streptomyces griseus strain No. 52-1. Acta Biol Acad Sci Hung 20, 85-92.[Medline]
Vitális, S., Szabó, G. & Vályi-Nagy, T. (1963). Comparison of the morphology of streptomycin producing and non-producing strains of Streptomyces griseus. Acta Biol Acad Sci Hung 14, 1-15.[Medline]
Vitális, S., Biró, S., Vargha, G., Békési, I. & Szabó, G. (1981). Differentiation and its regulation in submerged culture of Streptomyces griseus. In Actinomycetes (Zentbl Bakteriol Suppl 11), pp. 153-156. Edited by K. P. Schaal & G. Pulverer. New York: Springer.
Vitális, S., Valu, G., Békési, I., Szeszák, F. & Szabó, G. (1988). Changes of protein pattern of Streptomyces griseus strains that characterize differentiation of mycelia in submerged culture. J Basic Microbiol 28, 393-407.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13 mp18 and pUC19 vectors. Gene 33, 103-119.[Medline]
Received 5 March 1999;
revised 13 May 1999;
accepted 1 June 1999.