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- Disruption of SCO5461 Gene Coding for a Mono-ADP-Ribosyltransferase Enzyme Produces a Conditional Pleiotropic Phenotype Affecting Morphological Differentiation and Antibiotic Production in Streptomyces coelicolor
- Krisztina Szirák , Judit Keser&# , Sándor Biró , Iván Schmelczer , György Barabás , András Penyige
- J. Microbiol. 2012;50(3):409-418. Published online June 30, 2012
- DOI: https://doi.org/10.1007/s12275-012-1440-y
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Abstract
- The SCO5461 gene of Streptomyces coelicolor A3(2) codes for an ADP-ribosyltransferase enzyme that is predicted to be a transmembrane protein with an extracellular catalytic domain. PCR-targeted disruption of the gene resulted in a mutant that differentiated normally on complex SFM medium; however, morphological differentiation in minimal medium was significantly delayed and this phenotype was even more pronounced on osmotically enhanced minimal medium. The mutant did not sporulate when it was grown on R5 medium, however the normal morphological differentiation was restored when the strain was cultivated beside the wild-type S. coelicolor M145 strain. Comparison of the pattern of ADP-ribosylated proteins showed a difference between the mutant and the wild type, fewer modified proteins were present in the cellular crude extract of the mutant strain. These results support our previous suggestions that protein ADP-ribosylation is involved in the regulation of differentiation and antibiotic production and secretion in Streptomyces.
Research Support, Non-U.S. Gov't
- Characterization of Sgr3394 Produced only by the A-Factor-Producing Streptomyces griseus IFO 13350, not by the A-Factor Deficient Mutant HH1
- Won-Jae Chi , Xue-Mei Jin , Sung-Cheol Jung , Eun A Oh , Soon-Kwang Hong
- J. Microbiol. 2011;49(1):155-160. Published online March 3, 2011
- DOI: https://doi.org/10.1007/s12275-011-0330-z
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- 3 Citations
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Abstract
- Protein D (9.7 kDa) is an extracellular protein detected in the culture broth of A-factor-producing Streptomyces griseus IFO 13350, but not of the A-factor-deficient mutant strain S. griseus HH1. Comparison of the N-terminal amino acid sequence with the genomic sequencing data of S. griseus IFO 13350 identified protein D as Sgr3394, which encodes a putative secretory protein with unknown function. The premature Sgr3394 consisted of 128 amino acids (13.5 kDa), showed 87.5% identity with SACT1DRAFT-0503, from Streptomyces sp. ACT-1, and 68.8% identity with SrosN15-18634, from S. roseosporus NRRL15998, and was confirmed to be matured for secretion by a peptide cleavage between the Ala-38 and Ala-39 bond. RT-PCR anaylsis of Sgr3394 clearly showed that it can be transcribed in the wild-type strain, but not in the A-factor-deficient strain. However, a gel-mobility shift assay of the promoter region of sgr3394 with A-factor-dependent transcriptional regulator (AdpA) showed that AdpA could not specifically recognize the putative AdpA-binding site (5′-TCCCCCGAAT-3′). All of these data strongly suggest that the expression of sgr3394 is not directly induced by AdpA but is regulated indirectly by an A-factor dependent protein. Introduction of sgr3394 on a high-copy-numbered plasmid (pWHM3-sgr3394) into S. lividans TK21 induced massive production of actinorhodin (blue pigment) and undecylprodigiosin (red pigment). Compared to the control, production of each pigment increased by 6.1 and 2.6 times, respectively, on R2YE agar, and 3.1 and 1.4 times, respectively, in R2YE broth; there was little influence on morphogenesis. In S. coelicolor A3(2)/pWHM3-sgr3394, actinorhodin and undecylprodigiosin productions were enhanced to 1.8 and 1.1 times those observed in the control, respectively, suggesting that overexpression of sgr3394 can stimulate secondary metabolism, especially actinorhodin biosynthesis, in S. lividans and S. coelicolor.
- Overexpression of the sprD Gene Encoding Streptomyces griseus Protease D Stimulates Actinorhodin Production in Streptomyces lividans
- Si-Sun Choi , Won-Jae Chi , Jae Hag Lee , Sang-Soon Kang , Byeong Chul Jeong , Soon-Kwang Hong
- J. Microbiol. 2001;39(4):305-313.
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Abstract
- The sprD gene encoding Streptomyces griseus protease D (SGPD), a chymotrypsin-like protease, was cloned from Streptomyces griseus IFO13350 and sequenced. Most of the amino-acid sequence deduced from the nucleotide sequence is identical to that of Streptomyces griseus IMRU3499 except that one amino acid has been deleted and Trp369 has been substituted into Cys369 in the SGPD from S. griseus IFO13350 without affecting the protease activity. The sprD gene was overexpressed in Streptomyces lividans TK24 as a heterologous host. Various media with different compositions were also used to maximize the productivity of SGPD in the heterologous host. The SGPD productivity was best when the transformant of S. lividans TK24 was cultivated in R2YE medium. The relative chymotrypsin activity of the culture broth measured with an artificial chromogenic substrate, N-succinyl-ala-ala-pro-phe-[rho]-nitroanilide, was 16 units/ml. A high level of SGPD was also produced in YEME and SAAM media but it was relatively lower than in R2YE medium, and negligible amounts of SGPD were produced in GYE, GAE and Benedict media. The growth of S. lividans reached the maximum level of cell mass at days 3 and 4 of the culture, but SGPD production started in the stationary phase of cell growth and kept increasing till the 10th day of culture in R2YE and YEME medium, but in GYE media the productivity reached maximum level at 8 days of cultivation. The introduction of the sprD gene into S. lividans TK24 triggered biosynthesis of the pigmented antibiotic, actinorhodin, which implies some protease may play a very important role in secondary-metabolite formation in Streptomyces.
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