Journal:

Article Title: In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis

doi: 10.1128/JB.186.5.1345-1354.2004

Figure Lengend Snippet: Bacterial strains, plasmids, and phages used in this study

Article Snippet: Nystatin production was assessed by high-performance liquid chromatography ( 25 ) of the dimethylformamide extracts of cultures from 500-ml shake flask fermentations in 100 ml of semidefined SAO-23 medium ( 28 ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or phage Characteristics Reference or source a Strains S. noursei ATCC 11455 WT strain, nystatin producer ATCC SR12 WT with in-frame deletion in nysRI This work SR34 WT with in-frame deletion in nysRII This work SR56 WT with in-frame deletion in nysRIII This work NR4K WT with Km r insertion in nysRIV This work NR5D WT with deletion in orf3 This work DNR609 WT with Km r insertion in orf2 This work E. coli DH5α General cloning host BRL JM110 dam dcm strain Promega ET12567 (pUZ8002) Strain for intergeneric conjugation 13 DASHII phage vector derivatives N1 Recombinant phage (nystatin gene cluster) 7 N40 Recombinant phage (nystatin gene cluster) 7 N58 Recombinant phage (nystatin gene cluster) 7 N69 Recombinant phage (nystatin gene cluster) 7 N76 Recombinant phage (nystatin gene cluster) 7 Recombinant plasmids pGEM3Zf(−) Cloning vector Promega pGEM7Zf(−) Cloning vector Promega pGEM11Zf(−) Cloning vector Promega pGEM7ZfErmE*li pGEM7Zf(−) vector with ermE *p promoter C. R. Hutchinson pKTO2 VWB-based Streptomyces integrative vector 37 pSET152 E. coli-Streptomyces conjugative vector 6 pSOK101 E. coli-Streptomyces conjugative vector 41 pSOK201 E. coli-Streptomyces conjugative vector 41 pSOK804 2.3-kb Sph I- Hin dIII fragment from pKTO2 ligated with a 3.0-kb Sph I- Hin dIII fragment from pSET152 This work pSR12 Vector for nysRI in-frame deletion This work pSR34 Vector for nysRII in-frame deletion This work pSR56 Vector for nysRIII in-frame deletion This work pL58KX5 5.0-kb Kpn I- Xba I fragment from phage N58 cloned into pGEM3Zf(−) This work pNR4Km 1.3-kb Km r marker inserted into the Cla I site within the nysRIV gene in pL58KX5 after Klenow fill-in This work pNR4D 6.3-kb Kpn I- Sph I fragment from pNR4Km ligated with a 3.0-kb Kpn I- Sph I fragment from pSOK101 This work pNR5D Vector for orf3 inactivation This work pNR6 3.7-kb Xmn I- Fsp I fragment from phage N69 with orf2 cloned into the Sma I site of pGEM3Zf(−) This work pNR6K 1.3-kb Km r marker inserted into the Mlu I site within orf2 in pNR6 after Klenow fill-in This work pLDR6K 5.0-kb Eco RI- Xba I fragment from pNR6K ligated with a 3.0-kb Eco RI- Xba I fragment from pSOK804 This work pNRE2 Vector for nysRI expression This work pC3A1 Vector for nysRII expression This work pNR3T Vector for nysRIII expression This work pNR4EL Vector for expression of a 226-aa version of NysRIV This work pNR4ES Vector for expression of a 210-aa version of NysRIV This work pIJ4081 Vector with promoterless xylE gene J.

Techniques: Plasmid Preparation, Clone Assay, Conjugation Assay, Recombinant, Marker, Expressing

Journal:

Article Title: In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis

doi: 10.1128/JB.186.5.1345-1354.2004

Figure Lengend Snippet: XylE activities in the protein extracts of WT S. noursei and regulatory mutants expressing xylE from different promoters. The last diagram shows a comparative analysis of xylE expression from the nysAp and ermE*p promoters in S. noursei ATCC 11455. Variations from the mean in each data series are represented by error bars.

Article Snippet: Nystatin production was assessed by high-performance liquid chromatography ( 25 ) of the dimethylformamide extracts of cultures from 500-ml shake flask fermentations in 100 ml of semidefined SAO-23 medium ( 28 ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or phage Characteristics Reference or source a Strains S. noursei ATCC 11455 WT strain, nystatin producer ATCC SR12 WT with in-frame deletion in nysRI This work SR34 WT with in-frame deletion in nysRII This work SR56 WT with in-frame deletion in nysRIII This work NR4K WT with Km r insertion in nysRIV This work NR5D WT with deletion in orf3 This work DNR609 WT with Km r insertion in orf2 This work E. coli DH5α General cloning host BRL JM110 dam dcm strain Promega ET12567 (pUZ8002) Strain for intergeneric conjugation 13 DASHII phage vector derivatives N1 Recombinant phage (nystatin gene cluster) 7 N40 Recombinant phage (nystatin gene cluster) 7 N58 Recombinant phage (nystatin gene cluster) 7 N69 Recombinant phage (nystatin gene cluster) 7 N76 Recombinant phage (nystatin gene cluster) 7 Recombinant plasmids pGEM3Zf(−) Cloning vector Promega pGEM7Zf(−) Cloning vector Promega pGEM11Zf(−) Cloning vector Promega pGEM7ZfErmE*li pGEM7Zf(−) vector with ermE *p promoter C. R. Hutchinson pKTO2 VWB-based Streptomyces integrative vector 37 pSET152 E. coli-Streptomyces conjugative vector 6 pSOK101 E. coli-Streptomyces conjugative vector 41 pSOK201 E. coli-Streptomyces conjugative vector 41 pSOK804 2.3-kb Sph I- Hin dIII fragment from pKTO2 ligated with a 3.0-kb Sph I- Hin dIII fragment from pSET152 This work pSR12 Vector for nysRI in-frame deletion This work pSR34 Vector for nysRII in-frame deletion This work pSR56 Vector for nysRIII in-frame deletion This work pL58KX5 5.0-kb Kpn I- Xba I fragment from phage N58 cloned into pGEM3Zf(−) This work pNR4Km 1.3-kb Km r marker inserted into the Cla I site within the nysRIV gene in pL58KX5 after Klenow fill-in This work pNR4D 6.3-kb Kpn I- Sph I fragment from pNR4Km ligated with a 3.0-kb Kpn I- Sph I fragment from pSOK101 This work pNR5D Vector for orf3 inactivation This work pNR6 3.7-kb Xmn I- Fsp I fragment from phage N69 with orf2 cloned into the Sma I site of pGEM3Zf(−) This work pNR6K 1.3-kb Km r marker inserted into the Mlu I site within orf2 in pNR6 after Klenow fill-in This work pLDR6K 5.0-kb Eco RI- Xba I fragment from pNR6K ligated with a 3.0-kb Eco RI- Xba I fragment from pSOK804 This work pNRE2 Vector for nysRI expression This work pC3A1 Vector for nysRII expression This work pNR3T Vector for nysRIII expression This work pNR4EL Vector for expression of a 226-aa version of NysRIV This work pNR4ES Vector for expression of a 210-aa version of NysRIV This work pIJ4081 Vector with promoterless xylE gene J.

Techniques: Expressing

Journal:

Article Title: In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis

doi: 10.1128/JB.186.5.1345-1354.2004

Figure Lengend Snippet: (A) Organization of the regulatory gene locus associated with the nystatin biosynthetic gene cluster in S. noursei ATCC 11455 (GenBank accession number AF263912). The only restriction enzyme sites indicated are those used for vector construction as described in Materials and Methods and Table ​Table1.1. Heavy solid lines represent DNA fragments used in gene replacement experiments. (B) Putative functional features predicted for the NysRI, NysRII, NysRIII, and NysRIV proteins. WA and WB, Walker A and B NTP binding motifs; PAS, PAS-like domain.

Article Snippet: Nystatin production was assessed by high-performance liquid chromatography ( 25 ) of the dimethylformamide extracts of cultures from 500-ml shake flask fermentations in 100 ml of semidefined SAO-23 medium ( 28 ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or phage Characteristics Reference or source a Strains S. noursei ATCC 11455 WT strain, nystatin producer ATCC SR12 WT with in-frame deletion in nysRI This work SR34 WT with in-frame deletion in nysRII This work SR56 WT with in-frame deletion in nysRIII This work NR4K WT with Km r insertion in nysRIV This work NR5D WT with deletion in orf3 This work DNR609 WT with Km r insertion in orf2 This work E. coli DH5α General cloning host BRL JM110 dam dcm strain Promega ET12567 (pUZ8002) Strain for intergeneric conjugation 13 DASHII phage vector derivatives N1 Recombinant phage (nystatin gene cluster) 7 N40 Recombinant phage (nystatin gene cluster) 7 N58 Recombinant phage (nystatin gene cluster) 7 N69 Recombinant phage (nystatin gene cluster) 7 N76 Recombinant phage (nystatin gene cluster) 7 Recombinant plasmids pGEM3Zf(−) Cloning vector Promega pGEM7Zf(−) Cloning vector Promega pGEM11Zf(−) Cloning vector Promega pGEM7ZfErmE*li pGEM7Zf(−) vector with ermE *p promoter C. R. Hutchinson pKTO2 VWB-based Streptomyces integrative vector 37 pSET152 E. coli-Streptomyces conjugative vector 6 pSOK101 E. coli-Streptomyces conjugative vector 41 pSOK201 E. coli-Streptomyces conjugative vector 41 pSOK804 2.3-kb Sph I- Hin dIII fragment from pKTO2 ligated with a 3.0-kb Sph I- Hin dIII fragment from pSET152 This work pSR12 Vector for nysRI in-frame deletion This work pSR34 Vector for nysRII in-frame deletion This work pSR56 Vector for nysRIII in-frame deletion This work pL58KX5 5.0-kb Kpn I- Xba I fragment from phage N58 cloned into pGEM3Zf(−) This work pNR4Km 1.3-kb Km r marker inserted into the Cla I site within the nysRIV gene in pL58KX5 after Klenow fill-in This work pNR4D 6.3-kb Kpn I- Sph I fragment from pNR4Km ligated with a 3.0-kb Kpn I- Sph I fragment from pSOK101 This work pNR5D Vector for orf3 inactivation This work pNR6 3.7-kb Xmn I- Fsp I fragment from phage N69 with orf2 cloned into the Sma I site of pGEM3Zf(−) This work pNR6K 1.3-kb Km r marker inserted into the Mlu I site within orf2 in pNR6 after Klenow fill-in This work pLDR6K 5.0-kb Eco RI- Xba I fragment from pNR6K ligated with a 3.0-kb Eco RI- Xba I fragment from pSOK804 This work pNRE2 Vector for nysRI expression This work pC3A1 Vector for nysRII expression This work pNR3T Vector for nysRIII expression This work pNR4EL Vector for expression of a 226-aa version of NysRIV This work pNR4ES Vector for expression of a 210-aa version of NysRIV This work pIJ4081 Vector with promoterless xylE gene J.

Techniques: Plasmid Preparation, Functional Assay, Binding Assay

Journal:

Article Title: In Vivo Analysis of the Regulatory Genes in the Nystatin Biosynthetic Gene Cluster of Streptomyces noursei ATCC 11455 Reveals Their Differential Control Over Antibiotic Biosynthesis

doi: 10.1128/JB.186.5.1345-1354.2004

Figure Lengend Snippet: Nystatin production by recombinant S. noursei strains with inactivated or overexpressed regulatory genes

Article Snippet: Nystatin production was assessed by high-performance liquid chromatography ( 25 ) of the dimethylformamide extracts of cultures from 500-ml shake flask fermentations in 100 ml of semidefined SAO-23 medium ( 28 ). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain, plasmid, or phage Characteristics Reference or source a Strains S. noursei ATCC 11455 WT strain, nystatin producer ATCC SR12 WT with in-frame deletion in nysRI This work SR34 WT with in-frame deletion in nysRII This work SR56 WT with in-frame deletion in nysRIII This work NR4K WT with Km r insertion in nysRIV This work NR5D WT with deletion in orf3 This work DNR609 WT with Km r insertion in orf2 This work E. coli DH5α General cloning host BRL JM110 dam dcm strain Promega ET12567 (pUZ8002) Strain for intergeneric conjugation 13 DASHII phage vector derivatives N1 Recombinant phage (nystatin gene cluster) 7 N40 Recombinant phage (nystatin gene cluster) 7 N58 Recombinant phage (nystatin gene cluster) 7 N69 Recombinant phage (nystatin gene cluster) 7 N76 Recombinant phage (nystatin gene cluster) 7 Recombinant plasmids pGEM3Zf(−) Cloning vector Promega pGEM7Zf(−) Cloning vector Promega pGEM11Zf(−) Cloning vector Promega pGEM7ZfErmE*li pGEM7Zf(−) vector with ermE *p promoter C. R. Hutchinson pKTO2 VWB-based Streptomyces integrative vector 37 pSET152 E. coli-Streptomyces conjugative vector 6 pSOK101 E. coli-Streptomyces conjugative vector 41 pSOK201 E. coli-Streptomyces conjugative vector 41 pSOK804 2.3-kb Sph I- Hin dIII fragment from pKTO2 ligated with a 3.0-kb Sph I- Hin dIII fragment from pSET152 This work pSR12 Vector for nysRI in-frame deletion This work pSR34 Vector for nysRII in-frame deletion This work pSR56 Vector for nysRIII in-frame deletion This work pL58KX5 5.0-kb Kpn I- Xba I fragment from phage N58 cloned into pGEM3Zf(−) This work pNR4Km 1.3-kb Km r marker inserted into the Cla I site within the nysRIV gene in pL58KX5 after Klenow fill-in This work pNR4D 6.3-kb Kpn I- Sph I fragment from pNR4Km ligated with a 3.0-kb Kpn I- Sph I fragment from pSOK101 This work pNR5D Vector for orf3 inactivation This work pNR6 3.7-kb Xmn I- Fsp I fragment from phage N69 with orf2 cloned into the Sma I site of pGEM3Zf(−) This work pNR6K 1.3-kb Km r marker inserted into the Mlu I site within orf2 in pNR6 after Klenow fill-in This work pLDR6K 5.0-kb Eco RI- Xba I fragment from pNR6K ligated with a 3.0-kb Eco RI- Xba I fragment from pSOK804 This work pNRE2 Vector for nysRI expression This work pC3A1 Vector for nysRII expression This work pNR3T Vector for nysRIII expression This work pNR4EL Vector for expression of a 226-aa version of NysRIV This work pNR4ES Vector for expression of a 210-aa version of NysRIV This work pIJ4081 Vector with promoterless xylE gene J.

Techniques: Recombinant