Effects of growth phase and the developmentally significant bldA-specified tRNA on the membrane-associated proteome of Streptomyces coelicolor

Microbiology ◽  
2005 ◽  
Vol 151 (8) ◽  
pp. 2707-2720 ◽  
Author(s):  
Dae-Wi Kim ◽  
Keith F. Chater ◽  
Kye-Joon Lee ◽  
Andy Hesketh
Keyword(s):  
Minimal Medium ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Transmembrane Helix ◽  
Morphological Differentiation ◽  
Hypothetical Proteins ◽  
Membrane Proteome ◽  
Protein Mass ◽  
Associated Proteins ◽  
Transporter Systems

Previous proteomic analyses of Streptomyces coelicolor by two-dimensional electrophoresis and protein mass fingerprinting focused on extracts from total cellular material. Here, the membrane-associated proteome of cultures grown in a liquid minimal medium was partially characterized. The products of some 120 genes were characterized from the membrane fraction, with 70 predicted to possess at least one transmembrane helix. A notably high proportion of ABC transporter systems was represented; the specific types detected provided a snapshot of the nutritional requirements of the mycelium. The membrane-associated proteins did not change very much in abundance in different phases of growth in liquid minimal medium. Identification of gene products not expected to be present in membrane protein extracts led to a reconsideration of the genome annotation in two cases, and supplemented scarce information on 11 hypothetical/conserved hypothetical proteins of unknown function. The wild-type membrane proteome was compared with that of a bldA mutant lacking the only tRNA capable of efficient translation of the rare UUA (leucine) codon. Such mutants are unaffected in vegetative growth but are defective in many aspects of secondary metabolism and morphological differentiation. There were a few clear changes in the membrane proteome of the mutant. In particular, two hypothetical proteins (SCO4244 and SCO4252) were completely absent from the bldA mutant, and this was associated with the TTA-containing regulatory gene SCO4263. Evidence for the control of a cluster of function-unknown genes by the SCO4263 regulator revealed a new aspect of the pleiotropic bldA phenotype.

scholarly journals Identification of a Gene Negatively Affecting Antibiotic Production and Morphological Differentiation in Streptomyces coelicolor A3(2)

2006 ◽  
Vol 188 (24) ◽  
pp. 8368-8375 ◽  
Author(s):  
Wencheng Li ◽  
Xin Ying ◽  
Yuzheng Guo ◽  
Zhen Yu ◽  
Xiufen Zhou ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Morphological Differentiation ◽  
Aerial Mycelium ◽  
Protein Domain ◽  
Chromosomal Dna ◽  
Reading Frame ◽  
Directed Mutation ◽  
In The Wild

ABSTRACT SC7A1 is a cosmid with an insert of chromosomal DNA from Streptomyces coelicolor A3(2). Its insertion into the chromosome of S. coelicolor strains caused a duplication of a segment of ca. 40 kb and delayed actinorhodin antibiotic production and sporulation, implying that SC7A1 carried a gene negatively affecting these processes. The subcloning of SC7A1 insert DNA resulted in the identification of the open reading frame SCO5582 as nsdA, a gene n egatively affecting S treptomyces d ifferentiation. The disruption of chromosomal nsdA caused the overproduction of spores and of three of four known S. coelicolor antibiotics of quite different chemical types. In at least one case (that of actinorhodin), this was correlated with premature expression of a pathway-specific regulatory gene (actII-orf4), implying that nsdA in the wild-type strain indirectly repressed the expression of the actinorhodin biosynthesis cluster. nsdA expression was up-regulated upon aerial mycelium initiation and was strongest in the aerial mycelium. NsdA has DUF921, a Streptomyces protein domain of unknown function and a conserved SXR site. A site-directed mutation (S458A) in this site in NsdA abolished its function. Blast searching showed that NsdA homologues are present in some Streptomyces genomes. Outside of streptomycetes, NsdA-like proteins have been found in several actinomycetes. The disruption of the nsdA-like gene SCO4114 had no obvious phenotypic effects on S. coelicolor. The nsdA orthologue SAV2652 in S. avermitilis could complement the S. coelicolor nsdA-null mutant phenotype.

scholarly journals Changes in the Extracellular Proteome Caused by the Absence of the bldA Gene Product, a Developmentally Significant tRNA, Reveal a New Target for the Pleiotropic Regulator AdpA in Streptomyces coelicolor

2005 ◽  
Vol 187 (9) ◽  
pp. 2957-2966 ◽  
Author(s):  
Dae-Wi Kim ◽  
Keith Chater ◽  
Kye-Joon Lee ◽  
Andy Hesketh
Keyword(s):  
Stationary Phase ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Morphological Differentiation ◽  
Secreted Proteins ◽  
Two Dimensional Gel Electrophoresis ◽  
Extracellular Proteome ◽  
Peptide Mass ◽  
Subtilisin Inhibitor

ABSTRACT The extracellular proteome of Streptomyces coelicolor grown in a liquid medium was analyzed by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprint analysis. Culture supernatants became protein rich only after rapid growth had been completed, supporting the idea that protein secretion is largely a stationary phase phenomenon. Out of about 600 protein spots observed, 72 were characterized. The products of 47 genes were identified, with only 11 examples predicted to be secreted proteins. Mutation in bldA, previously known to impair the stationary phase processes of antibiotic production and morphological differentiation, also induced changes in the extracellular proteome, revealing even greater pleiotropy in the bldA phenotype than previously known. Four proteins increased in abundance in the bldA mutant, while the products of 11 genes, including four secreted proteins, were severely down-regulated. Although bldA encodes the only tRNA capable of efficiently translating the rare UUA (leucine) codon, none of the latter group of genes contains an in-frame TTA. SCO0762, a serine-protease inhibitor belonging to the Streptomyces subtilisin inhibitor family implicated in differentiation in other streptomycetes, was completely absent from the bldA mutant. This dependence was shown to be mediated via the TTA-containing regulatory gene adpA, also known as bldH, a developmental gene that is responsible for the effects of bldA on differentiation. Mutation of the SCO0762 gene abolished detectable trypsin-protease inhibitory activity but did not result in any obvious morphological defects.

scholarly journals Repression of Antibiotic Production and Sporulation in Streptomyces coelicolor by Overexpression of a TetR Family Transcriptional Regulator

2010 ◽  
Vol 76 (23) ◽  
pp. 7741-7753 ◽  
Author(s):  
Delin Xu ◽  
Nicolas Seghezzi ◽  
Catherine Esnault ◽  
Marie-Joelle Virolle
Keyword(s):  
Target Genes ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Morphological Differentiation ◽  
Promoter Sequence ◽  
Inhibitory Effect ◽  
Binding Motif

ABSTRACT The overexpression of a regulatory gene of the TetR family (SCO3201) originating either from Streptomyces lividans or from Streptomyces coelicolor was shown to strongly repress antibiotic production (calcium-dependent antibiotic [CDA], undecylprodigiosin [RED], and actinorhodin [ACT]) of S. coelicolor and of the ppk mutant strain of S. lividans. Curiously, the overexpression of this gene also had a strong inhibitory effect on the sporulation process of S. coelicolor but not on that of S. lividans. SCO3201 was shown to negatively regulate its own transcription, and its DNA binding motif was found to overlap its −35 promoter sequence. The interruption of this gene in S. lividans or S. coelicolor did not lead to any obvious phenotypes, indicating that when overexpressed SCO3201 likely controls the expression of target genes of other TetR regulators involved in the regulation of the metabolic and morphological differentiation process in S. coelicolor. The direct and functional interaction of SCO3201 with the promoter region of scbA, a gene under the positive control of the TetR-like regulator, ScbR, was indeed demonstrated by in vitro as well as in vivo approaches.

scholarly journals Regulation of Sporangium Formation by BldD in the Rare Actinomycete Actinoplanes missouriensis

2017 ◽  
Vol 199 (12) ◽  
Author(s):  
Yoshihiro Mouri ◽  
Kenji Konishi ◽  
Azusa Fujita ◽  
Takeaki Tezuka ◽  
Yasuo Ohnishi
Keyword(s):  
Vegetative Growth ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Transcriptional Regulator ◽  
Saccharopolyspora Erythraea ◽  
Transcriptional Analysis ◽  
Morphological Development ◽  
Sequencing Analysis ◽  
Content Type ◽  
Biological Studies

ABSTRACT The rare actinomycete Actinoplanes missouriensis forms sporangia, including hundreds of flagellated spores that start swimming as zoospores after their release. Under conditions suitable for vegetative growth, zoospores stop swimming and germinate. A comparative proteome analysis between zoospores and germinating cells identified 15 proteins that were produced in larger amounts in germinating cells. They include an orthologue of BldD (herein named AmBldD [BldD of A. missouriensis]), which is a transcriptional regulator involved in morphological development and secondary metabolism in Streptomyces. AmBldD was detected in mycelia during vegetative growth but was barely detected in mycelia during the sporangium-forming phase, in spite of the constant transcription of AmbldD throughout growth. An AmbldD mutant started to form sporangia much earlier than the wild-type strain, and the resulting sporangia were morphologically abnormal. Recombinant AmBldD bound a palindromic sequence, the AmBldD box, located upstream from AmbldD. 3′,5′-Cyclic di-GMP significantly enhanced the in vitro DNA-binding ability of AmBldD. A chromatin immunoprecipitation-sequencing analysis and an in silico search for AmBldD boxes revealed that AmBldD bound 346 genomic loci that contained the 19-bp inverted repeat 5′-NN(G/A)TNACN(C/G)N(G/C)NGTNA(C/T)NN-3′ as the consensus AmBldD-binding sequence. The transcriptional analysis of 27 selected AmBldD target gene candidates indicated that AmBldD should repress 12 of the 27 genes, including bldM, ssgB, whiD, ddbA, and wblA orthologues. These genes are involved in morphological development in Streptomyces coelicolor A3(2). Thus, AmBldD is a global transcriptional regulator that seems to repress the transcription of tens of genes during vegetative growth, some of which are likely to be required for sporangium formation. IMPORTANCE The rare actinomycete Actinoplanes missouriensis undergoes complex morphological differentiation, including sporangium formation. However, almost no molecular biological studies have been conducted on this bacterium. BldD is a key global regulator involved in the morphological development of streptomycetes. BldD orthologues are highly conserved among sporulating actinomycetes, but no BldD orthologues, except one in Saccharopolyspora erythraea, have been studied outside the streptomycetes. Here, it was revealed that the BldD orthologue AmBldD is essential for normal developmental processes in A. missouriensis. The AmBldD regulon seems to be different from the BldD regulon in Streptomyces coelicolor A3(2), but they share four genes that are involved in morphological differentiation in S. coelicolor A3(2).

scholarly journals Characterization of the Noncanonical Regulatory and Transporter Genes in Atratumycin Biosynthesis and Production in a Heterologous Host

Marine Drugs ◽  
2019 ◽  
Vol 17 (10) ◽  
pp. 560 ◽  
Author(s):  
Zhijie Yang ◽  
Xin Wei ◽  
Jianqiao He ◽  
Changli Sun ◽  
Jianhua Ju ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Biosynthetic Gene Cluster ◽  
Negative Regulator ◽  
Over Expression ◽  
Lead Compounds ◽  
Mycobacteria Tuberculosis ◽  
Abc Transporter Genes

Atratumycin is a cyclodepsipeptide with activity against Mycobacteria tuberculosis isolated from deep-sea derived Streptomyces atratus SCSIO ZH16NS-80S. Analysis of the atratumycin biosynthetic gene cluster (atr) revealed that its biosynthesis is regulated by multiple factors, including two LuxR regulatory genes (atr1 and atr2), two ABC transporter genes (atr29 and atr30) and one Streptomyces antibiotic regulatory gene (atr32). In this work, three regulatory and two transporter genes were unambiguously determined to provide positive, negative and self-protective roles during biosynthesis of atratumycin through bioinformatic analyses, gene inactivations and trans-complementation studies. Notably, an unusual Streptomyces antibiotic regulatory protein Atr32 was characterized as a negative regulator; the function of Atr32 is distinct from previous studies. Five over-expression mutant strains were constructed by rational application of the regulatory and transporter genes; the resulting strains produced significantly improved titers of atratumycin that were ca. 1.7–2.3 fold greater than wild-type (WT) producer. Furthermore, the atratumycin gene cluster was successfully expressed in Streptomyces coelicolor M1154, thus paving the way for the transfer and recombination of large DNA fragments. Overall, this finding sets the stage for understanding the unique biosynthesis of pharmaceutically important atratumycin and lays the foundation for generating anti-tuberculosis lead compounds possessing novel structures.

scholarly journals Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization.

1978 ◽  
Vol 76 (2) ◽  
pp. 547-555 ◽  
Author(s):  
N W Seeds ◽  
R B Maccioni
Keyword(s):  
High Speed ◽  
Gradient Centrifugation ◽  
Neuroblastoma Cells ◽  
Morphological Differentiation ◽  
Tubulin Polymerization ◽  
Associated Proteins ◽  
Neurite Formation ◽  
Brain Microtubules ◽  
Brain Tubulin

Clonal cells (N18) of the mouse neuroblastoma C-1300 can be induced to undergo a morphological differentiation characterized by the outgrowth of very long neurites (> 150 microns) that contain many microtubules. Because the marked increase in the number and length of microtubules is apparently not due to an increase in the concentration of tubulin subunits, the possible role of additional macromolecules in the regulation of tubulin polymerization during neurite formation by N18 cells was examined. Using an in vitro system where the polymerization of low concentrations (< 4 mg/ml) of purified brain tubulin requires microtubule-associated proteins (MAPs), high-speed supernates (250,000 g) from neuroblastoma and glioma cells were assayed for their ability to replace MAPs in the polymerization of brain tubulin. Only the supernates from "differentiated" N18 cells were polymerization competent. Electron microscope observations of these supernates failed to demonstrate the presence of nucleation structures (rings or disks). The active factor(s) sedimented at approximately 7S on sucrose gradient centrifugation and eluted from 4B Sepharose in the region of 170,000 mol wt proteins. Furthermore, the inactive supernates from other cells did not inhibit polymerization when tested in the presence of limiting MAPs. Thus, microtubule formation accompanying neurite outgrowth in neuroblastoma cells appears to be regulated by the presence of additional macromolecular factor(s) that may be functionally equivalent to the MAPs found with brain microtubules.

scholarly journals A Novel Insertion Mutation in Streptomyces coelicolor Ribosomal S12 Protein Results in Paromomycin Resistance and Antibiotic Overproduction

2008 ◽  
Vol 53 (3) ◽  
pp. 1019-1026 ◽  
Author(s):  
Guojun Wang ◽  
Takashi Inaoka ◽  
Susumu Okamoto ◽  
Kozo Ochi
Keyword(s):  
Transcriptional Activation ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Translation System ◽  
Insertion Mutation ◽  
Resistance Level ◽  
Double Mutation ◽  
Paromomycin Resistance ◽  
High Level

ABSTRACT We identified a novel paromomycin resistance-associated mutation in rpsL, caused by the insertion of a glycine residue at position 92, in Streptomyces coelicolor ribosomal protein S12. This insertion mutation (GI92) resulted in a 20-fold increase in the paromomycin resistance level. In combination with another S12 mutation, K88E, the GI92 mutation markedly enhanced the production of the blue-colored polyketide antibiotic actinorhodin and the red-colored antibiotic undecylprodigiosin. The gene replacement experiments demonstrated that the K88E-GI92 double mutation in the rpsL gene was responsible for the marked enhancement of antibiotic production observed. Ribosomes with the K88E-GI92 double mutation were characterized by error restrictiveness (i.e., hyperaccuracy). Using a cell-free translation system, we found that mutant ribosomes harboring the K88E-GI92 double mutation but not ribosomes harboring the GI92 mutation alone displayed sixfold greater translation activity relative to that of the wild-type ribosomes at late growth phase. This resulted in the overproduction of actinorhodin, caused by the transcriptional activation of the pathway-specific regulatory gene actII-orf4, possibly due to the increased translation of transcripts encoding activators of actII-orf4. The mutant with the K88E-GI92 double mutation accumulated a high level of ribosome recycling factor at late stationary phase, underlying the high level of protein synthesis activity observed.

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