AllR Controls the Expression of Streptomyces coelicolor Allantoin Pathway Genes

ABSTRACTStreptomycesspecies are native inhabitants of soil, a natural environment where nutrients can be scarce and competition fierce. They have evolved ways to metabolize unusual nutrients, such as purines and its derivatives, which are highly abundant in soil. Catabolism of these uncommon carbon and nitrogen sources needs to be tightly regulated in response to nutrient availability and environmental stimulus. Recently, the allantoin degradation pathway was characterized inStreptomyces coelicolor. However, there are questions that remained unanswered, particularly regarding pathway regulation. Here, using a combination of proteomics and genetic approaches, we identified the negative regulator of the allantoin pathway, AllR.In vitrostudies confirmed that AllR binds to the promoter regions of allantoin catabolic genes and determined the AllR DNA binding motif. In addition, effector studies showed that allantoic acid, and glyoxylate, to a lesser extent, inhibit the binding of AllR to the DNA. Inactivation of AllR repressor leads to the constitutive expression of the AllR regulated genes and intriguingly impairs actinorhodin and undecylprodigiosin production. Genetics and proteomics analysis revealed that among all genes from the allantoin pathway that are upregulated in theallRmutant, thehyigene encoding a hydroxypyruvate isomerase (Hyi) is responsible of the impairment of antibiotic production.

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Phosphorylated AbsA2 Negatively Regulates Antibiotic Production in Streptomyces coelicolor through Interactions with Pathway-Specific Regulatory Gene Promoters

Journal of Bacteriology ◽

10.1128/jb.00305-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5284-5292 ◽

Cited By ~ 67

Author(s):

Nancy L. McKenzie ◽

Justin R. Nodwell

Keyword(s):

Response Regulator ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Regulatory Gene ◽

Gene Clusters ◽

Negative Regulator ◽

Gene Promoters ◽

Promoter Regions ◽

Calcium Dependent

ABSTRACT The AbsA two-component signal transduction system, comprised of the sensor kinase AbsA1 and the response regulator AbsA2, acts as a negative regulator of antibiotic production in Streptomyces coelicolor, for which the phosphorylated form of AbsA2 (AbsA2∼P) is the agent of repression. In this study, we used chromatin immunoprecipitation to show that AbsA2 binds the promoter regions of actII-ORF4, cdaR, and redZ, which encode pathway-specific activators for actinorhodin, calcium-dependent antibiotic, and undecylprodigiosin, respectively. We confirm that these interactions also occur in vitro and that the binding of AbsA2 to each gene is enhanced by phosphorylation. Induced expression of actII-ORF4 and redZ in the hyperrepressive absA1 mutant (C542) brought about pathway-specific restoration of actinorhodin and undecylprodigiosin production, respectively. Our results suggest that AbsA2∼P interacts with as many as four sites in the region that includes the actII-ORF4 promoter. These data suggest that AbsA2∼P inhibits antibiotic production by directly interfering with the expression of pathway-specific regulators of antibiotic biosynthetic gene clusters.

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Deciphering the Regulon of Streptomyces coelicolor AbrC3, a Positive Response Regulator of Antibiotic Production

Applied and Environmental Microbiology ◽

10.1128/aem.03378-13 ◽

2014 ◽

Vol 80 (8) ◽

pp. 2417-2428 ◽

Cited By ~ 15

Author(s):

Sergio Rico ◽

Ramón I. Santamaría ◽

Ana Yepes ◽

Héctor Rodríguez ◽

Emma Laing ◽

...

Keyword(s):

Promoter Region ◽

Parent Strain ◽

Rational Design ◽

Genetic Manipulation ◽

Response Regulator ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Morphological Development ◽

Content Type

ABSTRACTThe atypical two-component system (TCS) AbrC1/C2/C3 (encoded bySCO4598,SCO4597, andSCO4596), comprising two histidine kinases (HKs) and a response regulator (RR), is crucial for antibiotic production inStreptomyces coelicolorand for morphological differentiation under certain nutritional conditions. In this study, we demonstrate that deletion of the RR-encoding gene,abrC3(SCO4596), results in a dramatic decrease in actinorhodin (ACT) and undecylprodiginine (RED) production and delays morphological development. In contrast, the overexpression ofabrC3in the parent strain leads to a 33% increase in ACT production in liquid medium. Transcriptomic analysis and chromatin immunoprecipitation with microarray technology (ChIP-chip) analysis of the ΔabrC3mutant and the parent strain revealed that AbrC3 directly controls ACT production by binding to theactII-ORF4promoter region; this was independently verified byin vitroDNA-binding assays. This binding is dependent on the sequence 5′-GAASGSGRMS-3′. In contrast, the regulation of RED production is not due to direct binding of AbrC3 to either theredZorredDpromoter region. This study also revealed other members of the AbrC3 regulon: AbrC3 is a positive autoregulator which also binds to the promoter regions ofSCO0736,bdtA(SCO3328),absR1(SCO6992), andSCO6809. The direct targets share the 10-base consensus binding sequence and may be responsible for some of the phenotypes of the ΔabrC3mutant. The identification of the AbrC3 regulon as part of the complex regulatory network governing antibiotic production widens our knowledge regarding TCS involvement in control of antibiotic synthesis and may contribute to the rational design of new hyperproducer host strains through genetic manipulation of such systems.

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Hydrolytic dehalogenation operon of 4-chlorobenzoate is transcriptionally regulated by the TetR-type repressor FcbR and its ligand 4-chlorobenzoyl-CoA

Applied and Environmental Microbiology ◽

10.1128/aem.02652-20 ◽

2021 ◽

Author(s):

Minggen Cheng ◽

Dongmei Pei ◽

Xiangrong He ◽

Yongchuang Liu ◽

Pingping Zhu ◽

...

Keyword(s):

Transcriptional Repressor ◽

Constitutive Expression ◽

Transcriptional Regulators ◽

Dna Probe ◽

Titration Calorimetry ◽

Binding Motif ◽

Catabolic Pathway ◽

Catabolic Genes ◽

Activation Type

The bacterial hydrolytic dehalogenation of 4-chlorobenzoate (4CBA) is a CoA-activation type catabolic pathway that is usually a common part of the microbial mineralization of chlorinated aromatic compounds. Previous studies have shown that the transport and dehalogenation genes for 4CBA are typically clustered as an fcbBAT1T2T3C operon and are inducibly expressed in response to 4CBA. However, the associated molecular mechanism remains unknown. In this study, a gene (fcbR) adjacent to the fcb operon was predicted to encode a TetR-type transcriptional regulator in strain Comamonas sediminis CD-2. The fcbR knockout strain exhibited constitutive expression of the fcb cluster. In the host E. coli, the expression of the Pfcb-fused gfp reporter was repressed by the introduction of the fcbR gene, and genetic studies combining various catabolic genes suggest that the FcbR ligand may be an intermediate metabolite. Purified FcbR could bind to the Pfcb DNA probe in vitro, and the metabolite 4-chlorobenzyl-CoA (4CBA-CoA) prevented FcbR binding to the Pfcb DNA probe. Isothermal titration calorimetry (ITC) measurements showed that 4CBA-CoA could bind to FcbR at a 1:1 mole ratio. DNase I footprinting showed that FcbR protected a 42-bp DNA motif (5′-GGAAATCAATAGGTCCATAGAAAATCTATTGACTAATCGAAT-3′) that consists of two sequence repeats containing four pseudo-palindromic sequences (5’-TCNATNGA-3’). This binding motif overlaps with the -35 box of Pfcb and was proposed to prevent the binding of RNA polymerase. This study identified a transcriptional repressor and its ligand of the fcb operon, extending halogenated benzoyl-CoA as a member of known ligands of transcriptional regulators. Importance The bacterial hydrolytic dehalogenation of 4CBA is a special CoA-activation type catabolic pathway, which plays an important role in the biodegradation of polychlorinated biphenyls and many certain herbicides. With genetic and biochemical approaches, the present study identified the transcriptional repressor and its cognate effector of a 4CBA hydrolytic dehalogenation operon. This work extends halogenated benzoyl-CoA as a new member of CoA-derived effector compounds that mediate allosteric regulation of transcriptional regulators.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00819-10 ◽

2010 ◽

Vol 76 (23) ◽

pp. 7741-7753 ◽

Cited By ~ 24

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.

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In VivoStudies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00523-13 ◽

2013 ◽

Vol 57 (9) ◽

pp. 4470-4480 ◽

Cited By ~ 29

Author(s):

Min Jung Kwun ◽

Gabriela Novotna ◽

Andrew R. Hesketh ◽

Lionel Hill ◽

Hee-Jeon Hong

Keyword(s):

Cell Wall ◽

Transcriptional Activation ◽

Streptomyces Coelicolor ◽

Constitutive Expression ◽

Transcriptional Response ◽

Vancomycin Resistance ◽

Content Type ◽

Expression Of Genes ◽

Peptidoglycan Cell Wall

ABSTRACTVanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system inStreptomyces coelicoloras a model, we have undertaken a series ofin vivostudies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with thed-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essentiald-Ala-d-Ala ligase activity by constitutive expression ofvanAencoding a bifunctionald-Ala-d-Ala andd-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containingd-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance ofd-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating ind-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask thed-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting withd-Ala-d-Ala residues, failed to inducevangene expression. Activation of resistance by a vancomycin–d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating ind-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.

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Novel Pathway for the Degradation of 2-Chloro-4-Nitrobenzoic Acid by Acinetobacter sp. Strain RKJ12

Applied and Environmental Microbiology ◽

10.1128/aem.00685-11 ◽

2011 ◽

Vol 77 (18) ◽

pp. 6606-6613 ◽

Cited By ~ 11

Author(s):

Dhan Prakash ◽

Ravi Kumar ◽

R. K. Jain ◽

B. N. Tiwary

Keyword(s):

Salicylic Acid ◽

Tricarboxylic Acid Cycle ◽

Sole Source ◽

Degradation Pathway ◽

Ring Cleavage ◽

Muconic Acid ◽

Content Type ◽

Nitrobenzoic Acid ◽

Catabolic Genes ◽

Catechol 1,2 Dioxygenase

ABSTRACTThe organismAcinetobactersp. RKJ12 is capable of utilizing 2-chloro-4-nitrobenzoic acid (2C4NBA) as a sole source of carbon, nitrogen, and energy. In the degradation of 2C4NBA by strain RKJ12, various metabolites were isolated and identified by a combination of chromatographic, spectroscopic, and enzymatic activities, revealing a novel assimilation pathway involving both oxidative and reductive catabolic mechanisms. The metabolism of 2C4NBA was initiated by oxidativeorthodehalogenation, leading to the formation of 2-hydroxy-4-nitrobenzoic acid (2H4NBA), which subsequently was metabolized into 2,4-dihydroxybenzoic acid (2,4-DHBA) by a mono-oxygenase with the concomitant release of chloride and nitrite ions. Stoichiometric analysis indicated the consumption of 1 mol O2per conversion of 2C4NBA to 2,4-DHBA, ruling out the possibility of two oxidative reactions. Experiments with labeled H218O and18O2indicated the involvement of mono-oxygenase-catalyzed initial hydrolytic dechlorination and oxidative denitration mechanisms. The further degradation of 2,4-DHBA then proceeds via reductive dehydroxylation involving the formation of salicylic acid. In the lower pathway, the organism transformed salicylic acid into catechol, which was mineralized by theorthoring cleavage catechol-1,2-dioxygenase tocis, cis-muconic acid, ultimately forming tricarboxylic acid cycle intermediates. Furthermore, the studies carried out on a 2C4NBA−derivative and a 2C4NBA+transconjugant demonstrated that the catabolic genes for the 2C4NBA degradation pathway possibly reside on the ∼55-kb transmissible plasmid present in RKJ12.

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Relationship between Glycopeptide Production and Resistance in the Actinomycete Nonomuraea sp. ATCC 39727

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02626-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5191-5201 ◽

Cited By ~ 16

Author(s):

Giorgia Letizia Marcone ◽

Elisa Binda ◽

Lucia Carrano ◽

Mervyn Bibb ◽

Flavia Marinelli

Keyword(s):

Antibiotic Resistance ◽

Gene Dosage ◽

Antibiotic Production ◽

Resistance Mechanism ◽

Biosynthetic Gene Cluster ◽

Penicillin G ◽

Glycopeptide Resistance ◽

Content Type ◽

High Level

ABSTRACTGlycopeptides and β-lactams inhibit bacterial peptidoglycan synthesis in Gram-positive bacteria; resistance to these antibiotics is studied intensively in enterococci and staphylococci because of their relevance to infectious disease. Much less is known about antibiotic resistance in glycopeptide-producing actinomycetes that are likely to represent the evolutionary source of resistance determinants found in bacterial pathogens.Nonomuraeasp. ATCC 39727, the producer of A40926 (the precursor for the semisynthetic dalbavancin), does not harbor the canonicalvanHAXgenes. Consequently, we investigated the role of the β-lactam-sensitived,d-peptidase/d,d-carboxypeptidase encoded byvanYn, the onlyvan-like gene found in the A40926 biosynthetic gene cluster, in conferring immunity to the antibiotic inNonomuraeasp. ATCC 39727. Taking advantage of the tools developed recently to genetically manipulate this uncommon actinomycete, we variedvanYngene dosage and expressedvanHatAatXatfrom the teicoplanin producerActinoplanes teichomyceticusinNonomuraeasp. ATCC 39727. Knocking outvanYn, complementing avanYnmutant, or duplicatingvanYnhad no effect on growth but influenced antibiotic resistance and, in the cases of complementation and duplication, antibiotic production.Nonomuraeasp. ATCC 39727 was found to be resistant to penicillins, but its glycopeptide resistance was diminished in the presence of penicillin G, which inhibits VanYnactivity. The heterologous expression ofvanHatAatXatincreased A40926 resistance inNonomuraeasp. ATCC 39727 but did not increase antibiotic production, indicating that the level of antibiotic production is not directly determined by the level of resistance. ThevanYn-based self-resistance inNonomuraeasp. ATCC 39727 resembles the glycopeptide resistance mechanism described recently in mutants ofEnterococcus faeciumselectedin vitrofor high-level resistance to glycopeptides and penicillins.

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Dramatic Activation of Antibiotic Production in Streptomyces coelicolor by Cumulative Drug Resistance Mutations

Applied and Environmental Microbiology ◽

10.1128/aem.02800-07 ◽

2008 ◽

Vol 74 (9) ◽

pp. 2834-2840 ◽

Cited By ~ 100

Author(s):

Guojun Wang ◽

Takeshi Hosaka ◽

Kozo Ochi

Keyword(s):

Drug Resistance ◽

Multiple Drug Resistance ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Strain Improvement ◽

Multiple Drug ◽

Resistance Mutations ◽

Drug Resistance Mutations ◽

Order Of Magnitude

ABSTRACT We recently described a new method to activate antibiotic production in bacteria by introducing a mutation conferring resistance to a drug such as streptomycin, rifampin, paromomycin, or gentamicin. This method, however, enhanced antibiotic production by only up to an order of magnitude. Working with Streptomyces coelicolor A3(2), we established a method for the dramatic activation of antibiotic production by the sequential introduction of multiple drug resistance mutations. Septuple and octuple mutants, C7 and C8, thus obtained by screening for resistance to seven or eight drugs, produced huge amounts (1.63 g/liter) of the polyketide antibiotic actinorhodin, 180-fold higher than the level produced by the wild type. This dramatic overproduction was due to the acquisition of mutant ribosomes, with aberrant protein and ppGpp synthesis activity, as demonstrated by in vitro protein synthesis assays and by the abolition of antibiotic overproduction with relA disruption. This new approach, called “ribosome engineering,” requires less time, cost, and labor than other methods and may be widely utilized for bacterial strain improvement.

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RNase III Is Required for Actinomycin Production in Streptomyces antibioticus

Applied and Environmental Microbiology ◽

10.1128/aem.02272-13 ◽

2013 ◽

Vol 79 (20) ◽

pp. 6447-6451 ◽

Cited By ~ 6

Author(s):

Jung-Hoon Lee ◽

Marcha L. Gatewood ◽

George H. Jones

Keyword(s):

Parental Strain ◽

Insertional Mutagenesis ◽

Southern Blotting ◽

Antibiotic Production ◽

Streptomyces Coelicolor ◽

Production Medium ◽

Wild Type ◽

Rnase Iii ◽

Content Type ◽

Streptomyces Antibioticus

ABSTRACTUsing insertional mutagenesis, we have disrupted the RNase III gene,rnc, of the actinomycin-producing streptomycete,Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of therncdisruption with the wild-typerncgene fromS. antibioticusrestored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case inStreptomyces coelicolor, RNase III is required for antibiotic production inS. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of theS. antibioticus rncmutant and its parental strain.

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An In Vitro TORC1 Kinase Assay That Recapitulates the Gtr-Independent Glutamine-Responsive TORC1 Activation Mechanism on Yeast Vacuoles

Molecular and Cellular Biology ◽

10.1128/mcb.00075-17 ◽

2017 ◽

Vol 37 (14) ◽

Cited By ~ 26

Author(s):

Mirai Tanigawa ◽

Tatsuya Maeda

Keyword(s):

Amino Acids ◽

Nitrogen Sources ◽

Small Gtpases ◽

Activation Mechanism ◽

Kinase Assay ◽

Content Type ◽

Vacuolar Protein ◽

Vacuolar Membranes ◽

First Time

ABSTRACT Evolutionarily conserved target of rapamycin (TOR) complex 1 (TORC1) responds to nutrients, especially amino acids, to promote cell growth. In the yeast Saccharomyces cerevisiae, various nitrogen sources activate TORC1 with different efficiencies, although the mechanism remains elusive. Leucine, and perhaps other amino acids, was reported to activate TORC1 via the heterodimeric small GTPases Gtr1-Gtr2, the orthologues of the mammalian Rag GTPases. More recently, an alternative Gtr-independent TORC1 activation mechanism that may respond to glutamine was reported, although its molecular mechanism is not clear. In studying the nutrient-responsive TORC1 activation mechanism, the lack of an in vitro assay hinders associating particular nutrient compounds with the TORC1 activation status, whereas no in vitro assay that shows nutrient responsiveness has been reported. In this study, we have developed a new in vitro TORC1 kinase assay that reproduces, for the first time, the nutrient-responsive TORC1 activation. This in vitro TORC1 assay recapitulates the previously predicted Gtr-independent glutamine-responsive TORC1 activation mechanism. Using this system, we found that this mechanism specifically responds to l-glutamine, resides on the vacuolar membranes, and involves a previously uncharacterized Vps34-Vps15 phosphatidylinositol (PI) 3-kinase complex and the PI-3-phosphate [PI(3)P]-binding FYVE domain-containing vacuolar protein Pib2. Thus, this system was proved to be useful for dissecting the glutamine-responsive TORC1 activation mechanism.

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