scholarly journals Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation

2013 ◽  
Vol 79 (7) ◽  
pp. 2349-2357 ◽  
Author(s):  
Li Li ◽  
Jun Wu ◽  
Zixin Deng ◽  
T. Mark Zabriskie ◽  
Xinyi He
Keyword(s):  
Gene Cluster ◽  
Genetic Manipulation ◽  
Biosynthetic Gene Cluster ◽  
Streptomyces Lividans ◽  
Dna Uptake ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Blasticidin S

ABSTRACTBlasticidin S is a peptidyl nucleoside antibiotic produced byStreptomyces griseochromogenesthat exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesisin vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome ofStreptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, forS. lividansblasticidin S deaminase) was identified inS. lividansand shown to govern thisin vivoconversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin Sin vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene inS. lividansLL2 led to successful production of active blasticidin S in the resultant mutant,S. lividansWJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster,blsE,blsF, andblsL, encoding a predicted radicalS-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis.

scholarly journals A Two-Step Mechanism for the Activation of Actinorhodin Export and Resistance in Streptomyces coelicolor

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Ye Xu ◽  
Andrew Willems ◽  
Catherine Au-yeung ◽  
Kapil Tahlan ◽  
Justin R. Nodwell
Keyword(s):  
Secondary Metabolites ◽  
Pathogenic Bacteria ◽  
Streptomyces Coelicolor ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistance Mechanisms ◽  
Biosynthetic Gene ◽  
Content Type

ABSTRACT Many microorganisms produce secondary metabolites that have antibiotic activity. To avoid self-inhibition, the producing cells often encode cognate export and/or resistance mechanisms in the biosynthetic gene clusters for these molecules. Actinorhodin is a blue-pigmented antibiotic produced by Streptomyces coelicolor. The actAB operon, carried in the actinorhodin biosynthetic gene cluster, encodes two putative export pumps and is regulated by the transcriptional repressor protein ActR. In this work, we show that normal actinorhodin yields require actAB expression. Consistent with previous in vitro work, we show that both actinorhodin and its 3-ring biosynthetic intermediates [e.g., (S)-DNPA] can relieve repression of actAB by ActR in vivo. Importantly, an ActR mutant that interacts productively with (S)-DNPA but not with actinorhodin responds to the actinorhodin biosynthetic pathway with the induction of actAB and normal yields of actinorhodin. This suggests that the intermediates are sufficient to trigger the export genes in actinorhodin-producing cells. We further show that actinorhodin-producing cells can induce actAB expression in nonproducing cells; however, in this case actinorhodin is the most important signal. Finally, while the “intermediate-only” ActR mutant permits sufficient actAB expression for normal actinorhodin yields, this expression is short-lived. Sustained culture-wide expression requires a subsequent actinorhodin-mediated signaling step, and the defect in this response causes widespread cell death. These results are consistent with a two-step model for actinorhodin export and resistance where intermediates trigger initial expression for export from producing cells and actinorhodin then triggers sustained export gene expression that confers culture-wide resistance. IMPORTANCE Understanding the links between antibiotic resistance and biosynthesis is important for our efforts to manipulate secondary metabolism. For example, many secondary metabolites are produced at low levels; our work suggests that manipulating export might be one way to enhance yields of these molecules. It also suggests that understanding resistance will be relevant to the generation of novel secondary metabolites through the creation of synthetic secondary metabolic gene clusters. Finally, these cognate resistance mechanisms are related to mechanisms that arise in pathogenic bacteria, and understanding them is relevant to our ability to control microbial infections clinically.

scholarly journals Heterologous Expression of Pseudouridimycin and Description of the Corresponding Minimal Biosynthetic Gene Cluster

Molecules ◽  
2021 ◽  
Vol 26 (2) ◽  
pp. 510
Author(s):  
Nils Böhringer ◽  
Maria A. Patras ◽  
Till F. Schäberle
Keyword(s):  
Heterologous Expression ◽  
Gene Cluster ◽  
Streptomyces Coelicolor ◽  
Biosynthetic Gene Cluster ◽  
Infection Model ◽  
Biosynthetic Gene ◽  
Mouse Infection Model ◽  
Production Platform

Pseudouridimycin (PUM) was recently discovered from Streptomyces sp. DSM26212 as a novel bacterial nucleoside analog that competes with UTP for access to the RNA polymerase (RNAP) active site, thereby inhibiting bacterial RNAP by blocking transcription. This represents a novel antibacterial mode of action and it is known that PUM inhibits bacterial RNAP in vitro, inhibits bacterial growth in vitro, and was active in vivo in a mouse infection model of Streptococcus pyogenes peritonitis. The biosynthetic gene cluster (BGC) was previously identified and characterized by knockout experiments. However, the minimal set of genes necessary for PUM production was not proposed. To identify the minimal BGC and to create a plug-and-play production platform for PUM and its biosynthetic precursors, several versions of a redesigned PUM BGC were generated and expressed in the heterologous host Streptomyces coelicolor M1146 under control of strong promotors. Heterologous expression allowed identification of the putative serine/threonine kinase PumF as an enzyme essential for heterologous PUM production and thus corroboration of the PUM minimal BGC.

scholarly journals Hawaiian Bobtail Squid Symbionts Inhibit Marine Bacteria via Production of Specialized Metabolites, Including New Bromoalterochromides BAC-D/D′

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Andrea M. Suria ◽  
Karen C. Tan ◽  
Allison H. Kerwin ◽  
Lucas Gitzel ◽  
Lydia Abini-Agbomson ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Gene Cluster ◽  
Marine Bacteria ◽  
Pathogenic Fungus ◽  
Biosynthetic Gene Cluster ◽  
No Production ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Egg Defense

ABSTRACT The Hawaiian bobtail squid, Euprymna scolopes, has a symbiotic bacterial consortium in the accessory nidamental gland (ANG), a female reproductive organ that protects eggs against fouling microorganisms. To test the antibacterial activity of ANG community members, 19 bacterial isolates were screened for their ability to inhibit Gram-negative and Gram-positive bacteria, of which two strains were inhibitory. These two antibacterial isolates, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, were subjected to further genomic characterization. Genomic analysis of Leisingera sp. ANG59 revealed a biosynthetic gene cluster encoding the antimicrobial compound indigoidine. The genome of Pseudoalteromonas sp. JC28 had a 14-gene cluster with >95% amino acid identity to a known bromoalterochromide (BAC) cluster. Chemical analysis confirmed production of known BACs, BAC-A/A′ (compounds 1a/1b), as well as two new derivatives, BAC-D/D′ (compounds 2a/2b). Extensive nuclear magnetic resonance (NMR) analyses allowed complete structural elucidation of compounds 2a/2b, and the absolute stereochemistry was unambiguously determined using an optimized Marfey’s method. The BACs were then investigated for in vitro antibacterial, antifungal, and nitric oxide (NO) inhibitory activity. Compounds 1a/1b were active against the marine bacteria Bacillus algicola and Vibrio fischeri, while compounds 2a/2b were active only against B. algicola. Compounds 1a/1b inhibited NO production via lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells and also inhibited the pathogenic fungus Fusarium keratoplasticum, which, coupled with their antibacterial activity, suggests that these polyketide-nonribosomal peptides may be used for squid egg defense against potential pathogens and/or fouling microorganisms. These results indicate that BACs may provide Pseudoalteromonas sp. JC28 an ecological niche, facilitating competition against nonsymbiotic microorganisms in the host’s environment. IMPORTANCE Animals that deposit eggs must protect their embryos from fouling and disease by microorganisms to ensure successful development. Although beneficial bacteria are hypothesized to contribute to egg defense in many organisms, the mechanisms of this protection are only recently being elucidated. Our previous studies of the Hawaiian bobtail squid focused on fungal inhibition by beneficial bacterial symbionts of a female reproductive gland and eggs. Herein, using genomic and chemical analyses, we demonstrate that symbiotic bacteria from this gland can also inhibit other marine bacteria in vitro. One bacterial strain in particular, Pseudoalteromonas sp. JC28, had broad-spectrum abilities to inhibit potential fouling bacteria, in part via production of novel bromoalterochromide metabolites, confirmed via genomic annotation of the associated biosynthetic gene cluster. Our results suggest that these bacterial metabolites may contribute to antimicrobial activity in this association and that such defensive symbioses are underutilized sources for discovering novel antimicrobial compounds.

scholarly journals Study of bicyclomycin biosynthesis in Streptomyces cinnamoneus by genetic and biochemical approaches

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jerzy Witwinowski ◽  
Mireille Moutiez ◽  
Matthieu Coupet ◽  
Isabelle Correia ◽  
Pascal Belin ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biological Activities ◽  
Large Family ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Bacterial Transcription ◽  
Bicyclic Structure ◽  
Transcription Termination Factor

AbstractThe 2,5-Diketopiperazines (DKPs) constitute a large family of natural products with important biological activities. Bicyclomycin is a clinically-relevant DKP antibiotic that is the first and only member in a class known to target the bacterial transcription termination factor Rho. It derives from cyclo-(l-isoleucyl-l-leucyl) and has an unusual and highly oxidized bicyclic structure that is formed by an ether bridge between the hydroxylated terminal carbon atom of the isoleucine lateral chain and the alpha carbon of the leucine in the diketopiperazine ring. Here, we paired in vivo and in vitro studies to complete the characterization of the bicyclomycin biosynthetic gene cluster. The construction of in-frame deletion mutants in the biosynthetic gene cluster allowed for the accumulation and identification of biosynthetic intermediates. The identity of the intermediates, which were reproduced in vitro using purified enzymes, allowed us to characterize the pathway and corroborate previous reports. Finally, we show that the putative antibiotic transporter was dispensable for the producing strain.

scholarly journals Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

2017 ◽  
Vol 114 (27) ◽  
pp. 7025-7030 ◽  
Author(s):  
Nicholas C. Harris ◽  
Michio Sato ◽  
Nicolaus A. Herman ◽  
Frederick Twigg ◽  
Wenlong Cai ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Acyl Chain ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Nonribosomal Peptide Synthetase ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.

scholarly journals Refactoring the formicamycin biosynthetic gene cluster to make high-level producing strains and new molecules

2020 ◽  
Author(s):  
Rebecca Devine ◽  
Hannah McDonald ◽  
Zhiwei Qin ◽  
Corinne Arnold ◽  
Katie Noble ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Large Scale ◽  
Liquid Culture ◽  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Resistant Bacteria ◽  
Drug Resistant ◽  
Biosynthetic Gene ◽  
Biosynthetic Genes

AbstractThe formicamycins are promising antibiotics with potent activity against Gram-positive pathogens including VRE and MRSA and display a high barrier to selection of resistant isolates. They were first identified in Streptomyces formicae KY5, which produces the formicamycins at low levels on solid agar but not in liquid culture, thus hindering further investigation of these promising antibacterial compounds. We hypothesised that by understanding the organisation and regulation of the for biosynthetic gene cluster, we could rationally refactor the cluster to increase production levels. Here we report that the for biosynthetic gene cluster consists of 24 genes expressed on nine transcripts. Seven of these transcripts, including those containing all the major biosynthetic genes, are repressed by the MarR-regulator ForJ which also controls the expression of the ForGF two-component system that initiates biosynthesis. A third cluster-situated regulator, ForZ, autoregulates and controls production of the putative MFS transporter ForAA. Consistent with these findings, deletion of forJ increased formicamycin biosynthesis 5-fold, while over-expression of forGF in the ΔforJ background increased production 10-fold compared to the wild-type. De-repression by deleting forJ also switched on biosynthesis in liquid-culture and induced the production of two novel formicamycin congeners. By combining mutations in regulatory and biosynthetic genes, six new biosynthetic precursors with antibacterial activity were also isolated. This work demonstrates the power of synthetic biology for the rational redesign of antibiotic biosynthetic gene clusters both to engineer strains suitable for fermentation in large scale bioreactors and to generate new molecules.ImportanceAntimicrobial resistance is a growing threat as existing antibiotics become increasingly ineffective against drug resistant pathogens. Here we determine the transcriptional organisation and regulation of the gene cluster encoding biosynthesis of the formicamycins, promising new antibiotics with activity against drug resistant bacteria. By exploiting this knowledge, we construct stable mutant strains which over-produce these molecules in both liquid and solid culture whilst also making some new compound variants. This will facilitate large scale purification of these molecules for further study including in vivo experiments and the elucidation of their mechanism of action. Our work demonstrates that understanding the regulation of natural product biosynthetic pathways can enable rational improvement of the producing strains.

Genetic localization and in vivo characterization of a Monascus azaphilone pigment biosynthetic gene cluster

2013 ◽  
Vol 97 (14) ◽  
pp. 6337-6345 ◽  
Author(s):  
Bijinu Balakrishnan ◽  
Suman Karki ◽  
Shih-Hau Chiu ◽  
Hyun-Ju Kim ◽  
Jae-Won Suh ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Genetic Localization ◽  
In Vivo Characterization ◽  
Azaphilone Pigment

scholarly journals Nonselective Persistence of a Rickettsia conorii Extrachromosomal Plasmid during Mammalian Infection

2016 ◽  
Vol 84 (3) ◽  
pp. 790-797 ◽  
Author(s):  
Sean P. Riley ◽  
Abigail I. Fish ◽  
Daniel A. Garza ◽  
Kaikhushroo H. Banajee ◽  
Emma K. Harris ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Genetic Manipulation ◽  
Fluorescent Proteins ◽  
Spotted Fever ◽  
Rickettsia Conorii ◽  
Mediterranean Spotted Fever ◽  
Extrachromosomal Dna ◽  
Content Type

Scientific analysis of the genusRickettsiais undergoing a rapid period of change with the emergence of viable genetic tools. The development of these tools for the mutagenesis of pathogenic bacteria will permit forward genetic analysis ofRickettsiapathogenesis. Despite these advances, uncertainty still remains regarding the use of plasmids to study these bacteria inin vivomammalian models of infection, namely, the potential for virulence changes associated with the presence of extrachromosomal DNA and nonselective persistence of plasmids in mammalian models of infection. Here, we describe the transformation ofRickettsia conoriiMalish 7 with the plasmid pRam18dRGA[AmTrCh]. TransformedR. conoriistably maintains this plasmid in infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth kinetics in cell culture similar to those of nontransformedR. conorii. Using a well-established murine model of fatal Mediterranean spotted fever, we demonstrate thatR. conorii(pRam18dRGA[AmTrCh]) elicits the same fatal outcomes in animals as its untransformed counterpart and, importantly, maintains the plasmid throughout infection in the absence of selective antibiotic pressure. Interestingly, plasmid-transformedR. conoriiwas readily observed both in endothelial cells and within circulating leukocytes. Together, our data demonstrate that the presence of an extrachromosomal DNA element in a pathogenic rickettsial species does not affect eitherin vitroproliferation orin vivoinfectivity in models of disease and that plasmids such as pRam18dRGA[AmTrCh] are valuable tools for the further genetic manipulation of pathogenic rickettsiae.

scholarly journals Regiospecificities and Prenylation Mode Specificities of the Fungal Indole Diterpene Prenyltransferases AtmD and PaxD

2013 ◽  
Vol 79 (23) ◽  
pp. 7298-7304 ◽  
Author(s):  
Chengwei Liu ◽  
Atsushi Minami ◽  
Motoyoshi Noike ◽  
Hiroaki Toshima ◽  
Hideaki Oikawa ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Biosynthetic Gene Cluster ◽  
Amino Acid Identity ◽  
Biosynthetic Gene ◽  
Content Type ◽  
Acid Identity ◽  
Dimethylallyl Diphosphate ◽  
Regular Type ◽  
Penicillium Paxilli ◽  
Related Compounds

ABSTRACTWe recently reported the function ofpaxD, which is involved in the paxilline (compound 1) biosynthetic gene cluster inPenicillium paxilli. Recombinant PaxD catalyzed a stepwise regular-type diprenylation at the 21 and 22 positions of compound 1 with dimethylallyl diphosphate (DMAPP) as the prenyl donor. In this study,atmD, which is located in the aflatrem (compound 2) biosynthetic gene cluster inAspergillus flavusand encodes an enzyme with 32% amino acid identity to PaxD, was characterized using recombinant enzyme. When compound 1 and DMAPP were used as substrates, two major products and a trace of minor product were formed. The structures of the two major products were determined to be reversely monoprenylated compound 1 at either the 20 or 21 position. Because compound 2 and β-aflatrem (compound 3), both of which are compound 1-related compounds produced byA. flavus, have the same prenyl moiety at the 20 and 21 position, respectively, AtmD should catalyze the prenylation in compound 2 and 3 biosynthesis. More importantly and surprisingly, AtmD accepted paspaline (compound 4), which is an intermediate of compound 1 biosynthesis that has a structure similar to that of compound 1, and catalyzed a regular monoprenylation of compound 4 at either the 21 or 22 position, though the reverse prenylation was observed with compound 1. This suggests that fungal indole diterpene prenyltransferases have the potential to alter their position and regular/reverse specificities for prenylation and could be applicable for the synthesis of industrially useful compounds.

scholarly journals An Orphan MbtH-Like Protein Interacts with Multiple Nonribosomal Peptide Synthetases inMyxococcus xanthusDK1622

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Karla J. Esquilín-Lebrón ◽  
Tye O. Boynton ◽  
Lawrence J. Shimkets ◽  
Michael G. Thomas
Keyword(s):  
Myxococcus Xanthus ◽  
Gene Clusters ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Nonribosomal Peptide ◽  
Content Type ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases

ABSTRACTOne mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 fromMyxococcus xanthusDK1622 for characterization. TheM. xanthusDK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination ofin vivoandin vitroassays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping ofM. xanthusDK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a “universal” MLP for generating functional hybrid NRPSs.IMPORTANCEMbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP fromMyxococcus xanthusDK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a “universal” MLP during the construction of functional hybrid NRPSs.

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