Antibiotics: opportunities for genetic manipulation

1989 ◽  
Vol 324 (1224) ◽  
pp. 549-562 ◽  
Keyword(s):  
Genetic Engineering ◽  
Genetic Manipulation ◽  
Antibiotic Production ◽  
Morphological Differentiation ◽  
Basic Structure ◽  
Antibiotic Biosynthesis ◽  
Full Potential ◽  
Metabolite Formation ◽  
Random Mutation

New antibiotics can still be discovered by the development of novel screening procedures. Notable successes over the last few years include the monobactams, β- lactamase inhibitors (clavulanic acid) and new glycopeptides in the antibacterial held; antiparasitic agents such as avermectins; and herbicidal antibiotics like bialaphos. In the future we can expect the engineering of genes from ‘difficult’ pathogens, including mycobacteria and fungi, and cancer cells, to provide increasingly useful in vitro targets for the screening of antibiotics that can kill pathogens and tumours. There will also be a greater awareness of the need to reveal the full potential for antibiotic production on the part of microorganisms by the physiologial and/or genetic awakening of ‘silent’ genes. Nevertheless, the supply of natural antibiotics for direct use or chemical modification is not infinite and there will be increasing scope for widening the range of available antibiotics by genetic engineering. ‘Hybrid’ antibiotics have been shown to be generated by the transfer of genes on suitable vectors between strains producing chemically related compounds. More exciting is the possibility of generating novelty by the genetic engineering of the synthases that determine the basic structure of antibiotics belonging to such classes as the β-lactams and polyketides. Research in this area will certainly yield knowledge of considerable scientific interest and probably also of potential applicability. In the improvement of antibiotic titre in actinomycetes, protoplast fusion between divergent selection lines has taken a place alongside random mutation and screening. In some cases the cloning of genes controlling metabolic ‘bottlenecks’ in fungi and actinomycetes will give an immediate benefit in the conversion of accumulated biosynthetic intermediates to the desired end product. However, the main impact of genetic engineering in titre improvement will probably come only after a further use of this technology to understand and manipulate the regulation of antibiotic biosynthesis as a facet of the general challenge of understanding differential gene expression. Streptomyces offers a particularly fertile field for such research, following the isolation of DNA segments that carry groups of closely linked operons for the biosynthesis of and resistance to particular antibiotics, and of genes with pleiotropic effects on morphological differentiation and secondary metabolite formation.

The Leeuwenhoek Lecture, 1987 - Towards an understanding of gene switching in Streptomyces , the basis of sporulation and antibiotic production

1988 ◽  
Vol 235 (1279) ◽  
pp. 121-138 ◽  
Keyword(s):  
Life Cycle ◽  
Secondary Metabolites ◽  
Vegetative Growth ◽  
Antibiotic Production ◽  
Morphological Differentiation ◽  
Aerial Mycelium ◽  
Secondary Phase ◽  
Antibiotic Biosynthesis ◽  
Regulatory Cascade ◽  
Pharmacologically Active

Streptomycetes are soil bacteria that differ from the genetically well-known Escherichia coli in two striking characteristics. (1) Instead of consisting of an alternation of growth and fission of morphologically simple, undifferentiated rods, the streptomycete life cycle involves the formation of a system of elongated, branching hyphae which, after a period of vegetative growth, respond to specific signals by producing specialized spore-bearing structures. (2) The streptomycetes produce an unrivalled range of chemically diverse ‘secondary metabolites’, which we recognize as antibiotics, herbicides and pharmacologically active molecules, and which presumably play an important role in the streptomycete life cycle in nature. This ‘physiological’ differentiation is often tem­porally associated with the morphological differentiation of sporulation and there are common elements in the regulation of the two sets of processes. In the model system provided by Streptomyces coelicolor A3(2), the isolation of several whole clusters of linked antibiotic biosynthetic pathway genes, and some key regulatory genes involved in sporulation, has made it possible to study the basis for the switching on and off of particular sets of genes during morphological and ‘physiological’ differen­tiation. Genetic analysis clearly reveals a regulatory cascade operating at several levels in a ‘physiological’ branch of the differentiation control system. At the lowest level, within individual clusters of antibiotic biosynthesis genes are genes with a role as activators of the structural genes for the pathway enzymes, and also resistance genes. It is attractive to speculate that the latter play a dual role: protecting the organism from self-destruction by its own potentially lethal product, and forming an essential component of a regulatory circuit that activates the biosyn­thetic genes, thus ensuring that resistance is established before any antibiotic is made. A next higher level of regulation is revealed by the isolation of mutations in a gene ( afsB ) required for expression (probably at the level of transcription) of all five known secondary metabolic pathways in the organism. At a higher level still, the bldA gene, whose product seems to be a tRNA essential to translate the rare (in high [G + C] Streptomyces DNA) TTA leucine codon, controls or influences the whole gamut of morphological and ‘physiological’ differentiation, because bldA mutants fail to produce either secondary metabolites or aerial mycelium and spores, while growing normally in the vegetative phase. Thus a decision to switch from vegetative growth to the secondary phase of colonial development may be taken at the level of translation. In the ‘morphological’ branch of the proposed regulatory cascade, a key gene is whiG whose product, essential for the earliest known step in the metamorphosis of aerial hyphae into spore chains, appears to be an RNA polymerase sigma factor which is not needed for transcription of vegetative genes, but seems to control, at the level of transcription, the decision to sporulate.

scholarly journals The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

2011 ◽  
Vol 77 (21) ◽  
pp. 7586-7594 ◽  
Author(s):  
Fernando Santos-Beneit ◽  
Mónica Barriuso-Iglesias ◽  
Lorena T. Fernández-Martínez ◽  
Miriam Martínez-Castro ◽  
Alberto Sola-Landa ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Response Regulator ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Luciferase Reporter ◽  
Antibiotic Biosynthesis ◽  
Binding Assays ◽  
Content Type ◽  
Phosphate Depletion

ABSTRACTThe RNA polymerase (RNAP) omega factor (ω) forms a complex with the α2ββ′ core of this enzyme in bacteria. We have characterized therpoZgene ofStreptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of therpoZgene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ΔrpoZstrain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ΔrpoZmutant with the wild-typerpoZallele restored both phenotype and antibiotic production. Interestingly, therpoZgene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed thatrpoZpromoter activity was increased in a ΔphoPbackground, it can be concluded thatrpoZis controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation inStreptomyces.

scholarly journals Genetic Engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete

2021 ◽  
Author(s):  
Emily Romeis ◽  
Lauren Tantalo ◽  
Nicole Lieberman ◽  
Quynh Phung ◽  
Alex Greninger ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Vaccine Development ◽  
Genetic Manipulation ◽  
Treponema Pallidum ◽  
Gene Promoter ◽  
Digital Pcr ◽  
Kanamycin Resistance ◽  
In Vitro Cultivation

Background.  Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum ( T. pallidum ), the causative of agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA ( tp0009 ) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance ( kan R ) cassette.               Principal findings. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kan R gene was cloned downstream of the tp0574 gene promoter. The tp0574 prom- kan R cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kan R cassette into T. pallidum chromosome, in vitro -cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl 2 -based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kan R cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole genome sequencing (WGS) of transformed treponemes propagated in vitro and in vivo . ddPCR analysis of RNA and mass spectrometry confirmed expression of the kan R message and protein in treponemes propagated in vitro . Moreover, tprA knockout ( tprA ko -SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin.             Conclusion. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

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

2014 ◽  
Vol 80 (8) ◽  
pp. 2417-2428 ◽  
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.

scholarly journals The relA/spoT-Homologous Gene inStreptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

1998 ◽  
Vol 180 (16) ◽  
pp. 4123-4132 ◽  
Author(s):  
Oscar H. Martínez-Costa ◽  
Miguel A. Fernández-Moreno ◽  
Francisco Malpartida
Keyword(s):  
Amino Acids ◽  
Deletion Mutant ◽  
Streptomyces Coelicolor ◽  
Stringent Response ◽  
Morphological Differentiation ◽  
Antibiotic Biosynthesis ◽  
Homologous Gene ◽  
E Coli

ABSTRACT Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined.S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence thatS. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of anS. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. colitruncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, arel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition,E. coli relA in an S. coelicolor reldeletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-typerel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.

scholarly journals Cas9-mediated genome editing in Giardia intestinalis

2021 ◽  
Author(s):  
Vendula Horáčková ◽  
Luboš Voleman ◽  
Markéta Petrů ◽  
Martina Vinopalová ◽  
Filip Weisz ◽  
...  
Keyword(s):  
Genome Editing ◽  
Cell Lines ◽  
Genetic Manipulation ◽  
Protozoan Parasite ◽  
Giardia Intestinalis ◽  
Full Potential ◽  
Knock Out ◽  
Genome Modifications ◽  
First Time

CRISPR/Cas9 system is an extremely powerful technique that is extensively used for different genome modifications in various organisms including parasitic protists. Giardia intestinalis, a protozoan parasite infecting large number of people around the world each year, has been eluding the use of CRISPR/Cas9 technique so far which may be caused by its rather complicated genome containing four copies of each gene in its two nuclei. Apart from only single exception (Ebneter et al., 2016), without the use of CRISPR/Cas9 technology in its full potential, researchers in the field have not been able to establish knock-out cell lines to study the functional aspect of Giardia genes. In this work, we show the ability of in-vitro developed CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. Moreover, we used self-propagating CRISPR/Cas9 system to establish full knock out cell lines for mem, cwp1 and mlf1 genes. We also show that the system function even for essential genes, as we knocked-down tom40, lowering the amount of Tom40 protein by more than 90%. Further, we tested the length of homologous arms needed for successful integration of homology recombination cassette used for genome editing. Taken together, our work introduces CRISPR/Cas9 to Giardia for routine use in the lab, further extending the catalogue of molecular tolls available for genetic manipulation of the protist and allowing researchers to study the function of Giardia genes properly for the first time.

scholarly journals CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool

Antibiotics ◽  
2019 ◽  
Vol 8 (1) ◽  
pp. 18 ◽  
Author(s):  
Alexandre Loureiro ◽  
Gabriela da Silva
Keyword(s):  
Genetic Engineering ◽  
Genetic Manipulation ◽  
State Of The Art ◽  
Defence Mechanism ◽  
Engineering Technology ◽  
Defence System ◽  
In Vivo Experiments ◽  
Genetic Engineering Technology

Bacteriophages are pervasive viruses that infect bacteria, relying on their genetic machinery to replicate. In order to protect themselves from this kind of invader, bacteria developed an ingenious adaptive defence system, clustered regularly interspaced short palindromic repeats (CRISPR). Researchers soon realised that a specific type of CRISPR system, CRISPR-Cas9, could be modified into a simple and efficient genetic engineering technology, with several improvements over currently used systems. This discovery set in motion a revolution in genetics, with new and improved CRISPR systems being used in plenty of in vitro and in vivo experiments in recent years. This review illustrates the mechanisms behind CRISPR-Cas systems as a means of bacterial immunity against phage invasion and how these systems were engineered to originate new genetic manipulation tools. Newfound CRISPR-Cas technologies and the up-and-coming applications of these systems on healthcare and other fields of science are also discussed.

scholarly journals Genetic engineering of Treponema pallidum subsp. pallidum, the Syphilis Spirochete

2021 ◽  
Vol 17 (7) ◽  
pp. e1009612
Author(s):  
Emily Romeis ◽  
Lauren Tantalo ◽  
Nicole Lieberman ◽  
Quynh Phung ◽  
Alex Greninger ◽  
...  
Keyword(s):  
Genetic Engineering ◽  
Vaccine Development ◽  
Genetic Manipulation ◽  
Treponema Pallidum ◽  
Gene Promoter ◽  
Digital Pcr ◽  
Kanamycin Resistance ◽  
Suicide Vector

Despite more than a century of research, genetic manipulation of Treponema pallidum subsp. pallidum (T. pallidum), the causative agent of syphilis, has not been successful. The lack of genetic engineering tools has severely limited understanding of the mechanisms behind T. pallidum success as a pathogen. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible to experiment with transformation and selection protocols in this pathogen. Here, we describe an approach that successfully replaced the tprA (tp0009) pseudogene in the SS14 T. pallidum strain with a kanamycin resistance (kanR) cassette. A suicide vector was constructed using the pUC57 plasmid backbone. In the vector, the kanR gene was cloned downstream of the tp0574 gene promoter. The tp0574prom-kanR cassette was then placed between two 1-kbp homology arms identical to the sequences upstream and downstream of the tprA pseudogene. To induce homologous recombination and integration of the kanR cassette into the T. pallidum chromosome, in vitro-cultured SS14 strain spirochetes were exposed to the engineered vector in a CaCl2-based transformation buffer and let recover for 24 hours before adding kanamycin-containing selective media. Integration of the kanR cassette was demonstrated by qualitative PCR, droplet digital PCR (ddPCR), and whole-genome sequencing (WGS) of transformed treponemes propagated in vitro and/or in vivo. ddPCR analysis of RNA and mass spectrometry confirmed expression of the kanR message and protein in treponemes propagated in vitro. Moreover, tprA knockout (tprAko-SS14) treponemes grew in kanamycin concentrations that were 64 times higher than the MIC for the wild-type SS14 (wt-SS14) strain and in infected rabbits treated with kanamycin. We demonstrated that genetic manipulation of T. pallidum is attainable. This discovery will allow the application of functional genetics techniques to study syphilis pathogenesis and improve syphilis vaccine development.

scholarly journals Incipient genome erosion and metabolic streamlining for antibiotic production in a defensive symbiont

2021 ◽  
Vol 118 (17) ◽  
pp. e2023047118
Author(s):  
Taras Y. Nechitaylo ◽  
Mario Sandoval-Calderón ◽  
Tobias Engl ◽  
Natalie Wielsch ◽  
Diane M. Dunn ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Early Stage ◽  
Antibiotic Production ◽  
Axenic Culture ◽  
Building Blocks ◽  
In Vitro Assays ◽  
Antibiotic Biosynthesis ◽  
Protein Coding ◽  
Post Transcriptional Regulation

Genome erosion is a frequently observed result of relaxed selection in insect nutritional symbionts, but it has rarely been studied in defensive mutualisms. Solitary beewolf wasps harbor an actinobacterial symbiont of the genus Streptomyces that provides protection to the developing offspring against pathogenic microorganisms. Here, we characterized the genomic architecture and functional gene content of this culturable symbiont using genomics, transcriptomics, and proteomics in combination with in vitro assays. Despite retaining a large linear chromosome (7.3 Mb), the wasp symbiont accumulated frameshift mutations in more than a third of its protein-coding genes, indicative of incipient genome erosion. Although many of the frameshifted genes were still expressed, the encoded proteins were not detected, indicating post-transcriptional regulation. Most pseudogenization events affected accessory genes, regulators, and transporters, but “Streptomyces philanthi” also experienced mutations in central metabolic pathways, resulting in auxotrophies for biotin, proline, and arginine that were confirmed experimentally in axenic culture. In contrast to the strong A+T bias in the genomes of most obligate symbionts, we observed a significant G+C enrichment in regions likely experiencing reduced selection. Differential expression analyses revealed that—compared to in vitro symbiont cultures—“S. philanthi” in beewolf antennae showed overexpression of genes for antibiotic biosynthesis, the uptake of host-provided nutrients and the metabolism of building blocks required for antibiotic production. Our results show unusual traits in the early stage of genome erosion in a defensive symbiont and suggest tight integration of host–symbiont metabolic pathways that effectively grants the host control over the antimicrobial activity of its bacterial partner.

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.

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