Marker-Free Genome Engineering in Amycolatopsis Using the pSAM2 Site-Specific Recombination System

Actinobacteria of the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as bioconversion or bioremediation. Despite their importance, tools and methods for their genetic manipulation are less developed than in other actinobacteria such as Streptomyces. We report here the use of the pSAM2 site-specific recombination system to delete antibiotic resistance cassettes used in gene replacement experiments or to create large genomic deletions. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the integrase and the excisionase from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the attL and attR sites on each side of a large region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei by obtaining with high efficiency a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster. These robust and simple tools enrich the toolbox for genome engineering in Amycolatopsis

Transcriptional and preliminary functional analysis of the six genes located in divergence of phoR/phoP in Streptomyces lividans

International audienc

Regulation of the Biosynthesis of the Macrolide Antibiotic Spiramycin in Streptomyces ambofaciens ▿ †

Streptomyces ambofaciens synthesizes the macrolide antibiotic spiramycin. The biosynthetic gene cluster for spiramycin has been characterized for S. ambofaciens. In addition to the regulatory gene srmR (srm22), previously identified (M. Geistlich et al., Mol. Microbiol. 6:2019-2029, 1992), three putative regulatory genes had been identified by sequence analysis. Gene expression analysis and gene inactivation experiments showed that only one of these three genes, srm40, plays a major role in the regulation of spiramycin biosynthesis. The disruption of srm22 or srm40 eliminated spiramycin production while their overexpression increased spiramycin production. Expression analysis was performed by reverse transcription-PCR (RT-PCR) for all the genes of the cluster in the wild-type strain and in the srm22 (srmR) and srm40 deletion mutants. The results from the expression analysis, together with the ones from the complementation experiments, indicated that Srm22 is required for srm40 expression, Srm40 being a pathway-specific activator that controls most, if not all, of the spiramycin biosynthetic genes

The Generation of an Artificial ATP Deficit Triggers Antibiotic Production in Streptomyces lividans

International audienceIn most Streptomyces species, antibiotic production is triggered in a condition of phosphate limitation, a condition that is known to be correlated with a low intracellular ATP content compared to growth in a condition of phosphate proficiency. This observation suggests that a low ATP content might be a direct trigger of antibiotic biosynthesis. In order to test this hypothesis, we introduced into the model strain Streptomyces lividans, a functional and a non-functional ATPase cloned into the replicative vector pOSV206 and expressed under the control of the strong ErmE* promoter. The functional ATPase was constituted by the α (AtpA), β (AtpB) and γ (AtpD) sub-units of the native F1 part of the ATP synthase of S. lividans that, when separated from the membrane-bound F0 part, bears an ATPase activity. The non-functional ATPase was a mutated version of the latter, bearing a 12 amino acids deletion encompassing the active site of the AtpD sub-unit. S. lividans was chosen to test our hypothesis since this strain hardly produces any antibiotics. However, it possesses the same biosynthetic pathways of various specialized metabolites as S. coelicolor, a phylogenetically closely related strain that produces these metabolites in abundance. Our results demonstrated that the over-expression of the functional ATPase, but not that of its mutated version, indeed correlated with the production of the bioactive metabolites of the CDA, RED and ACT clusters. These results confirmed the long known and mysterious link existing between a phosphate limitation leading to an ATP deficit and the triggering of antibiotic biosynthesis. Based on this work and the previous published results of our group, we propose an entirely novel conception of the nature of this lin

Cloning and Sequencing of two Enterococcal glpK Genes and Regulation of the Encoded Glycerol Kinases by Phosphoenolpyruvate-dependent, Phosphotransferase System-catalyzed Phosphorylation of a Single Histidyl Residue

International audienc

Transcription Regulators Potentially Controlled by HPr Kinase/Phosphorylase in Gram-Negative Bacteria

International audiencePhosphorylation and dephosphorylation at Ser-46 in HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is controlled by the bifunctional HPr kinase/phosphorylase (HprK/P). In Gram-positive bacteria, P-Ser-HPr controls (1) sugar uptake via the PTS; (2) catabolite control protein A (CcpA)-mediated carbon catabolite repression, and (3) inducer exclusion. Genome sequencing revealed that HprK/P is absent from Gram-negative enteric bacteria, but present in many other proteobacteria. These organisms also possess (1) HPr, the substrate for HprK/P; (2) enzyme I, which phosphorylates HPr at His-15, and (3) one or several enzymes IIA, which receive the phosphoryl group from P∼His-HPr. The genes encoding the PTS proteins are often organized in an operon with hprK. However, most of these organisms miss CcpA and a functional PTS, as enzymes IIB and membrane-integrated enzymes IIC seem to be absent. HprK/P and the rudimentary PTS phosphorylation cascade in Gram-negative bacteria must therefore carry out functions different from those in Gram-positive organisms. The gene organization in many HprK/P-containing Gram-negative bacteria as well as some preliminary experiments suggest that HprK/P might control transcription regulators implicated in cell adhesion and virulence. In α-proteobacteria, hprK is located downstream of genes encoding a two-component system of the EnvZ/OmpR family. In several other proteobacteria, hprK is organized in an operon together with genes from the rpoN region of Escherichia coli (rpoN encodes a σ54). We propose that HprK/P might control the phosphorylation state of HPr and EIIAs, which in turn could control the transcription regulators

Excisable Cassettes: New Tools for Functional Analysis of Streptomyces Genomes

The functional analysis of microbial genomes often requires gene inactivation. We constructed a set of cassettes consisting of single antibiotic resistance genes flanked by the attL and attR sites resulting from site-specific integration of the Streptomyces pSAM2 element. These cassettes can easily be used to inactivate genes by in-frame deletion in Streptomyces by a three-step strategy. In the first step, in Escherichia coli, the cassette is inserted into a cloned copy of the gene to be inactivated. In the second step, the gene is replaced by homologous recombination in Streptomyces, allowing substitution of the wild-type target gene with its inactivated counterpart. In the third step, the cassette can be removed by expression of the pSAM2 genes xis and int. The resulting strains are marker-free and contain an “attB-like” sequence of 33, 34, or 35 bp with no stop codon if the cassette is correctly chosen. Thus, a gene can be disrupted by creating an in-frame deletion, avoiding polar effects if downstream genes are cotranscribed with the target gene. A set of cassettes was constructed to contain a hygromycin or gentamicin resistance gene flanked by the attL and attR sites. The initial constructions carrying convenient cloning sites allow the insertion of any other marker gene. We tested insertion and excision by inserting a cassette into orf3, the third gene of an operon involved in spiramycin biosynthesis. We verified that the cassette exerted a polar effect on the transcription of downstream genes but that, after excision, complementation with orf3 alone restored spiramycin production

La séquence complète du génome de la bactérie de la viande Lactobacillus sakei

National audienc

The Phosin PptA plays a negative role in the regulation of antibiotic production in Streptomyces lividansStreptomyces\ lividans

International audienceIn Streptomyces, antibiotic biosynthesis is triggered in phosphate limitation that is usually correlated with energetic stress. Polyphosphates constitute an important reservoir of phosphate and energy and a better understanding of their role in the regulation of antibiotic biosynthesis is of crucial importance. We previously characterized a gene, SLI_4384/ppk, encoding a polyphosphate kinase, whose disruption greatly enhanced the weak antibiotic production of Streptomyces lividans. In the condition of energetic stress, Ppk utilizes polyP as phosphate and energy donor, to generate ATP from ADP. In this paper, we established that ppk is co-transcribed with its two downstream genes, SLI_4383, encoding a phosin called PptA possessing a CHAD domain constituting a polyphosphate binding module and SLI_4382 encoding a nudix hydrolase. The expression of the ppk/pptA/SLI_4382 operon was shown to be under the positive control of the two-component system PhoR/PhoP and thus mainly expressed in condition of phosphate limitation. However, pptA and SLI_4382 can also be transcribed alone from their own promoter. The deletion of pptA resulted into earlier and stronger actinorhodin production and lower lipid content than the disruption of ppk, whereas the deletion of SLI_4382 had no obvious phenotypical consequences. The disruption of ppk was shown to have a polar effect on the expression of pptA, suggesting that the phenotype of the ppk mutant might be linked, at least in part, to the weak expression of pptA in this strain. Interestingly, the expression of phoR/phoP and that of the genes of the pho regulon involved in phosphate supply or saving were strongly up-regulated in pptA and ppk mutants, revealing that both mutants suffer from phosphate stress. Considering the presence of a polyphosphate binding module in PptA, but absence of similarities between PptA and known exo-polyphosphatases, we proposed that PptA constitutes an accessory factor for exopolyphosphatases or general phosphatases involved in the degradation of polyphosphates into phosphate