Control of Genome Stability by EndoMS/NucS-Mediated Non-Canonical Mismatch Repair.

The DNA repair endonuclease EndoMS/NucS is highly conserved in Archaea and Actinobacteria. This enzyme is able to recognize and cleave dsDNA carrying a mismatched base pair, and its activity is enhanced by the interaction with the sliding clamp of the replisome. Today, EndoMS/NucS has been established as the key protein of a non-canonical mismatch repair (MMR) pathway, acting specifically in the repair of transitions and being essential for maintaining genome stability. Despite having some particularities, such as its lower activity on transversions and the inability to correct indels, EndoMS/NucS meets the main hallmarks of a MMR. Its absence leads to a hypermutator phenotype, a transition-biased mutational spectrum and an increase in homeologous recombination. Interestingly, polymorphic EndoMS/NucS variants with a possible effect in mutation rate have been detected in clinical isolates of the relevant actinobacterial pathogen Mycobacterium tuberculosis. Considering that MMR defects are often associated with the emergence of resistant bacteria, the existence of EndoMS/NucS-defective mutators could have an important role in the acquisition of antibiotic resistance in M. tuberculosis. Therefore, a further understanding of the EndoMS/NucS-mediated non-canonical MMR pathway may reveal new strategies to predict and fight drug resistance. This review is focused on the recent progress in NucS, with special emphasis on its effect on genome stability and evolvability in Actinobacteria.

Dynamic metabolic modelling of overproduced protein secretion in Streptomyces lividans using adaptive DFBA.

BACKGROUND: Streptomyces lividans is an appealing host for the production of proteins of biotechnological interest due to its relaxed exogenous DNA restriction system and its ability to secrete proteins directly to the medium through the major Sec or the minor Tat routes. Often, protein secretion displays non-uniform time-dependent patterns. Understanding the associated metabolic changes is a crucial step to engineer protein production. Dynamic Flux Balance Analysis (DFBA) allows the study of the interactions between a modelled organism and its environment over time. Existing methods allow the specification of initial model and environment conditions, but do not allow introducing arbitrary modifications in the course of the simulation. Living organisms, however, display unexpected adaptive metabolic behaviours in response to unpredictable changes in their environment. Engineering the secretion of products of biotechnological interest has systematically proven especially difficult to model using DFBA. Accurate time-dependent modelling of complex and/or arbitrary, adaptive metabolic processes demands an extended approach to DFBA. RESULTS: In this work, we introduce Adaptive DFBA, a novel, versatile simulation approach that permits inclusion of changes in the organism or the environment at any time in the simulation, either arbitrary or interactively responsive to environmental changes. This approach extends traditional DFBA to allow steering arbitrarily complex simulations of metabolic dynamics. When applied to Sec- or Tat-dependent secretion of overproduced proteins in S. lividans, Adaptive DFBA can overcome the limitations of traditional DFBA to reproduce experimental data on plasmid-free, plasmid bearing and secretory protein overproducing S. lividans TK24, and can yield useful insights on the behaviour of systems with limited experimental knowledge such as agarase or amylase overproduction in S. lividans TK21. CONCLUSIONS: Adaptive DFBA has allowed us to overcome DFBA limitations and to generate more accurate models of the metabolism during the overproduction of secretory proteins in S. lividans, improving our understanding of the underlying processes. Adaptive DFBA is versatile enough to permit dynamical metabolic simulations of arbitrarily complex biotechnological processes.

Four thiol-oxidoreductases involved in the formation of disulphide bonds in the Streptomyces lividans TK21 secretory proteins.

BACKGROUND: Bacterial secretory proteins often require the formation of disulphide bonds outside the cell to acquire an active conformation. Thiol-disulphide oxidoreductases are enzymes that catalyse the formation of disulphide bonds. The bacterium Streptomyces lividans is a well-known host for the efficient secretion of overproduced homologous and heterologous secretory proteins of industrial application. Therefore, the correct conformation of these extracellular proteins is of great importance when engineering that overproduction. RESULTS: We have identified four acting thiol-disulphide oxidoreductases (TDORs) in S. lividans TK21, mutants in all TDOR candidates affect the secretion and activity of the Sec-dependent alpha-amylase, which contains several disulphide bonds, but the effect was more drastic in the case of the Sli-DsbA deficient strain. Thus, the four TDOR are required to obtain active alpha-amylase. Additionally, only mutations in Sli-DsbA and Sli-DsbB affect the secretion and activity of the Tat-dependent agarase, which does not form a disulphide bond, when it is overproduced. This suggests a possible role of the oxidised Sli-DsbA as a chaperone in the production of active agarase. CONCLUSIONS: Enzymes involved in the production of the extracellular mature active proteins are not fully characterised yet in Streptomyces lividans. Our results suggest that the role of thiol-disulphide oxidoreductases must be considered when engineering Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application, irrespective of their secretion route, in order to obtain active, correctly folded proteins.

Modelling the metabolism of protein secretion through the Tat route in Streptomyces lividans.

BACKGROUND: Streptomyces lividans has demonstrated its value as an efficient host for protein production due to its ability to secrete functional proteins directly to the media. Secretory proteins that use the major Sec route need to be properly folded outside the cell, whereas secretory proteins using the Tat route appear outside the cell correctly folded. This feature makes the Tat system very attractive for the production of natural or engineered Tat secretory proteins. S. lividans cells are known to respond differently to overproduction and secretion of Tat versus Sec proteins. Increased understanding of the impact of protein secretion through the Tat route can be obtained by a deeper analysis of the metabolic impact associated with protein production, and its dependence on protein origin, composition, secretion mechanisms, growth phases and nutrients. Flux Balance Analysis of Genome-Scale Metabolic Network models provides a theoretical framework to investigate cell metabolism under different constraints. RESULTS: We have built new models for various S. lividans strains to better understand the mechanisms associated with overproduction of proteins secreted through the Tat route. We compare models of an S. lividans Tat-dependent agarase overproducing strain with those of the S. lividans wild-type, an S. lividans strain carrying the multi-copy plasmid vector and an α-amylase Sec-dependent overproducing strain. Using updated genomic, transcriptomic and experimental data we could extend existing S. lividans models and produce a new model which produces improved results largely extending the coverage of S. lividans strains, the number of genes and reactions being considered, the predictive behaviour and the dependence on specification of exchange constraints. Comparison of the optimized solutions obtained highlights numerous changes between Tat- and Sec-dependent protein secreting strains affecting the metabolism of carbon, amino acids, nucleotides, lipids and cofactors, and variability analysis predicts a large potential for protein overproduction. CONCLUSIONS: This work provides a detailed look to metabolic changes associated to Tat-dependent protein secretion reproducing experimental observations and identifying changes that are specific to each secretory route, presenting a novel, improved, more accurate and strain-independent model of S. lividans, thus opening the way for enhanced metabolic engineering of protein overproduction in S. lividans.

The Cellular Mechanisms that Ensure an Efficient Secretion in Streptomyces.

Gram-positive soil bacteria included in the genus Streptomyces produce a large variety of secondary metabolites in addition to extracellular hydrolytic enzymes. From the industrial and commercial viewpoints, the S. lividans strain has generated greater interest as a host bacterium for the overproduction of homologous and heterologous hydrolytic enzymes as an industrial application, which has considerably increased scientific interest in the characterization of secretion routes in this bacterium. This review will focus on the secretion machinery in S. lividans.

The Three Streptomyces lividans HtrA-Like Proteases Involved in the Secretion Stress Response Act in a Cooperative Manner.

Overproduction of Sec-proteins in S. lividans accumulates misfolded proteins outside of the cytoplasmic membrane where the accumulated proteins interfere with the correct functioning of the secretion machinery and with the correct cell functionality, triggering the expression in S. lividans of a CssRS two-component system which regulates the degradation of the accumulated protein, the so-called secretion stress response. Optimization of secretory protein production via the Sec route requires the identification and characterisation of quality factors involved in this process. The phosphorylated regulator (CssR) interacts with the regulatory regions of three genes encoding three different HtrA-like proteases. Individual mutations in each of these genes render degradation of the misfolded protein inoperative, and propagation in high copy number of any of the three proteases encoding genes results on indiscriminate alpha-amylase degradation. None of the proteases could complement the other two deficiencies and only propagation of each single copy protease gene can restore its own deficiency. The obtained results strongly suggest that the synthesis of the three HtrA-like proteases needs to be properly balanced to ensure the effective degradation of misfolded overproduced secretory proteins and, at the same time, avoid negative effects in the secreted proteins and the secretion machinery. This is particularly relevant when considering the optimisation of Streptomyces strains for the overproduction of homologous or heterologous secretory proteins of industrial application.

Looking for Rhizobacterial Ecological Indicators in Agricultural Soils Using 16S rRNA metagenomic Amplicon Data.

INTRODUCTION: Biological communities present in soil are essential to sustainable and productive agricultural practices; however, an accurate determination of the ecological status of agricultural soils remains to date an elusive task. An ideal indicator should be pervasive, play a relevant role in the ecosystem, show a rapid and proportional answer to external perturbations and be easily and economically measurable. Rhizobacteria play a major role in determining soil properties, becoming an attractive candidate for the detection of ecological indicators. The application of massive sequencing technologies to metagenomic analysis is providing an increasingly more precise view of the structure and composition of soil communities. In this work, we analyse soil rhizobacterial composition under various stress levels to search for potential ecological indicators. GENERAL BIODIVERSITY INDICATORS: Our results suggest that the Shannon index requires observation of a relatively large number of individuals to be representative of the true population diversity, and that the Simpson index may underestimate rare taxa in rhizobacterial environments. TAXONOMICAL CLASSIFICATION METHODS: Detection of indicator taxa requires comparison of taxonomical classification of sequences. We have compared RDP classifier, RTAX and similarity-based taxonomical classification and selected the latter for taxonomical assignment because it provides larger detail. TAXONOMY-BASED ECOLOGICAL INDICATORS: The study of significant variations in common, clearly identified, taxa, using paired datasets allows minimization of non-treatment effects and avoidance of false positives. We have identified taxa associated to specific perturbations as well as taxa generally affected in treated soils. Changes in these taxa, or combinations of them, may be used as ecological indicators of soil health. The overall number and magnitude of changes detected in taxonomic groups does also increase with stress. These changes constitute an alternative indicator to measuring specific taxa, although their determination requires large sample sizes, better obtained by massive sequencing. SUMMARY: The main ecological indicators available are the Shannon index, OTU counts and estimators, overall detection of the number and proportion of changes, and changes of specific indicator taxa. Massive sequencing remains the most accurate tool to measure rhizobacterial ecological indicators. When massive sequencing is not an option, various cultivable taxonomic groups, such as specific groups in the Actinobacteria tree, are attractive as potential indicators of large disruptions to the rhizobiome.

Exploring the Feasibility of the Sec Route to Secrete Proteins Using the Tat Route in Streptomyces lividans.

Streptomyces lividans uses mainly two pathways to target secretory proteins to the cytoplasmic membrane. The major pathway (Sec pathway) transports pre-proteins using the signal recognition particle, and the minor Tat pathway is responsible for the secretion using a folded conformation of a relatively low number of proteins. The signal peptides of the Sec-dependent alpha-amylase and the Tat-dependent agarase were interchanged and fused in-frame to the corresponding mature part of the other enzyme. Alpha-amylase was unable to use the Tat route when fused to the agarase signal peptide, while agarase used the Sec route when it was targeted by the alpha-amylase signal peptide. In addition to the signal peptide some yet unidentified parts of the secreted proteins may play a role in selecting the secretory route. Structure predictions for the Tat- and Sec-dependent proteins suggest that less structured proteins are more likely to be candidates for the Tat route.

Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans.

Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients' depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host.

Transcriptional characterisation of the negative effect exerted by a deficiency in type II signal peptidase on extracellular protein secretion in Streptomyces lividans.

Bacterial lipoproteins are a specialised class of membrane proteins that represent a small percentage of the proteome of Gram-positive bacteria, yet these lipoproteins have been reported to play important roles in nutrient scavenging, cell envelope assembly, protein folding, environmental signalling, host cell adhesion and virulence. Upon translocation of lipoproteins, the type II signal peptidase (Lsp) cleaves the signal peptide, leaving the lipoproteins bound to the outer face of the cytoplasmic membrane by means of linking lipid molecule to their +1 cysteine residue. We have studied the role played by Lsp in Streptomyces lividans cellular metabolism, particularly, in secretory protein production, and found that the absence of functional Lsp, apparently produces a translocase blockage, diminishes the synthesis of secretory proteins and triggers a stringent response. These findings could be particularly relevant when optimising S. lividans for the overproduction of secretory proteins of industrial application.

A novel two-component system involved in secretion stress response in Streptomyces lividans.

BACKGROUND: Misfolded proteins accumulating outside the bacterial cytoplasmic membrane can interfere with the secretory machinery, hence the existence of quality factors to eliminate these misfolded proteins is of capital importance in bacteria that are efficient producers of secretory proteins. These bacteria normally use a specific two-component system to respond to the stress produced by the accumulation of the misfolded proteins, by activating the expression of HtrA-like proteases to specifically eliminate the incorrectly folded proteins. METHODOLOGY/PRINCIPAL FINDINGS: Overproduction of alpha-amylase in S. lividans causing secretion stress permitted the identification of a two-component system (SCO4156-SCO4155) that regulates three HtrA-like proteases which appear to be involved in secretion stress response. Mutants in each of the genes forming part of the two-genes operon that encodes the sensor and regulator protein components accumulated misfolded proteins outside the cell, strongly suggesting the involvement of this two-component system in the S. lividans secretion stress response. CONCLUSIONS/SIGNIFICANCE: To our knowledge this is the first time that a specific secretion stress response two-component system is found to control the expression of three HtrA-like protease genes in S. lividans, a bacterium that has been repeatedly used as a host for the synthesis of homologous and heterologous secretory proteins of industrial application.

Translocase and major signal peptidase malfunctions affect aerial mycelium formation in Streptomyces lividans.

Deficiency in the translocase complex (SecG mutant strain) or in the major type I signal peptidase (SipY mutant strain) function in Streptomyces lividans resulted, as expected, in a drastic reduction of secretory protein production and in a bald phenotype. The transcriptional profiling of both strains showed that the expression of a set of genes involved in the morphological differentiation process was down regulated in both mutant strains (bldG, bldN and bldM), whereas bldA and bldH were only down-regulated in the SipY mutant strain. Consistently, low temperature scanning electron microscopy revealed that the disruption of sipY had a more noticeable effect in the growth/morphological aspect of the mycelium than that of secG, suggesting that in the sipY mutant, the blockage of the export process might have more severe consequences than in the secG mutant. In both cases, the likely degradation of the proteins that cannot be secreted might provide nutrients that might be responsible for the lack of induction of the bald cascade, which is thought to be triggered under conditions of nutritional limitation.

A novel two-component system involved in the transition to secondary metabolism in Streptomyces coelicolor.

BACKGROUND: Bacterial two-component signal transduction regulatory systems are the major set of signalling proteins frequently mediating responses to changes in the environment. They typically consist of a sensor, a membrane-associated histidine kinase and a cytoplasmic response regulator. The membrane-associated sensor detects the environmental signal or stress, whereas the cytoplasmic regulatory protein controls the cellular response usually by gene transcription modulation. METHODOLOGY/PRINCIPALFINDINGS: The Streptomyces coelicolor two genes operon SCO5784-SCO5785 encodes a two-component system, where SCO5784 encodes a histidine-kinase sensor and SCO5785 encodes a response regulator protein. When the expression level of the regulator gene decreases, the antibiotic synthesis and sporulation is delayed temporarily in addition to some ribosomal genes became up regulated, whereas the propagation of the regulatory gene in high copy number results in the earlier synthesis of antibiotics and sporulation, as well as the down regulation of some ribosomal genes and, moreover, in the overproduction of several extracellular proteins. Therefore, this two-component system in S. coelicolor seems to influence various processes characterised by the transition from primary to secondary metabolism, as determined by proteomic and transcriptomic analyses. CONCLUSIONS/SIGNIFICANCE: Propagation of SCO5785 in multicopy enhances the production of antibiotics as well as secretory proteins. In particular, the increase in the expression level of secretory protein encoding genes, either as an artefactual or real effect of the regulator, could be of potential usefulness when using Streptomyces strains as hosts for homologous or heterologous extracellular protein production.

Glycosylated derivatives of steffimycin: insights into the role of the sugar moieties for the biological activity.

Expression of the steffimycin gene cluster in Steptomyces albus in combination with plasmids directing the biosynthesis of different neutral and branched-chain deoxyhexoses led to the identification of twelve new glycosylated derivatives of steffimycin with different degrees of decoration in the tetracyclic core. These experiments demonstrate the flexibility of L-rhamnosyltransferase StfG for recognition of a variety of D- and L-deoxyhexoses, harboring different degrees of deoxygenation as 2-deoxyhexoses, 2,6-deoxyhexoses, and 2,3,6-deoxyhexoses, and their attachment to 8-demethoxy-10-deoxysteffimycinone. In addition, the flexibility of 3'-O-methyltransferase OleY, from Streptomyces, for the methylation of deoxyhexoses attached to the steffimycin aglycone is shown by expression of oleY in Streptomyces steffisburgensis, leading to the isolation of 3'-O-methylsteffimycin. Analysis of the biological activities of these compounds against three human tumor cell lines-breast adenocarcinoma, non-small cell lung cancer, and colon adenocarcinoma-revealed two of them, 3'-O-methylsteffimycin and D-digitoxosyl-8-demethoxy-10-deoxysteffimycinone, to possess improved antitumor activities, showing GI50 values below 1.0 microM, while steffimycin's GI50 values fluctuate between 2.61 to 6.79 microM depending upon the cell line used. The antitumor activity data provide some insights into the structure-activity relationships of the new steffimycin derivatives, in relation to the configuration of hydroxy groups at positions C-3' and C-4' of the sugar moiety and positions C-8 and C-10 of the tetracyclic core.

Isolation, characterization, and heterologous expression of the biosynthesis gene cluster for the antitumor anthracycline steffimycin.

The biosynthetic gene cluster for the aromatic polyketide steffimycin of the anthracycline family has been cloned and characterized from "Streptomyces steffisburgensis" NRRL 3193. Sequence analysis of a 42.8-kbp DNA region revealed the presence of 36 open reading frames (ORFs) (one of them incomplete), 24 of which, spanning 26.5 kb, are probably involved in steffimycin biosynthesis. They code for all the activities required for polyketide biosynthesis, tailoring, regulation, and resistance but show no evidence of genes involved in L-rhamnose biosynthesis. The involvement of the cluster in steffimycin biosynthesis was confirmed by expression of a region of about 15 kb containing 15 ORFS, 11 of them forming part of the cluster, in the heterologous host Streptomyces albus, allowing the isolation of a biosynthetic intermediate. In addition, the expression in S. albus of the entire cluster, contained in a region of 34.8 kb, combined with the expression of plasmid pRHAM, directing the biosynthesis of L-rhamnose, led to the production of steffimycin. Inactivation of the stfX gene, coding for a putative cyclase, revealed that this enzymatic activity participates in the cyclization of the fourth ring, making the final steps in the biosynthesis of the steffimycin aglycon similar to those in the biosynthesis of jadomycin or rabelomycin. Inactivation of the stfG gene, coding for a putative glycosyltransferase involved in the attachment of L-rhamnose, allowed the production of a new compound corresponding to the steffimycin aglycon compound also observed in S. albus upon expression of the entire cluster.