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Background Next generation sequencing (NGS) enables a more comprehensive analysis of bacterial diversity from complex environmental samples. NGS data can be analysed using a variety of workflows. We test several simple and complex workflows, including frequently used as well as recently published tools, and report on their respective accuracy and efficiency under various conditions covering different sequence lengths, number of sequences and real world experimental data from rhizobacterial populations of glyphosate-tolerant maize treated or untreated with two different herbicides representative of differential diversity studies. Results Alignment and distance calculations affect OTU estimations, and multiple sequence alignment exerts a major impact on the computational time needed. Generally speaking, most of the analyses produced consistent results that may be used to assess differential diversity changes, however, dataset characteristics dictate which workflow should be preferred in each case. Conclusions When estimating bacterial diversity, ESPRIT as well as the web-based workflow, RDP pyrosequencing pipeline, produced good results in all circumstances, however, its computational requirements can make method-combination workflows more attractive, depending on sequence variability, number and length.

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. 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.

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.

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.

Bt-maize is a transgenic variety of maize expressing the Cry toxin from Bacillus turingiensis. The potential accumulation of the relative effect of the transgenic modification and the cry toxin on the rhizobacterial communities of Bt-maize has been monitored over a period of four years. The accumulative effects of the cultivation of this transgenic plant have been monitored by means of high throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region from rhizobacterial communities. The obtained sequences were subjected to taxonomic, phylogenetic and taxonomic-independent diversity studies. The results obtained were consistent, indicating that variations detected in the rhizobacterial community structure were possibly due to climatic factors rather than to the presence of the Bt-gene. No variations were observed in the diversity estimates between non-Bt and Bt-maize. The cultivation of Bt-maize during the four-year period did not change the maize rhizobacterial communities when compared to those of the non-Bt maize. This is the first study to be conducted with Bt-maize during such a long cultivation period and the first evaluation of rhizobacterial communities to be performed in this transgenic plant using Next Generation Sequencing.

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.

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 .

Glyphosate is a herbicide that is liable to be used in the extensive cultivation of glyphosate-tolerant cultivars. The potential accumulation of the relative effect of glyphosate on the rhizobacterial communities of glyphosate-tolerant maize has been monitored over a period of three years. The composition of rhizobacterial communities is known to vary with soil texture, hence, the analyses have been performed in two agricultural fields with a different soil texture. The accumulative effects of glyphosate have been monitored by means of high throughput DNA pyrosequencing of the bacterial DNA coding for the 16S rRNA hypervariable V6 region from rhizobacterial communities. The relative composition of the rhizobacterial communities does vary in each field over the three-year period. The overall distribution of the bacterial phyla seems to change from one year to the next similarly in the untreated and glyphosate-treated soils in both fields. The two methods used to estimate bacterial diversity offered consistent results and are equally suitable for diversity assessment. The glyphosate treatment during the three-year period of seasonal cultivation in two different fields did not seem to significantly change the maize rhizobacterial communities when compared to those of the untreated soil. This may be particularly relevant with respect to a potential authorisation to cultivate glyphosate-tolerant maize in the European Union.

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.

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. 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. 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.