SummaryBy the time the complete genome sequence of the soil bacterium Pseudomonas putida KT2440 was published in (Nelson et al., 2002 this bacterium was considered a potential agent for environmental bioremediation of industrial waste and a good colonizer of the rhizosphere. However, neither the annotation tools available at that time nor the scarcely available omics data-let alone metabolic modeling and other nowadays common systems biology approaches-allowed them to anticipate the astonishing capacities that are encoded in the genetic complement of this unique microorganism. In this work we have adopted a suite of state-of-the-art genomic analysis tools to revisit the functional and metabolic information encoded in the chromosomal sequence of strain KT2440. We identified 242 new protein-coding genes and re-annotated the functions of 1548 genes, which are linked to almost 4900 PubMed references. Catabolic pathways for 92 compounds (carbon, nitrogen and phosphorus sources) that could not be accommodated by the previously constructed metabolic models were also predicted. The resulting examination not only accounts for some of the known stress tolerance traits known in P. putida but also recognizes the capacity of this bacterium to perform difficult redox reactions, thereby multiplying its value as a platform microorganism for industrial biotechnology.

Titania (TiO2)-based nanocomposites subjected to light excitation are remarkably effective in eliciting microbial death. However, the mechanism by which these materials induce microbial death and the effects that they have on microbes are poorly understood. Here, we assess the low dose radical-mediated TiO2 photocatalytic action of such nanocomposites and evaluate the genome/proteome-wide expression profiles of Pseudomonas aeruginosa PAO1 cells after two minutes of intervention. The results indicate that the impact on the gene-wide flux distribution and metabolism is moderate in the analysed time span. Rather, the photocatalytic action triggers the decreased expression of a large array of genes/proteins specific for regulatory, signalling and growth functions in parallel with subsequent selective effects on ion homeostasis, coenzyme-independent respiration and cell wall structure. The present work provides the first solid foundation for the biocidal action of titania and may have an impact on the design of highly active photobiocidal nanomaterials.

Macroscopic growths at geographically separated acid mine drainages (AMDs) exhibit distinct populations. Yet, local heterogeneities are poorly understood. To gain novel mechanistic insights into this, we used OMICs tools to profile microbial populations coexisting in a single pyrite gallery AMD (pH ∼2) in three distinct compartments: two from a stratified streamer (uppermost oxic and lowermost anoxic sediment-attached strata) and one from a submerged anoxic non-stratified mat biofilm. The communities colonising pyrite and those in the mature formations appear to be populated by the greatest diversity of bacteria and archaea (including ‘ARMAN' (archaeal Richmond Mine acidophilic nano-organisms)-related), as compared with the known AMD, with ∼44.9% unclassified sequences. We propose that the thick polymeric matrix may provide a safety shield against the prevailing extreme condition and also a massive carbon source, enabling non-typical acidophiles to develop more easily. Only 1 of 39 species were shared, suggesting a high metabolic heterogeneity in local microenvironments, defined by the O2 concentration, spatial location and biofilm architecture. The suboxic mats, compositionally most similar to each other, are more diverse and active for S, CO2, CH4, fatty acid and lipopolysaccharide metabolism. The oxic stratum of the streamer, displaying a higher diversity of the so-called ‘ARMAN'-related Euryarchaeota, shows a higher expression level of proteins involved in signal transduction, cell growth and N, H2, Fe, aromatic amino acids, sphingolipid and peptidoglycan metabolism. Our study is the first to highlight profound taxonomic and functional shifts in single AMD formations, as well as new microbial species and the importance of H2 in acidic suboxic macroscopic growths.

BackgroundThe human pathogen Mycobacterium tuberculosis has the capacity to escape eradication by professional phagocytes. During infection, M. tuberculosis resists the harsh environment of phagosomes and actively manipulates macrophages and dendritic cells to ensure prolonged intracellular survival. In contrast to other intracellular pathogens, it has remained difficult to capture the transcriptome of mycobacteria during infection due to an unfavorable host-to-pathogen ratio.ResultsWe infected the human macrophage-like cell line THP-1 with the attenuated M. tuberculosis surrogate M. bovis Bacillus Calmette–Guérin (M. bovis BCG). Mycobacterial RNA was up to 1000-fold underrepresented in total RNA preparations of infected host cells. We employed microbial enrichment combined with specific ribosomal RNA depletion to simultaneously analyze the transcriptional responses of host and pathogen during infection by dual RNA sequencing. Our results confirm that mycobacterial pathways for cholesterol degradation and iron acquisition are upregulated during infection. In addition, genes involved in the methylcitrate cycle, aspartate metabolism and recycling of mycolic acids were induced. In response to M. bovis BCG infection, host cells upregulated de novo cholesterol biosynthesis presumably to compensate for the loss of this metabolite by bacterial catabolism.ConclusionsDual RNA sequencing allows simultaneous capture of the global transcriptome of host and pathogen, during infection. However, mycobacteria remained problematic due to their relatively low number per host cell resulting in an unfavorable bacterium-to-host RNA ratio. Here, we use a strategy that combines enrichment for bacterial transcripts and dual RNA sequencing to provide the most comprehensive transcriptome of intracellular mycobacteria to date. The knowledge acquired into the pathogen and host pathways regulated during infection may contribute to a solid basis for the deployment of novel intervention strategies to tackle infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-014-1197-2) contains supplementary material, which is available to authorized users.

BackgroundGlobal climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from Aspergillus niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes.ResultsA hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization.ConclusionsThis study revealed the existence of a variety of xylose transporters in the cell factories A. niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0564-4) contains supplementary material, which is available to authorized users.

Summary Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.

Systems-level metabolic network reconstructions and the derived constraint-based (CB) mathematical models are efficient tools to explore bacterial metabolism. Approximately one-fourth of the Mycobacterium tuberculosis (Mtb) genome contains genes that encode proteins directly involved in its metabolism. These represent potential drug targets that can be systematically probed with CB models through the prediction of genes essential (or the combination thereof) for the pathogen to grow. However, gene essentiality depends on the growth conditions and, so far, no in vitro model precisely mimics the host at the different stages of mycobacterial infection, limiting model predictions. These limitations can be circumvented by combining expression data from in vivo samples with a validated CB model, creating an accurate description of pathogen metabolism in the host. To this end, we present here a thoroughly curated and extended genome-scale CB metabolic model of Mtb quantitatively validated using 13C measurements. We describe some of the efforts made in integrating CB models and high-throughput data to generate condition specific models, and we will discuss challenges ahead. This knowledge and the framework herein presented will enable to identify potential new drug targets, and will foster the development of optimal therapeutic strategies.

BackgroundPseudomonas putida is a metabolically versatile, genetically accessible, and stress-robust species with outstanding potential to be used as a workhorse for industrial applications. While industry recognises the importance of robustness under micro-oxic conditions for a stable production process, the obligate aerobic nature of P. putida, attributed to its inability to produce sufficient ATP and maintain its redox balance without molecular oxygen, severely limits its use for biotechnology applications.ResultsHere, a combination of genome-scale metabolic modelling and comparative genomics is used to pinpoint essential -dependent processes. These explain the inability of the strain to grow under anoxic conditions: a deficient ATP generation and an inability to synthesize essential metabolites. Based on this, several P. putida recombinant strains were constructed harbouring acetate kinase from Escherichia coli for ATP production, and a class I dihydroorotate dehydrogenase and a class III anaerobic ribonucleotide triphosphate reductase from Lactobacillus lactis for the synthesis of essential metabolites. Initial computational designs were fine-tuned by means of adaptive laboratory evolution.ConclusionsWe demonstrated the value of combining in silico approaches, experimental validation and adaptive laboratory evolution for microbial design by making the strictly aerobic Pseudomonas putida able to grow under micro-oxic conditions.