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Graphical abstract, Highlights • Limitations in molecular resources for gene expression influence bacterial physiology. • Bacteria optimise trade-offs between resource allocation and growth. • Resource allocation plays a role in the emergence of phenotypic heterogeneity. • Trade-offs between bet-hedging and growth can be harnessed in biotechnology., Bacterial cells have a limited number of resources that can be allocated for gene expression. The intracellular competition for these resources has an impact on the cell physiology. Bacteria have evolved mechanisms to optimize resource allocation in a variety of scenarios, showing a trade-off between the resources used to maximise growth (e.g. ribosome synthesis) and the rest of cellular functions. Limitations in gene expression also play a role in generating phenotypic diversity, which is advantageous in fluctuating environments, at the expenses of decreasing growth rates. Our current understanding of these trade-offs can be exploited for biotechnological applications benefiting from the selective manipulation of the allocation of resources.

Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second most common cause of cancer death in the USA by 2030, yet progress continues to lag behind that of other cancers, with only 9% of patients surviving beyond 5 years. Long-term survivorship of PDAC and improving survival has, until recently, escaped our understanding. One recent frontier in the cancer field is the microbiome. The microbiome collectively refers to the extensive community of bacteria and fungi that colonise us. It is estimated that there is one to ten prokaryotic cells for each human somatic cell, yet, the significance of this community in health and disease has, until recently, been overlooked. This review examines the role of the microbiome in PDAC and how it may alter survival outcomes. We evaluate the possibility of employing microbiomic signatures as biomarkers of PDAC. Ultimately this review analyses whether the microbiome may be amenable to targeting and consequently altering the natural history of PDAC.

Non-metal, metal and metalloid oxyanions occur naturally in minerals and rocks of the Earth’s crust and are mostly found in low concentrations or confined in specific regions of the planet. However, anthropogenic activities including urban development, mining, agriculture, industrial activities and new technologies have increased the release of oxyanions to the environment, which threatens the sustainability of natural ecosystems, in turn affecting human development. For these reasons, the implementation of new methods that could allow not only the remediation of oxyanion contaminants but also the recovery of valuable elements from oxyanions of the environment is imperative. From this perspective, the use of microorganisms emerges as a strategy complementary to physical, mechanical and chemical methods. In this review, we discuss the opportunities that the Pseudomonas genus offers for the bioremediation of oxyanions, which is derived from its specialized central metabolism and the high number of oxidoreductases present in the genomes of these bacteria. Finally, we review the current knowledge on the transport and metabolism of specific oxyanions in Pseudomonas species. We consider that the Pseudomonas genus is an excellent starting point for the development of biotechnological approaches for the upcycling of oxyanions into added-value metal and metalloid byproducts. UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA) UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Química

SummaryBacteria regulate their cellular resource allocation to enable fast growth-adaptation to a variety of environmental niches. We studied the ribosomal allocation, growth and expression profiles of two sets of fast-growing mutants of Escherichia coli K-12 MG1655 in glucose minimal medium. Mutants with only 3 of the seven copies of ribosomal RNA operons grew faster than the wild-type strain in minimal media and show similar phenotype to previously studied rpoB mutants. Higher growth rates due to increased ribosome content affected resource allocation. Expression profiles of fast-growing mutants shared downregulation of hedging functions and upregulated growth functions. Mutants showed longer diauxic shifts and reduced activity of gluconeogenic promoters during glucose-acetate shifts, suggesting reduced availability of the RNA Polymerase for expressing hedging proteome. These results show that the regulation of ribosomal allocation underlies the growth/hedging phenotypes obtained from laboratory evolution experiments. We show how two different regulatory perturbations (rRNA promoters or rpoB mutations) reshape the proteome for growth with a concomitant fitness costHighlightsMutants with only 3 ribosomal operons grow faster than wild-type in minimal mediumΔ4 rrn and rpoB mutants share phenotypic traitsFaster growth of mutants is achieved by increased ribosome contentFast-growing mutants display reduced hedging expression and adaptation trade-offsDespite similar ribosomal content in rich medium the mutants present growth defects

The gene expression capacity of bacterial cells depends on the interplay between growth and the availability of the transcriptional and translational machinery. Growth rate is widely accepted as the global physiological parameter controlling the allocation of cell resources. This allocation has an impact on the ability of the cell to produce both host and heterologous proteins required for synthetic circuits and pathways. Understanding the relationship between growth and resources is key for the efficient design of artificial genetic constructs, however, it is obscured by the mutual dependence of growth and gene expression on each other. In this work, we investigate the individual contributions of molecular factors, growth rate and metabolism to gene expression by investigating the behaviour of bacterial cells growing in chemostats in growth-limited conditions. We develop a model of the whole cell that captures trade-offs in gene expression arising from the individual contributions of different factors, and validate it by analysing gene couplings which emerge from competition for the gene expression machinery. Our results show that while growth rate and molecular factors, such as the number of rRNA operons, set the abundance of transcriptional and translational machinery available, it is metabolism that governs the usage of those resources by tuning elongation rates. We show that synthetic gene expression capacity can be maximised by using low growth in a high-quality medium. These findings provide valuable insights into fundamental trade-offs in microbial physiology that will inform future strain and bioprocesses optimisation.

In this article we have summarised our findings resulting from analysing the complete genome sequence of Pseudomonas umsongensis GO16 and we have determined experimentally its potential for the upcycling of monomers resulting from PET hydrolysis. Besides a general genomic characterisation, we have identified and validated the set of genes required for terephthalate and ethylene glycol degradation as well as the ability of the strain for producing short and medium‐chain length polyhydroxyalkanoates., Summary The throwaway culture related to the single‐use materials such as polyethylene terephthalate (PET) has created a major environmental concern. Recycling of PET waste into biodegradable plastic polyhydroxyalkanoate (PHA) creates an opportunity to improve resource efficiency and contribute to a circular economy. We sequenced the genome of Pseudomonas umsongensis GO16 previously shown to convert PET‐derived terephthalic acid (TA) into PHA and performed an in‐depth genome analysis. GO16 can degrade a range of aromatic substrates in addition to TA, due to the presence of a catabolic plasmid pENK22. The genetic complement required for the degradation of TA via protocatechuate was identified and its functionality was confirmed by transferring the tph operon into Pseudomonas putida KT2440, which is unable to utilize TA naturally. We also identified the genes involved in ethylene glycol (EG) metabolism, the second PET monomer, and validated the capacity of GO16 to use EG as a sole source of carbon and energy. Moreover, GO16 possesses genes for the synthesis of both medium and short chain length PHA and we have demonstrated the capacity of the strain to convert mixed TA and EG into PHA. The metabolic versatility of GO16 highlights the potential of this organism for biotransformations using PET waste as a feedstock.

Function of bacterial small non-coding RNAs (sRNAs) and overall RNA metabolism is largely shaped by a vast diversity of RNA-protein interactions. However, in non-model bacteria with defined non-coding transcriptomes the sRNA interactome remains almost unexplored. We used affinity chromatography to capture proteins associated in vivo with MS2-tagged trans-sRNAs that regulate nutrient uptake (AbcR2 and NfeR1) and cell cycle (EcpR1) mRNAs by antisense-based translational inhibition in the nitrogen-fixing α-rhizobia Sinorhizobium meliloti. The three proteomes were rather distinct, with that of EcpR1 particularly enriched in cell cycle-related enzymes, whilst sharing several transcription/translation-related proteins recurrently identified associated with sRNAs. Strikingly, MetK, the synthetase of the major methyl donor S-adenosylmethionine, was reliably recovered as a binding partner of the three sRNAs, which reciprocally co-immunoprecipitated with a FLAG-tagged MetK variant. Induced (over)expression of the trans-sRNAs and MetK depletion did not influence canonical riboregulatory traits, `for example, protein titration or sRNA stability, respectively. An in vitro filter assay confirmed binding of AbcR2, NfeR1 and EcpR1 to MetK and further revealed interaction of the protein with other non-coding and coding transcripts but not with the 5S rRNA. These findings uncover a broad specificity for RNA binding as an unprecedented feature of this housekeeping prokaryotic enzyme., This work was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades under ERDF-cofinanced grants [BFU2013- 48282-C2-2-P and BFU2017-82645-P to J.I.J.-Z.], a contract of the program ‘Formación Post-doctoral’ (Juan de la Cierva) [FPDI-2013-16255 to M.R.], and an FPU fellowship [FPU16/01275 to N.I.G.-T.].

Bacteria regulate their cellular resource allocation to enable their fast growth-adaptation to a variety of environmental niches. We studied the ribosomal allocation, growth and expression profiles of two sets of fast-growing mutants of Escherichia coli K-12 MG1655. Mutants with only 3 copies of the strongest ribosomal RNA operons grew faster than the wild-type strain in minimal media and show similar phenotype to previously studied rpoB mutants. Both fast-growing strains showed higher ribosomal content rather than higher ribosomal efficiency. Expression profiles of fast-growing mutants shared downregulation of hedging functions and upregulated growth functions. Mutants showed longer diauxic shifts and reduced activity of gluconeogenic promoters during glucose-acetate shifts, suggesting reduced availability of the RNA Polymerase for expressing hedging proteome. These results show that the regulation of ribosomal allocation underlies the growth/hedging phenotypes obtained from laboratory evolution experiments. We show how two different regulatory perturbations (rRNA promoters or rpoB mutations) optimize the proteome for growth with a concomitant fitness cost.

We report here the complete genome sequence of the salt-tolerant Sinorhizobium meliloti strain AK21, isolated from nodules of Medicago sativa L. subsp. ambigua inhabiting the northern Aral Sea Region. This genome (7.36 Mb) consists of a chromosome and four accessory plasmids, two of which are the symbiotic megaplasmids pSymA and pSymB., Strain SmeAK21 was provided by Marina Roumiantseva and Boris Simarov to the partners of the International Cooperation-Cooperation with the Countries of Central Europe and with the New Independent States of the Former Soviet Union (INCO-COPERNICUS) program (grant ICA2-CT-2001-10001), and it is available at the All-Russia Research Institute for Agricultural Microbiology (ARRIAM; strain collection number RCAM05222). This work was funded by European Regional Development Fund (ERDF)-cofinanced grants from the Spanish Ministerio de Ciencia, Innovación y Universidades (grant CSD2009-00006 to N.T. and grants AGL2009-07925 and BFU2017-82645-P to J.I.J.-Z.).

Extensive work in model enterobacteria has evidenced that the RNA chaperone Hfq and several endoribonucleases, such as RNase E or RNase III, serve pivotal roles in small RNA-mediated post-transcriptional silencing of gene expression. Characterization of these protein hubs commonly provide global functional and mechanistic insights into complex sRNA regulatory networks. The legume endosymbiont Sinorhizobium meliloti is a non-classical model bacterium with a very complex lifestyle in which riboregulation is expected to play important adaptive functions. Here, we discuss current knowledge about RNA silencing in S. meliloti from the perspective of the activity of Hfq and a recently discovered endoribonuclease (YbeY) exhibiting unprecedented catalytic versatility for the cleavage of single- and double-stranded RNA molecules., This work was funded by the Spanish Ministerio de Economía y Competitividad ERDF-cofinanced grant BFU2013–48282-C2–2-P. M.R. was funded by a contract of the program of “Formacion Post-doctoral” from the same Ministry.

Microorganisms are widely used in synthetic biology for applications which require the simultaneous expression of multiple genes. However, gene expression in these microbes is limited by the number of free ribosomes. This resource limitation can result in the emergence of hidden regulatory interactions between co-expressed genes, such as gene coupling, where for example the expression of one gene decreases as another increases. In synthetic biology, where circuits utilising multiple new genes may be introduced into microbes, such hidden interactions can have serious consequences, since resource competition can introduce coupling which in turn causes circuit performance degradation or even failure. Here, we propose a novel approach that allows the decoupling of genes in the presence of resource limitations by dynamically controlling the allocation of translational resources between the host and circuit genes. We develop a complete mechanistic model that captures the key resource limitations in the system, and show how this model may be approximated by a reduced model that can be used for the purposes of controller design. An optimal resource allocation controller which decouples circuit genes is designed and used to guide the design of an experimentally feasible controller. Simulation results verify the ability of the controller to effectively remove the regulatory interactions imposed by translational resource limitations.

Small non-coding RNAs (sRNAs) are expected to have pivotal roles in the adaptive responses underlying symbiosis of nitrogen-fixing rhizobia with legumes. Here, we provide primary insights into the function and activity mechanism of the Sinorhizobium meliloti trans-sRNA NfeR1 (Nodule Formation Efficiency RNA). Northern blot probing and transcription tracking with fluorescent promoter-reporter fusions unveiled high nfeR1 expression in response to salt stress and throughout the symbiotic interaction. The strength and differential regulation of nfeR1 transcription are conferred by a motif, which is conserved in nfeR1 promoter regions in α-proteobacteria. NfeR1 loss-of-function compromised osmoadaptation of free-living bacteria, whilst causing misregulation of salt-responsive genes related to stress adaptation, osmolytes catabolism and membrane trafficking. Nodulation tests revealed that lack of NfeR1 affected competitiveness, infectivity, nodule development and symbiotic efficiency of S. meliloti on alfalfa roots. Comparative computer predictions and a genetic reporter assay evidenced a redundant role of three identical NfeR1 unpaired anti Shine-Dalgarno motifs for targeting and downregulation of translation of multiple mRNAs from transporter genes. Our data provide genetic evidence of the hyperosmotic conditions of the endosymbiotic compartments. NfeR1-mediated gene regulation in response to this cue could contribute to coordinate nutrient uptake with the metabolic reprogramming concomitant to symbiotic transitions.

Plastics have become an important environmental concern due to their durability and resistance to degradation. Out of all plastic materials, polyesters such as polyethylene terephthalate (PET) are amenable to biological degradation due to the action of microbial polyester hydrolases. The hydrolysis products obtained from PET can thereby be used for the synthesis of novel PET as well as becoming a potential carbon source for microorganisms. In addition, microorganisms and biomass can be used for the synthesis of the constituent monomers of PET from renewable sources. The combination of both biodegradation and biosynthesis would enable a completely circular bio-PET economy beyond the conventional recycling processes. Circular strategies like this could contribute to significantly decrease the environmental impact of our dependence on this polymer. Here we review the efforts made towards turning PET into a viable feedstock for microbial transformations. We highlight current bottlenecks in the degradation of the polymer and the metabolism of the monomers and we showcase fully biological or semisynthetic processes leading to the synthesis of PET from sustainable substrates.

Primary infection of legumes by rhizobia involves the controlled localized enzymatic breakdown of cell walls at root hair tips. Previous studies determined the role of rhizobial CelC2 cellulase in different steps of the symbiotic interaction Rhizobium leguminosarum-Trifolium repens. Recent findings also showed that CelC2 influences early signalling events in the Ensifer meliloti-Medicago truncatula interaction. Here, we have monitored the root hair phenotypes of two legume plants, T. repens and M. sativa, upon inoculation with strains of their cognate and non-cognate rhizobial species, R. leguminosarum bv trifolii and E. meliloti, (over)expressing the CelC2 coding gene, celC. Regardless of the host, CelC2 specifically elicited ‘hole-on-the-tip’ events (Hot phenotype) in the root hair apex, consistent with the role of this endoglucanase in eroding the noncrystalline cellulose found in polarly growing cell walls. Overproduction of CelC2 also increased root hair tip redirections (RaT phenotype) events in both cognate and non-cognate hosts. Interestingly, heterologous celC expression also induced non-canonical alterations in ROS (Reactive Oxygen Species) homeostasis at root hair tips of Trifolium and Medicago. These results suggest the concurrence of shared unspecific and host-related plant responses to CelC2 during early steps of symbiotic rhizobial infection. Our data thus identify CelC2 cellulase as an important determinant of events underlying early infection of the legume host by rhizobia., This work was supported by Grant AGL2015-70510-R from MINECO (Spanish Ministry of Economy, Industry and Competitiveness). E.M. and M.R. were supported by PhD fellowships from the Spanish government.

International audience; Plastics have become an important environmental concern due to their durability and resistance to degradation. Out of all plastic materials, polyesters such as polyethylene terephthalate (PET) are amenable to biological degradation due to the action of microbial polyester hydrolases. The hydrolysis products obtained from PET can thereby be used for the synthesis of novel PET as well as become a potential carbon source for microorganisms. In addition, microorganisms and biomass can be used for the synthesis of the constituent monomers of PET from renewable sources. The combination of both biodegradation and biosynthesis would enable a completely circular bio-PET economy beyond the conventional recycling processes. Circular strategies like this could contribute to significantly decreasing the environmental impact of our dependence on this polymer. Here we review the efforts made towards turning PET into a viable feedstock for microbial transformations. We highlight current bottlenecks in degradation of the polymer and metabolism of the monomers, and we showcase fully biological or semisynthetic processes leading to the synthesis of PET from sustainable substrates.

Members of the ribonuclease (RNase) III family of enzymes are metal-dependent double-strand specific endoribonucleases. They are ubiquitously found and eukaryotic RNase III-like enzymes include Dicer and Drosha, involved in RNA processing and RNA interference. In this work, we have addressed the primary characterization of RNase III from the symbiotic nitrogen-fixing a-proteobacterium Sinorhizobium meliloti. The S. meliloti rnc gene does encode an RNase III-like protein (SmRNase III), with recognizable catalytic and double-stranded RNA (dsRNA)-binding domains that clusters in a branch with its a-proteobacterial counterparts. Purified SmRNase III dimerizes, is active at neutral to alkaline pH and behaves as a strict metal cofactor-dependent double-strand endoribonuclease, with catalytic features distinguishable from those of the prototypical member of the family, the Escherichia coli ortholog (EcRNase III). SmRNase III prefers Mn2+ rather than Mg as metal cofactor, cleaves the generic structured R1.1 substrate at a site atypical for RNase III cleavage, and requires higher cofactor concentrations and longer dsRNA substrates than EcRNase III for optimal activity. Furthermore, the ultraconserved E125 amino acid was shown to play a major role in the metal-dependent catalysis of SmRNase III. SmRNase III degrades endogenous RNA substrates of diverse biogenesis with different efficiency, and is involved in the maturation of the 23S rRNA. SmRNase III loss-of-function neither compromises viability nor alters morphology of S. meliloti cells, but influences growth, nodulation kinetics, the onset of nitrogen fixation and the overall symbiotic efficiency of this bacterium on the roots of its legume host, alfalfa, which ultimately affects plant growth. Our results support an impact of SmRNase III on nodulation and symbiotic nitrogen fixation in plants., This work was funded by project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by FCT – Fundação para a Ciência e a Tecnologia to CMA; project PTDC/BIA-MIC/1399/2014. In addition, FCT provides post-doctoral grants ref SFRH/BPD/109464/2015 to MS and ref SFRH/BPD/75887/2011 to RGM. This work was also funded by the ERDF-cofinanced grants BFU2013-48282-C2-2-P and BFU2017-82645-P from the Spanish Ministerio de Economía, Industria y Competitividad (MINEICO) to JJ-Z; MR was supported by a contract of the Programa de Formación Post-Doctoral Juan de la Cierva from MINEICO.

The identification of the protein partners of bacterial small noncoding RNAs (sRNAs) is essential to understand the mechanistic principles and functions of riboregulation in prokaryotic cells. Here, we describe an optimized affinity chromatography protocol that enables purification of in vivo formed sRNA-protein complexes in Sinorhizobium meliloti, a genetically tractable nitrogen-fixing plant symbiotic bacterium. The procedure requires the tagging of the desired sRNA with the MS2 aptamer, which is affinity-captured by the MS2-MBP protein conjugated to an amylose resin. As proof of principle, we show recovery of the RNA chaperone Hfq associated to the strictly Hfq-dependent AbcR2 trans-sRNA. This method can be applied for the investigation of sRNA-protein interactions on a broad range of genetically tractable α-proteobacteria.

The infection of legume plants by rhizobia is tightly regulated to ensure accurate bacterial penetration, infection, and development of functionally efficient nitrogen-fixing root nodules. Rhizobial Nod factors (NF) have key roles in the elicitation of nodulation signaling. Infection of white clover roots also involves the tightly regulated specific breakdown of the noncrystalline apex of cell walls in growing root hairs, which is mediated by Rhizobium leguminosarum bv. trifolii cellulase CelC2. Here, we have analyzed the impact of this endoglucanase on symbiotic signaling in the model legume Medicago truncatula. Ensifer meliloti constitutively expressing celC gene exhibited delayed nodulation and elicited aberrant ineffective nodules, hampering plant growth in the absence of nitrogen. Cotreatment of roots with NF and CelC2 altered Ca spiking in root hairs and induction of the early nodulin gene ENOD11. Our data suggest that CelC2 alters early signaling between partners in the rhizobia-legume interaction., Ministerio de Economía y Competitividad Grant/Award Number: AGL2015-70510-R

The identification of the protein partners of bacterial small noncoding RNAs (sRNAs) is essential to understand the mechanistic principles and functions of riboregulation in prokaryotic cells. Here, we describe an optimized affinity chromatography protocol that enables purification of in vivo formed sRNA-protein complexes in Sinorhizobium meliloti, a genetically tractable nitrogen-fixing plant symbiotic bacterium. The procedure requires the tagging of the desired sRNA with the MS2 aptamer, which is affinity-captured by the MS2-MBP protein conjugated to an amylose resin. As proof of principle, we show recovery of the RNA chaperone Hfq associated to the strictly Hfq-dependent AbcR2 trans-sRNA. This method can be applied for the investigation of sRNA-protein interactions on a broad range of genetically tractable α-proteobacteria.

The use of orthogonal ribosomes in combination with dynamic resource allocation controllers is a promising approach for relieving the negative effects of cellular resource limitations on the modularity of synthetic gene circuits. Here, we develop a detailed mechanistic model of gene expression and resource allocation, which when simplified to a tractable level of complexity, allows the rational design of translational resource allocation controllers. Analysis of this model reveals a fundamental design trade-off; that reducing coupling acts to decrease gene expression. Through a sensitivity analysis of the experimentally tuneable controller parameters, we identify how each controller design parameter affects the overall closed-loop behaviour of the system, leading to a detailed set of design guidelines for optimally managing this trade-off. Based on our designs, we evaluated a number of alternative potential experimental implementations of the proposed system using commonly available biological components. Finally, we show that the controller is capable of dynamically allocating ribosomes as needed to restore modularity in a number of more complex synthetic circuits, such as the repressilator, and activation cascades composed of multiple interacting modules.

High-throughput transcriptome profiling (RNAseq) has uncovered large and heterogeneous populations of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated and rely on protein-assisted antisense interactions to trans-encoded target mRNAs to fine-tune posttranscriptional reprogramming of gene expression in response to external cues. However, annotation and function of sRNAs are still largely overlooked in nonmodel bacteria with complex lifestyles. Here, we describe experimental protocols successfully applied for the accurate annotation, expression profiling and target mRNA identification of trans-acting sRNAs in the nitrogen-fixing α-rhizobium Sinorhizobium meliloti. The protocols presented here can be similarly applied for the characterization of trans-sRNAs in genetically tractable α-proteobacteria of agronomical or clinical relevance interacting with eukaryotic hosts.

High-throughput transcriptome profiling (RNAseq) has uncovered large and heterogeneous populations of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated and rely on protein-assisted antisense interactions to trans-encoded target mRNAs to fine-tune posttranscriptional reprogramming of gene expression in response to external cues. However, annotation and function of sRNAs are still largely overlooked in nonmodel bacteria with complex lifestyles. Here, we describe experimental protocols successfully applied for the accurate annotation, expression profiling and target mRNA identification of trans-acting sRNAs in the nitrogen-fixing α-rhizobium Sinorhizobium meliloti. The protocols presented here can be similarly applied for the characterization of trans-sRNAs in genetically tractable α-proteobacteria of agronomical or clinical relevance interacting with eukaryotic hosts.

Transcripts that do not encode proteins (ncRNAs) have emerged as a widespread type of regulators in all kingdoms of life. They usually fine-tune gene expression in response to environmental changes by modulating mRNA translation and/or stability through base pairing. Given that bacterial adaptation during the transition from free-living to host-dependent conditions demands a tightly and coordinated regulation of gene expression upon perception of a variety of signals, symbioses, as well as pathogenesis, present a set of characteristics expected to be modulated by ncRNAs. However, little is known about the relevance of ncRNAs in the biology of nitrogen-fixing endosymbiotic bacteria. Here, we mined published RNAseq data from the legume symbiont Sinorhizobium meliloti to search for ncRNAs transcribed opposite to the coding strand (antisense ncRNAs; asRNAs) of genuine symbiotic genes, i.e. required for the synthesis of nodulation signal factors (nod genes) and the nitrogenase components (nif genes). Further characterization of selected nodD2, nifK and nifE asRNAs revealed that some transcripts accumulate differentially in response to different environmental factors, and seem to repress nitrogen fixation genes under conditions in which functional nitrogenase is dispensable, suggesting regulatory functions. Furthermore, Medicago sativa plants inoculated with genetically modified S. meliloti strains overexpressing one of these asRNAs showed slight differences in nitrogen fixation ability with respect to the controls. These experiments provide the first evidences of symbiotically relevant antisense riboregulation in rhizobia.

High-throughput transcriptome profiling (RNAseq) has uncovered large and heterogeneous populations of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. These sRNAs act mostly by protein-assisted base-pairing with target mRNAs to fine-tune post-transcriptional reprogramming of gene expression underlying bacterial responses to changing environments. Riboregulation impacts virtually any physiological process, and has been shown to largely influence virulence of pathogenic bacteria. Here, we review our current knowledge on the structure, conservation and function of the noncoding transcriptome of the α-rhizobia Sinorhizobium meliloti, the nitrogen-fixing symbiotic partner of alfalfa and related medics. Several RNAseq-based surveys in S. meliloti have shown abundant transcription from hitherto regarded as noncoding intergenic regions (IGRs), strikingly high numbers of mRNA-derived RNAs and pervasive antisense transcription of protein-coding genes. sRNAs encoded within IGRs constitute the most extensively studied group of bacterial riboregulators. They are differentially expressed and modulate translation and/or stability of trans-encoded target mRNAs by short antisense interactions that, in enteric model bacteria, are facilitated by the RNA chaperone Hfq. Among symbiotic rhizobia, regulatory sRNAs have been functionally characterized only in S. meliloti to date. The trans-sRNAs AbcR1 and AbcR2 are examples of Hfq-dependent sRNAs whereas EcpR1 does not bind Hfq. We will provide insights into the transcriptional regulation and activity mechanisms of these sRNAs for the targeting and control of multiple mRNAs involved in nutrient uptake (AbcR1/2) and cell cycle progression (EcpR1).

Surveying the dynamics of metabolic networks of Gram-negative bacteria often requires the conditional shutdown of enzymatic activities once the corresponding proteins have been produced. We show that given biochemical functions can be entirely suppressed in vivo with camel antibodies (VHHs, nanobodies) that target active sites of cognate enzymes expressed in the cytoplasm. As a proof of principle, we raised VHHs against 2,5-dihydroxypyridine dioxygenase (NicX) of Pseudomonas putida, involved in nicotinic acid metabolism. Once fused to a thioredoxin domain, the corresponding nanobodies inhibited the enzyme both in Escherichia coli and in P. putida cells, which then accumulated the metabolic substrate of NicX. VHHs were further engineered to track the antigen in vivo by C-terminal fusion to a fluorescent protein. Conditional expression of the resulting VHHs allows simultaneously to track and target proteins of interest and enables the design of transient phenotypes without mutating the genetic complement of the bacteria under study.

We study the dynamics of a flexibly-mounted cylinder having a control rod in its near wake, when subject to a uniform cross-flow. The objective is to investigate the effect of a localized perturbation introduced in the near wake of the cylinder. The work consists of a parametric study with a rigid cylinder mounted in an air bearing rig, with its motion constrained to move in the direction transverse to the flow. The cylinder system is characterised by a low combined mass-damping parameter. During the experiments, the location of the rod will be fixed, but its size and the free stream velocity will be varied. The dynamic response of the cylinder will be measured using non-intrusive techniques. The vortex dynamics in the near wake of the system will also be described after measurements obtained using Digital Image Particle Velocimetry (DPIV). The objective is to understand the physical mechanisms of the fluid-structure interaction phenomena involved in the changes of the dynamic response of the cylinder when using the control rod.

A common approach to design genetic circuits is to compose gene expression cassettes together. While appealing, this modular approach is challenged by the fact that expression of each gene depends on the availability of transcriptional/translational resources, which is in turn determined by the presence of other genes in the circuit. This raises the question of how competition for resources by different genes affects a circuit’s behavior. Here, we create a library of genetic activation cascades in bacteriaE. coli, where we explicitly tune the resource demand by each gene. We develop a general Hill-function-based model that incorporates resource competition effects through resource demand coefficients. These coefficients lead to non-regulatory interactions among genes that reshape circuit’s behavior. For the activation cascade, such interactions result in surprising biphasic or monotonically decreasing responses. Finally, we use resource demand coefficients to guide the choice of ribosome binding site (RBS) and DNA copy number to restore the cascade’s intended monotonically increasing response. Our results demonstrate how unintended circuit’s behavior arises from resource competition and provide a model-guided methodology to minimize the resulting effects.

Myxococcus xanthus is a social bacterium that preys on prokaryotic and eukaryotic microorganisms. Co-culture of M. xanthus with reference laboratory strains and field isolates of the legume symbiont Sinorhizobium meliloti revealed two different predatory patterns that resemble frontal and wolf-pack attacks. Use of mutants impaired in the two types of M. xanthus surface motility (A or adventurous and S or social motility) and a csgA mutant, which is unable to form macroscopic travelling waves known as ripples, has demonstrated that both motility systems but not rippling are required for efficient predation. To avoid frontal attack and reduce killing rates, rhizobial cells require a functional expR gene. ExpR regulates expression of genes involved in a variety of functions. The use of S. meliloti mutants impaired in several of these functions revealed that the exopolysaccharide galactoglucan (EPS II) is the major determinant of the M. xanthus predatory pattern. The data also suggest that this biopolymer confers an ecological advantage to rhizobial survival in soil, which may have broad environmental implications.

Significance Evolution by natural selection is driven by fitness differences, which define a “fitness landscape” in the space of all possible genetic sequences. Understanding the landscape is critical for understanding and predicting natural selection, yet very little is known about the structure of a real fitness landscape. Here we experimentally determine the complete fitness landscape of small RNA selected to interact with GTP, a building block for early life. We find that the landscape is composed of largely disconnected islands of active sequences. This scenario suggests that natural selection under these conditions would be constrained to local exploration of sequence space but that replaying the initial emergence of a functional RNA could lead to a different outcome.

34 p.-4 fig., Our view on the bacterial responses to the aerobic degradation of aromatic compounds has been enriched considerably by the current omic methodologies and systems biology approaches,revealing the participation of intricate metabolic and regulatory networks. New enzymes, transporters, and specific/global regulatory systems have been recently characterized, and reveal that the widespread biodegradation capabilities extend to unexpected substrates such as lignin. A completely different biochemical strategy based on the formation of aryl-CoA epoxide intermediates has been unraveled for aerobic hybrid pathways, such as those involved in benzoate and phenylacetate degradation. Aromatic degradation pathways are also an important source of metabolic exchange factors and, therefore, they play a previously unrecognized biological role in cell-to-cell communication. Beyond the native bacterial biodegradation capabilities, pathway evolution as well as computational and synthetic biology approaches are emerging as powerful tools to design novel strain-specific pathways for degradation of xenobiotic compounds., The work in E. Díaz laboratory was supported by grants BIO2009-10438 and CSD2007-00005 from the Spanish Ministry of Science and Innovation.

Microbial communities are increasingly utilised in biotechnology. Efficiency and productivity in many of these applications depends on the presence of cooperative interactions between members of the community. Two key processes underlying these interactions are the production of public goods and metabolic crossfeeding, which can be understood in the general framework of ecological and evolutionary (eco-evo) dynamics. In this review we illustrate the relevance of cooperative interactions in microbial biotechnological processes, discuss their mechanistic origins, and analyse their evolutionary resilience. Cooperative behaviours can be damaged by the emergence of ‘cheating' cells that benefit from the cooperative interactions but do not contribute to them. Despite this, cooperative interactions can be stabilized by spatial segregation, by the presence of feedbacks between the evolutionary dynamics and the ecology of the community, by the role of regulatory systems coupled to the environmental conditions and by the action of horizontal gene transfer. Cooperative interactions enrich microbial communities with a higher degree of robustness against environmental stress and can facilitate the evolution of more complex traits. Therefore, the evolutionary resilience of microbial communities and their ability to constraint detrimental mutants should be considered in order to design robust biotechnological applications. This article is protected by copyright. All rights reserved.

In vitro selection experiments in biochemistry allow for the discovery of novel molecules capable of specific desired biochemical functions. However, this is not the only benefit we can obtain from such selection experiments. Since selection from a random library yields an unprecedented, and sometimes comprehensive, view of how a particular biochemical function is distributed across sequence space, selection experiments also provide data for creating and analyzing molecular fitness landscapes, which directly map function (phenotypes) to sequence information (genotypes). Given the importance of understanding the relationship between sequence and functional activity, reliable methods to build and analyze fitness landscapes are needed. Here, we present some statistical methods to extract this information from pools of RNA molecules. We also provide new computational tools to construct and study molecular fitness landscapes.

The chassis is the cellular host used as a recipient of engineered biological systems in synthetic biology. They are required to propagate the genetic information and to express the genes encoded in it. Despite being an essential element for the appropriate function of genetic circuits, the chassis is rarely considered in their design phase. Consequently, the circuits are transferred to model organisms commonly used in the laboratory, such as Escherichia coli, that may be suboptimal for a required function. In this review, we discuss some of the properties desirable in a versatile chassis and summarize some examples of alternative hosts for synthetic biology amenable for engineering. These properties include a suitable life style, a robust cell wall, good knowledge of its regulatory network as well as of the interplay of the host components with the exogenous circuits, and the possibility of developing whole-cell models and tuneable metabolic fluxes that could allow a better distribution of cellular resources (metabolites, ATP, nucleotides, amino acids, transcriptional and translational machinery). We highlight Pseudomonas putida, widely used in many different biotechnological applications as a prominent organism for synthetic biology due to its metabolic diversity, robustness and ease of manipulation.

Experimental evolution of RNA (or DNA) is a powerful method to isolate sequences with useful function (e.g., catalytic RNA), discover fundamental features of the sequence-activity relationship (i.e., the fitness landscape), and map evolutionary pathways or functional optimization strategies. However, the limitations of current sequencing technology create a significant undersampling problem which impedes our ability to measure the true distribution of unique sequences. In addition, synthetic sequence pools contain a non-random distribution of nucleotides. Here, we present and analyze simple models to approximate the true sequence distribution. We also provide tools that compensate for sequencing errors and other biases that occur during sample processing. We describe our implementation of these algorithms in the Galaxy bioinformatics platform.