Synthetic Biology Toolbox for Antarctic Pseudomonas sp. Strains: Toward a Psychrophilic Nonmodel Chassis for Function-Driven Metagenomics.

One major limitation of function-driven metagenomics is the ability of the host to express the metagenomic DNA correctly. Differences in the transcriptional, translational, and post-translational machinery between the organism to which the DNA belongs and the host strain are all factors that influence the success of a functional screening. For this reason, the use of alternative hosts is an appropriate approach to favor the identification of enzymatic activities in function-driven metagenomics. To be implemented, appropriate tools should be designed to build the metagenomic libraries in those hosts. Moreover, discovery of new chassis and characterization of synthetic biology toolbox in nonmodel bacteria is an active field of research to expand the potential of these organisms in processes of industrial interest. Here, we assessed the suitability of two Antarctic psychrotolerant Pseudomonas strains as putative alternative hosts for function-driven metagenomics using pSEVA modular vectors as scaffold. We determined a set of synthetic biology tools suitable for these hosts and, as a proof of concept, we demonstrated their fitness for heterologous protein expression. These hosts represent a step forward for the prospection and identification of psychrophilic enzymes of biotechnological interest.

Paenibacillus farraposensis sp. nov., isolated from a root nodule of Arachis villosa.

Strain UY79T was isolated from a root nodule of Arachis villosa, collected at the Esteros de Farrapos National Park, Río Negro, Uruguay. Cells were non-motile Gram-variable rods with central to subterminal oval to ellipsoidal endospores that swell the sporangia. Growth was observed in the range of 15-42 °C (optimum, 30 °C), pH 5.0-9.0 (optimum, pH 7.0-8.0) and with up to 3 % (w/v) NaCl (optimum, 1-2 %). Strain UY79T was facultative anaerobic, catalase-positive and oxidase-negative. According to the results of 16S rRNA gene sequence analysis, UY79T belongs to the genus Paenibacillus and is closely related to P. ottowii MS2379T, P. peoriae BD-57T, P. polymyxa ATCC 842T and P. brasilensis PB172T, exhibiting 99.4, 99.0, 99.0 and 98.9% sequence identity, respectively. Average nucleotide identity and digital DNA-DNA hybridization values with the most closely related type strains were 74.3-88.6% and 38.2-48.7 %, respectively. Major fatty acids (>10 %) were anteiso-C15:0, iso-C15:0, and C16 : 0. Menaquinones MK-7 and MK-6 were the only isoprenoid quinones detected. Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified glycolipid. Spermidine was the predominant polyamine. The DNA G+C content based on the draft genome sequence was 46.34 mol%. Based on the current polyphasic study, UY79T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus farraposensis sp. nov. is proposed. The type strain is UY79T (=CCM 9147T=CGMCC 1.19038T).

Paenibacillus sp. Strain UY79, Isolated from a Root Nodule of Arachis villosa, Displays a Broad Spectrum of Antifungal Activity.

A nodule-inhabiting Paenibacillus sp. strain (UY79) isolated from wild peanut (Arachis villosa) was screened for its antagonistic activity against diverse fungi and oomycetes (Botrytis cinerea, Fusarium verticillioides, Fusarium oxysporum, Fusarium graminearum, Fusarium semitectum, Macrophomina phaseolina, Phomopsis longicolla, Pythium ultimum, Phytophthora sojae, Rhizoctonia solani, Sclerotium rolfsii, and Trichoderma atroviride). The results obtained show that Paenibacillus sp. UY79 was able to antagonize these fungi/oomycetes and that agar-diffusible compounds and volatile compounds (different from HCN) participate in the antagonism exerted. Acetoin, 2,3-butanediol, and 2-methyl-1-butanol were identified among the volatile compounds produced by strain UY79 with possible antagonistic activity against fungi/oomycetes. Paenibacillus sp. strain UY79 did not affect symbiotic association or growth promotion of alfalfa plants when coinoculated with rhizobia. By whole-genome sequence analysis, we determined that strain UY79 is a new species of Paenibacillus within the Paenibacillus polymyxa complex. Diverse genes putatively involved in biocontrol activity were identified in the UY79 genome. Furthermore, according to genome mining and antibiosis assays, strain UY79 would have the capability to modulate the growth of bacteria commonly found in soil/plant communities. IMPORTANCE Phytopathogenic fungi and oomycetes are responsible for causing devastating losses in agricultural crops. Therefore, there is enormous interest in the development of effective and complementary strategies that allow the control of the phytopathogens, reducing the input of agrochemicals in croplands. The discovery of new strains with expanded antifungal activities and with a broad spectrum of action is challenging and of great future impact. Diverse strains belonging to the P. polymyxa complex have been reported to be effective biocontrol agents. Results presented here show that the novel discovered strain of Paenibacillus sp. presents diverse traits involved in antagonistic activity against a broad spectrum of pathogens and is a potential and valuable strain to be further assessed for the development of biofungicides.

Genetically Engineered Proteins to Improve Biomass Conversion: New Advances and Challenges for Tailoring Biocatalysts.

Protein engineering emerged as a powerful approach to generate more robust and efficient biocatalysts for bio-based economy applications, an alternative to ecologically toxic chemistries that rely on petroleum. On the quest for environmentally friendly technologies, sustainable and low-cost resources such as lignocellulosic plant-derived biomass are being used for the production of biofuels and fine chemicals. Since most of the enzymes used in the biorefinery industry act in suboptimal conditions, modification of their catalytic properties through protein rational design and in vitro evolution techniques allows the improvement of enzymatic parameters such as specificity, activity, efficiency, secretability, and stability, leading to better yields in the production lines. This review focuses on the current application of protein engineering techniques for improving the catalytic performance of enzymes used to break down lignocellulosic polymers. We discuss the use of both classical and modern methods reported in the literature in the last five years that allowed the boosting of biocatalysts for biomass degradation.

Microbiology can be comic.

For microbiologists, the importance of microorganisms in our daily lives and their impact on our well-being is evident. However, microbiology literacy in our society is far from being enough for individuals to make informed choices and to demand actions based on that information. The vaccine hesitation movement and the alarming increase in antimicrobial resistance due to overuse and misuse of antibiotics are just two examples of how much work is needed to make our society literate in topics related to microbiology. Considering the challenges of communicating a discipline surrounded by misconceptions, which studies the role of living organisms that cannot be seen in plain sight, we need to explore different strategies to effectively contribute to microbiology literacy in our society. Here, we will comment on the use of comics for such a task.

Comics as an Educational Resource To Teach Microbiology in the Classroom.

New educational resources are being implemented as an initiative to foster learning. In order to contribute to the toolkit of innovative educational resources, we developed a microbiology comic book. The aim of this comic is to provide educators with a fun, accessible, and rigorous way to generate awareness of the invisible world that surrounds us and that inhabits us. Bacteria have a reputation as harmful and disgusting entities. Mass media, with advertisements of disinfectants, soaps, and house cleaning products, are sending a distorted message about microbes. We must debunk these misconceptions and emphasize the importance of microorganisms, and particularly bacteria, in the environment and our lives. Education is the means to this end, and therefore this comic is intended to help educators teach microbiology in an attractive, accurate, and straightforward way. Here, we present this educational tool and give some tips on the different themes that can be addressed in the classroom using this resource.

La implementación de nuevos recursos educativos como iniciativa para fomentar el aprendizaje es cada vez más frecuente, y es con esta finalidad que desarrollamos un cómic sobre microbiología. A lo largo de la historia las bacterias han sido concebidas como entidades dañinas y repugnantes. Los medios de comunicación, con sus anuncios de desinfectantes, jabones y productos de limpieza, envían continuamente un mensaje distorsionado sobre los microbios. En este sentido, creemos que es importante refutar los conceptos erróneos y enfatizar la importancia de los microorganismos, y en particular de las bacterias, en el medio ambiente y en nuestras vidas. La manera de incorporar conceptos certeros en el conocimiento es mediante la educación. Por ello, creamos este cómic destinado a ayudar a los educadores en la enseñanza de la microbiología de una manera atractiva, divertida y accesible para los alumnos, y a la vez precisa y rigurosa, como forma de generar conciencia del mundo invisible que nos rodea y que nos habita. En este trabajo presentamos nuestro comic como herramienta educativa y brindamos recomendaciones sobre los diferentes temas que se pueden abordar en el aula con él.

Expanding the Toolbox of Broad Host-Range Transcriptional Terminators for Proteobacteria through Metagenomics.

As the field of synthetic biology moves toward the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacterium Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria, for instance Pseudomonas putida. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in P. putida. Further characterization allowed the identification of 4 unique sequences ranging from 58 to 181 bp long that efficiently terminate transcription in P. putida, E. coli, Burkholderia phymatum, and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

Highly conserved nucleotide motifs present in the 5'UTR of the heme-receptor gene shmR are required for HmuP-dependent expression of shmR in Ensifer meliloti.

Heme may represent a major iron-source for bacteria. In the symbiotic nitrogen-fixing bacterium Ensifer meliloti 1021, iron acquisition from heme depends on the outer-membrane heme-receptor ShmR. Expression of shmR gene is repressed by iron in a RirA dependent manner while under iron-limitation its expression requires the small protein HmuP. In this work, we identified highly conserved nucleotide motifs present upstream the shmR gene. These motifs are widely distributed among Alpha and Beta Proteobacteria, and correlate with the presence of HmuP coding sequences in bacterial genomes. According to data presented in this work, we named these new motifs as HmuP-responsive elements (HPREs). In the analyzed genomes, the HPREs were always present upstream of genes encoding putative heme-receptors. Moreover, in those Alpha and Beta Proteobacteria where transcriptional start sites for shmR homologs are known, HPREs were located in the 5'UTR region. In this work we show that in E. meliloti 1021, HPREs are involved in HmuP-dependent shmR expression. Moreover, we show that changes in sequence composition of the HPREs correlate with changes in a predicted RNA secondary structure element and affect shmR gene expression.

Converting a Periplasmic Binding Protein into a Synthetic Biosensing Switch through Domain Insertion.

All biosensing platforms rest on two pillars: specific biochemical recognition of a particular analyte and transduction of that recognition into a readily detectable signal. Most existing biosensing technologies utilize proteins that passively bind to their analytes and therefore require wasteful washing steps, specialized reagents, and expensive instruments for detection. To overcome these limitations, protein engineering strategies have been applied to develop new classes of protein-based sensor/actuators, known as protein switches, responding to small molecules. Protein switches change their active state (output) in response to a binding event or physical signal (input) and therefore show a tremendous potential to work as a biosensor. Synthetic protein switches can be created by the fusion between two genes, one coding for a sensor protein (input domain) and the other coding for an actuator protein (output domain) by domain insertion. The binding of a signal molecule to the engineered protein will switch the protein function from an "off" to an "on" state (or vice versa) as desired. The molecular switch could, for example, sense the presence of a metabolite, pollutant, or a biomarker and trigger a cellular response. The potential sensing and response capabilities are enormous; however, the recognition repertoire of natural switches is limited. Thereby, bioengineers have been struggling to expand the toolkit of molecular switches recognition repertoire utilizing periplasmic binding proteins (PBPs) as protein-sensing components. PBPs are a superfamily of bacterial proteins that provide interesting features to engineer biosensors, for instance, immense ligand-binding diversity and high affinity, and undergo large conformational changes in response to ligand binding. The development of these protein switches has yielded insights into the design of protein-based biosensors, particularly in the area of allosteric domain fusions. Here, recent protein engineering approaches for expanding the versatility of protein switches are reviewed, with an emphasis on studies that used PBPs to generate novel switches through protein domain insertion.

The Irr and RirA Proteins Participate in a Complex Regulatory Circuit and Act in Concert To Modulate Bacterioferritin Expression in Ensifer meliloti 1021.

In this work we found that the bfr gene of the rhizobial species Ensifer meliloti, encoding a bacterioferritin iron storage protein, is involved in iron homeostasis and the oxidative stress response. This gene is located downstream of and overlapping the smc03787 open reading frame (ORF). No well-predicted RirA or Irr boxes were found in the region immediately upstream of the bfr gene although two presumptive RirA boxes and one presumptive Irr box were present in the putative promoter of smc03787 We demonstrate that bfr gene expression is enhanced under iron-sufficient conditions and that Irr and RirA modulate this expression. The pattern of bfr gene expression as well as the response to Irr and RirA is inversely correlated to that of smc03787 Moreover, our results suggest that the small RNA SmelC759 participates in RirA- and Irr-mediated regulation of bfr expression and that additional unknown factors are involved in iron-dependent regulation.IMPORTANCEE. meliloti belongs to the Alphaproteobacteria, a group of bacteria that includes several species able to associate with eukaryotic hosts, from mammals to plants, in a symbiotic or pathogenic manner. Regulation of iron homeostasis in this group of bacteria differs from that found in the well-studied Gammaproteobacteria In this work we analyzed the effect of rirA and irr mutations on bfr gene expression. We demonstrate the effect of an irr mutation on iron homeostasis in this bacterial genus. Moreover, results obtained indicate a complex regulatory circuit where multiple regulators, including RirA, Irr, the small RNA SmelC759, and still unknown factors, act in concert to balance bfr gene expression.

Improved glycerol to ethanol conversion by E. coli using a metagenomic fragment isolated from an anaerobic reactor.

Crude glycerol obtained as a by-product of biodiesel production is a reliable feedstock with the potential to be converted into reduced chemicals with high yields. It has been previously shown that ethanol is the primary product of glycerol fermentation by Escherichia coli. However, few efforts were made to enhance this conversion by means of the expression of heterologous genes with the potential to improve glycerol transport or metabolism. In this study, a fosmid-based metagenomic library constructed from an anaerobic reactor purge sludge was screened for genetic elements that promote the use and fermentation of crude glycerol by E. coli. One clone was selected based on its improved growth rate on this feedstock. The corresponding fosmid, named G1, was fully sequenced (41 kbp long) and the gene responsible for the observed phenotype was pinpointed by in vitro insertion mutagenesis. Ethanol production from both pure and crude glycerol was evaluated using the parental G1 clone harboring the ethanologenic plasmid pLOI297 or the industrial strain LY180 complemented with G1. In mineral salts media containing 50 % (v/v) pure glycerol, ethanol concentrations increased two-fold on average when G1 was present in the cells reaching up to 20 g/L after 24 h fermentation. Similar fermentation experiments were done using crude instead of pure glycerol. With an initial OD620 of 8.0, final ethanol concentrations after 24 h were much higher reaching 67 and 75 g/L with LY180 cells carrying the control fosmid or the G1 fosmid, respectively. This translates into a specific ethanol production rate of 0.39 g h(-1) OD(-1) L(-1).

HmuS and HmuQ of Ensifer/Sinorhizobium meliloti degrade heme in vitro and participate in heme metabolism in vivo.

Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a Kd value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-ß and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.

Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation.

With the aim of improving current ethanologenic Escherichia coli strains, we screened a metagenomic library from bovine ruminal fluid for cellulolytic enzymes. We isolated one fosmid, termed Csd4, which was able to confer to E. coli the ability to grow on complex cellulosic material as the sole carbon source such as avicel, carboxymethyl cellulose, filter paper, pretreated sugarcane bagasse, and xylan. Glucanolytic activity obtained from E. coli transformed with Csd4 was maximal at 24 h of incubation and was inhibited when glucose or xylose were present in the media. The 34,406-bp DNA fragment of Csd4 was completely sequenced, and a putative endoglucanase, a xylosidase/arabinosidase, and a laccase gene were identified. Comparison analysis revealed that Csd4 derived from an organism closely related to Prevotella ruminicola, but no homologies were found with any of the genomes already sequenced. Csd4 was introduced into the ethanologenic E. coli MS04 strain and ethanol production from CMC, avicel, sugarcane bagasse, or filter paper was observed. Exogenously expressed ß-glucosidase had a positie effect on cell growth in agreement with the fact that no putative ß-glucosidase was found in Csd4. Ethanol production from sugarcane bagasse was improved threefold by Csd4 after saccharification by commercial Trichoderma reesei cellulases underlining the ability of Csd4 to act as a saccharification enhancer to reduce the enzymatic load and time required for cellulose deconstruction.

Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes.

A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40 °C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases.

A new small regulatory protein, HmuP, modulates haemin acquisition in Sinorhizobium meliloti.

Sinorhizobium meliloti has multiple systems for iron acquisition, including the use of haem as an iron source. Haem internalization involves the ShmR haem outer membrane receptor and the hmuTUV locus, which participates in haem transport across the cytoplasmic membrane. Previous studies have demonstrated that expression of the shmR gene is negatively regulated by iron through RirA. Here, we identify hmuP in a genetic screen for mutants that displayed aberrant control of shmR. The normal induction of shmR in response to iron limitation was lost in the hmuP mutant, showing that this gene positively affects shmR expression. Moreover, the HmuP protein is not part of the haemin transporter system. Analysis of gene expression and siderophore production indicates that disruption of hmuP does not affect other genes related to the iron-restriction response. Our results strongly indicate that the main function of HmuP is the transcriptional regulation of shmR. Sequence alignment of HmuP homologues and comparison with the NMR structure of Rhodopseudomonas palustris CGA009 HmuP protein revealed that certain amino acids localized within predicted beta-sheets are well conserved. Our data indicate that at least one of the beta-sheets is important for HmuP activity.

ShmR is essential for utilization of heme as a nutritional iron source in Sinorhizobium meliloti.

The bacterium Sinorhizobium meliloti is able to use heme as a nutritional iron source. Here, we show that the iron-regulated shmR gene encodes an outer membrane protein required for growth on heme. Furthermore, an shmR mutant is resistant to the toxic heme analog gallium protoporphyrin. Thus, the receptor protein of the heme transport system has been identified in S. meliloti.