A directed genome evolution method to enhance hydrogen production in Rhodobacter capsulatus.

Nitrogenase-dependent H2 production by photosynthetic bacteria, such as Rhodobacter capsulatus, has been extensively investigated. An important limitation to increase H2 production using genetic manipulation is the scarcity of high-throughput screening methods to detect possible overproducing mutants. Previously, we engineered R. capsulatus strains that emitted fluorescence in response to H2 and used them to identify mutations in the nitrogenase Fe protein leading to H2 overproduction. Here, we used ultraviolet light to induce random mutations in the genome of the engineered H2-sensing strain, and fluorescent-activated cell sorting to detect and isolate the H2-overproducing cells from libraries containing 5 × 105 mutants. Three rounds of mutagenesis and strain selection gradually increased H2 production up to 3-fold. The whole genomes of five H2 overproducing strains were sequenced and compared to that of the parental sensor strain to determine the basis for H2 overproduction. No mutations were present in well-characterized functions related to nitrogen fixation, except for the transcriptional activator nifA2. However, several mutations mapped to energy-generating systems and to carbon metabolism-related functions, which could feed reducing power or ATP to nitrogenase. Time-course experiments of nitrogenase depression in batch cultures exposed mismatches between nitrogenase protein levels and their H2 and ethylene production activities that suggested energy limitation. Consistently, cultivating in a chemostat produced up to 19-fold more H2 than the corresponding batch cultures, revealing the potential of selected H2 overproducing strains.

Functional Nitrogenase Cofactor Maturase NifB in Mitochondria and Chloroplasts of Nicotiana benthamiana.

Engineering plants to synthesize nitrogenase and assimilate atmospheric N2 will reduce crop dependency on industrial N fertilizers. This technology can be achieved by expressing prokaryotic nitrogen fixation gene products for the assembly of a functional nitrogenase in plants. NifB is a critical nitrogenase component since it catalyzes the first committed step in the biosynthesis of all types of nitrogenase active-site cofactors. Here, we used a library of 30 distinct nifB sequences originating from different phyla and ecological niches to restore diazotrophic growth of an Azotobacter vinelandii nifB mutant. Twenty of these variants rescued the nifB mutant phenotype despite their phylogenetic distance to A. vinelandii. Because multiple protein interactions are required in the iron-molybdenum cofactor (FeMo-co) biosynthetic pathway, the maturation of nitrogenase in a heterologous host can be divided in independent modules containing interacting proteins that function together to produce a specific intermediate. Therefore, nifB functional modules composed of a nifB variant, together with the A. vinelandii NifS and NifU proteins (for biosynthesis of NifB [Fe4S4] clusters) and the FdxN ferredoxin (for NifB function), were expressed in Nicotiana benthamiana chloroplasts and mitochondria. Three archaeal NifB proteins accumulated at high levels in soluble fractions of chloroplasts (Methanosarcina acetivorans and Methanocaldococcus infernus) or mitochondria (M. infernus and Methanothermobacter thermautotrophicus). These NifB proteins were shown to accept [Fe4S4] clusters from NifU and were functional in FeMo-co synthesis in vitro. The accumulation of significant levels of soluble and functional NifB proteins in chloroplasts and mitochondria is critical to engineering biological nitrogen fixation in plants. IMPORTANCE Biological nitrogen fixation is the conversion of inert atmospheric dinitrogen gas into nitrogen-reactive ammonia, a reaction catalyzed by the nitrogenase enzyme of diazotrophic bacteria and archaea. Because plants cannot fix their own nitrogen, introducing functional nitrogenase in cereals and other crop plants would reduce our strong dependency on N fertilizers. NifB is required for the biosynthesis of the active site cofactors of all nitrogenases, which arguably makes it the most important protein in global nitrogen fixation. NifB functionality is therefore a requisite to engineer a plant nitrogenase. The expression of nifB genes from a wide range of prokaryotes into the model diazotroph Azotobacter vinelandii shows a surprising level of genetic complementation suggestive of plasticity in the nitrogenase biosynthetic pathway. In addition, we obtained NifB proteins from both mitochondria and chloroplasts of tobacco that are functional in vitro after reconstitution by providing [Fe4S4] clusters from NifU, paving the way to nitrogenase cofactor biosynthesis in plants.

Hydrogen overproducing nitrogenases obtained by random mutagenesis and high-throughput screening.

When produced biologically, especially by photosynthetic organisms, hydrogen gas (H2) is arguably the cleanest fuel available. An important limitation to the discovery or synthesis of better H2-producing enzymes is the absence of methods for the high-throughput screening of H2 production in biological systems. Here, we re-engineered the natural H2 sensing system of Rhodobacter capsulatus to direct the emission of LacZ-dependent fluorescence in response to nitrogenase-produced H2. A lacZ gene was placed under the control of the hupA H2-inducible promoter in a strain lacking the uptake hydrogenase and the nifH nitrogenase gene. This system was then used in combination with fluorescence-activated cell sorting flow cytometry to screen large libraries of nitrogenase Fe protein variants generated by random mutagenesis. Exact correlation between fluorescence emission and H2 production levels was found for all automatically selected strains. One of the selected H2-overproducing Fe protein variants lacked 40% of the wild-type amino acid sequence, a surprising finding for a protein that is highly conserved in nature. We propose that this method has great potential to improve microbial H2 production by allowing powerful approaches such as the directed evolution of nitrogenases and hydrogenases.

Pseudomonas fluorescens F113 Can Produce a Second Flagellar Apparatus, Which Is Important for Plant Root Colonization.

The genomic sequence of Pseudomonas fluorescens F113 has shown the presence of a 41 kb cluster of genes that encode the production of a second flagellar apparatus. Among 2,535 pseudomonads strains with sequenced genomes, these genes are only present in the genomes of F113 and other six strains, all but one belonging to the P. fluorescens cluster of species, in the form of a genetic island. The genes are homologous to the flagellar genes of the soil bacterium Azotobacter vinelandii. Regulation of these genes is mediated by the flhDC master operon, instead of the typical regulation in pseudomonads, which is through fleQ. Under laboratory conditions, F113 does not produce this flagellum and the flhDC operon is not expressed. However, ectopic expression of the flhDC operon is enough for its production, resulting in a hypermotile strain. This flagellum is also produced under laboratory conditions by the kinB and algU mutants. Genetic analysis has shown that kinB strongly represses the expression of the flhDC operon. This operon is activated by the Vfr protein probably in a c-AMP dependent way. The strains producing this second flagellum are all hypermotile and present a tuft of polar flagella instead of the single polar flagellum produced by the wild-type strain. Phenotypic variants isolated from the rhizosphere produce this flagellum and mutation of the genes encoding it, results in a defect in competitive colonization, showing its importance for root colonization.

Identification of flgZ as a flagellar gene encoding a PilZ domain protein that regulates swimming motility and biofilm formation in Pseudomonas.

Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC) and one phosphodiesterase (BifA) implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ.

Genome sequence reveals that Pseudomonas fluorescens F113 possesses a large and diverse array of systems for rhizosphere function and host interaction.

BACKGROUND: Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) isolated from the sugar-beet rhizosphere. This bacterium has been extensively studied as a model strain for genetic regulation of secondary metabolite production in P. fluorescens, as a candidate biocontrol agent against phytopathogens, and as a heterologous host for expression of genes with biotechnological application. The F113 genome sequence and annotation has been recently reported. RESULTS: Comparative analysis of 50 genome sequences of strains belonging to the P. fluorescens group has revealed the existence of five distinct subgroups. F113 belongs to subgroup I, which is mostly composed of strains classified as P. brassicacearum. The core genome of these five strains is highly conserved and represents approximately 76% of the protein-coding genes in any given genome. Despite this strong conservation, F113 also contains a large number of unique protein-coding genes that encode traits potentially involved in the rhizocompetence of this strain. These features include protein coding genes required for denitrification, diterpenoids catabolism, motility and chemotaxis, protein secretion and production of antimicrobial compounds and insect toxins. CONCLUSIONS: The genome of P. fluorescens F113 is composed of numerous protein-coding genes, not usually found together in previously sequenced genomes, which are potentially decisive during the colonisation of the rhizosphere and/or interaction with other soil organisms. This includes genes encoding proteins involved in the production of a second flagellar apparatus, the use of abietic acid as a growth substrate, the complete denitrification pathway, the possible production of a macrolide antibiotic and the assembly of multiple protein secretion systems.

Genome sequence of the biocontrol strain Pseudomonas fluorescens F113.

Pseudomonas fluorescens F113 is a plant growth-promoting rhizobacterium (PGPR) that has biocontrol activity against fungal plant pathogens and is a model for rhizosphere colonization. Here, we present its complete genome sequence, which shows that besides a core genome very similar to those of other strains sequenced within this species, F113 possesses a wide array of genes encoding specialized functions for thriving in the rhizosphere and interacting with eukaryotic organisms.

The Gac-Rsm and SadB signal transduction pathways converge on AlgU to downregulate motility in Pseudomonas fluorescens.

Flagella mediated motility in Pseudomonas fluorescens F113 is tightly regulated. We have previously shown that motility is repressed by the GacA/GacS system and by SadB through downregulation of the fleQ gene, encoding the master regulator of the synthesis of flagellar components, including the flagellin FliC. Here we show that both regulatory pathways converge in the regulation of transcription and possibly translation of the algU gene, which encodes a sigma factor. AlgU is required for multiple functions, including the expression of the amrZ gene which encodes a transcriptional repressor of fleQ. Gac regulation of algU occurs during exponential growth and is exerted through the RNA binding proteins RsmA and RsmE but not RsmI. RNA immunoprecipitation assays have shown that the RsmA protein binds to a polycistronic mRNA encoding algU, mucA, mucB and mucD, resulting in lower levels of algU. We propose a model for repression of the synthesis of the flagellar apparatus linking extracellular and intracellular signalling with the levels of AlgU and a new physiological role for the Gac system in the downregulation of flagella biosynthesis during exponential growth.

Pseudomonas fluorescens F113 mutant with enhanced competitive colonization ability and improved biocontrol activity against fungal root pathogens.

Motility is one of the most important traits for efficient rhizosphere colonization by Pseudomonas fluorescens F113rif (F113). In this bacterium, motility is a polygenic trait that is repressed by at least three independent pathways, including the Gac posttranscriptional system, the Wsp chemotaxis-like pathway, and the SadB pathway. Here we show that the kinB gene, which encodes a signal transduction protein that together with AlgB has been implicated in alginate production, participates in swimming motility repression through the Gac pathway, acting downstream of the GacAS two-component system. Gac mutants are impaired in secondary metabolite production and are unsuitable as biocontrol agents. However, the kinB mutant and a triple mutant affected in kinB, sadB, and wspR (KSW) possess a wild-type phenotype for secondary metabolism. The KSW strain is hypermotile and more competitive for rhizosphere colonization than the wild-type strain. We have compared the biocontrol activity of KSW with those of the wild-type strain and a phenotypic variant (F113v35 [V35]) which is hypermotile and hypercompetitive but is affected in secondary metabolism since it harbors a gacS mutation. Biocontrol experiments in the Fusarium oxysporum f. sp. radicis-lycopersici/Lycopersicum esculentum (tomato) and Phytophthora cactorum/Fragaria vesca (strawberry) pathosystems have shown that the three strains possess biocontrol activity. Biocontrol activity was consistently lower for V35, indicating that the production of secondary metabolites was the most important trait for biocontrol. Strain KSW showed improved biocontrol compared with the wild-type strain, indicating that an increase in competitive colonization ability resulted in improved biocontrol and that the rational design of biocontrol agents by mutation is feasible.

Efficient rhizosphere colonization by Pseudomonas fluorescens f113 mutants unable to form biofilms on abiotic surfaces.

Motility is a key trait for rhizosphere colonization by Pseudomonas fluorescens. Mutants with reduced motility are poor competitors, and hypermotile, more competitive phenotypic variants are selected in the rhizosphere. Flagellar motility is a feature associated to planktonic, free-living single cells, and although it is necessary for the initial steps of biofilm formation, bacteria in biofilm lack flagella. To test the correlation between biofilm formation and rhizosphere colonization, we have used P. fluorescens F113 hypermotile derivatives and mutants affected in regulatory genes which in other bacteria modulate biofilm development, namely gacS (G), sadB (S) and wspR (W). Mutants affected in these three genes and a hypermotile variant (V35) isolated from the rhizosphere were impaired in biofilm formation on abiotic surfaces, but colonized the alfalfa root apex as efficiently as the wild-type strain, indicating that biofilm formation on abiotic surfaces and rhizosphere colonization follow different regulatory pathways in P. fluorescens. Furthermore, a triple mutant gacSsadBwspR (GSW) and V35 were more competitive than the wild-type strain for root-tip colonization, suggesting that motility is more relevant in this environment than the ability to form biofilms on abiotic surfaces. Microscopy showed the same root colonization pattern for P. fluorescens F113 and all the derivatives: extensive microcolonies, apparently held to the rhizoplane by a mucigel that seems to be plant produced. Therefore, the ability to form biofilms on abiotic surfaces does not necessarily correlates with efficient rhizosphere colonization or competitive colonization.

Three independent signalling pathways repress motility in Pseudomonas fluorescens F113.

Motility is one of the most important traits for rhizosphere colonization by pseudomonads. Despite this importance, motility is severely repressed in the rhizosphere-colonizing strain Pseudomonas fluorescens F113. This bacterium is unable to swarm under laboratory conditions and produce relatively small swimming haloes. However, phenotypic variants with the ability to swarm and producing swimming haloes up to 300% larger than the wild-type strain, arise during rhizosphere colonization. These variants harbour mutations in the genes encoding the GacA/GacS two-component system and in other genes. In order to identify genes and pathways implicated in motility repression, we have used generalized mutagenesis with transposons. Analysis of the mutants has shown that besides the Gac system, the Wsp system and the sadB gene, which have been previously implicated in cyclic di-GMP turnover, are implicated in motility repression: mutants in the gacS, sadB or wspR genes can swarm and produce swimming haloes larger than the wild-type strain. Epistasis analysis has shown that the pathways defined by each of these genes are independent, because double and triple mutants show an additive phenotype. Furthermore, GacS, SadB and WspR act at different levels. Expression of the fleQ gene, encoding the master regulator of flagella synthesis is higher in the gacS(-) and sadB(-) backgrounds than in the wild-type strain and this differential expression is reflected by a higher secretion of the flagellin protein FliC. Conversely, no differences in fleQ expression or FliC secretion were observed between the wild-type strain and the wspR(-) mutant.

Transcriptional organization of the region encoding the synthesis of the flagellar filament in Pseudomonas fluorescens.

Pseudomonas fluorescens F113 is motile by means of type b flagella. Analysis of the region encoding the synthesis of the flagellar filament has shown a transcriptional organization different from that of type a flagella. Additionally to the promoters driving fliC, fliD, and fleQ expression, we have found promoters upstream of the flaG gene and the fliST operon. These promoters were functional in vivo. Both promoters have been mapped and appear to be dependent on the vegetative sigma factor and independent of FleQ, the master regulator of flagellum synthesis.