Search

Engineering signalling between plants and microbes could be exploited to establish host-specificity between plant-growth-promoting bacteria and target crops in the environment. We previously engineered rhizopine-signalling circuitry facilitating exclusive signalling between rhizopine-producing (RhiP) plants and model bacterial strains. Here, we conduct an in-depth analysis of rhizopine-inducible expression in bacteria. We characterize two rhizopine-inducible promoters and explore the bacterial host-range of rhizopine biosensor plasmids. By tuning the expression of rhizopine uptake genes, we also construct a new biosensor plasmid pSIR05 that has minimal impact on host cell growth in vitro and exhibits markedly improved stability of expression in situ on RhiP barley roots compared to the previously described biosensor plasmid pSIR02. We demonstrate that a sub-population of Azorhizobium caulinodans cells carrying pSIR05 can sense rhizopine and activate gene expression when colonizing RhiP barley roots. However, these bacteria were mildly defective for colonization of RhiP barley roots compared to the wild-type parent strain. This work provides advancement towards establishing more robust plant-dependent control of bacterial gene expression and highlights the key challenges remaining to achieve this goal.

Funder: University of Massachusetts Amherst; Id: http://dx.doi.org/10.13039/100008975, The plant common symbiosis signalling (SYM) pathway has shared function between interactions with rhizobia and arbuscular mycorrhizal fungi, the two most important symbiotic interactions between plants and microorganisms that are crucial in plant and agricultural yields. Here, we determine the role of the plant SYM pathway in the structure and abundance of the microbiota in the model legume Medicago truncatula and whether this is controlled by the nitrogen or phosphorus status of the plant. We show that SYM mutants (dmi3) differ substantially from the wild type (WT) in the absolute abundance of the root microbiota, especially under nitrogen limitation. Changes in the structure of the microbiota were less pronounced and depended on both plant genotype and nutrient status. Thus, the SYM pathway has a major impact on microbial abundance in M. truncatula and also subtly alters the composition of the microbiota.

Biological nitrogen fixation in rhizobium-legume symbioses is of major importance for sustainable agricultural practices. To establish a mutualistic relationship with their plant host, rhizobia transition from free-living bacteria in soil to growth down infection threads inside plant roots and finally differentiate into nitrogen-fixing bacteroids. We reconstructed a genome-scale metabolic model for Rhizobium leguminosarum and integrated the model with transcriptome, proteome, metabolome and gene essentiality data to investigate nutrient uptake and metabolic fluxes characteristic of these different lifestyles. Synthesis of leucine, polyphosphate and AICAR is predicted to be important in the rhizosphere, while myo-inositol catabolism is active in undifferentiated nodule bacteria in agreement with experimental evidence. The model indicates that bacteroids utilize xylose and glycolate in addition to dicarboxylates, which could explain previously described gene expression patterns. Histidine is predicted to be actively synthesized in bacteroids, consistent with transcriptome and proteome data for several rhizobial species. These results provide the basis for targeted experimental investigation of metabolic processes specific to the different stages of the rhizobium-legume symbioses.ImportanceRhizobia are soil bacteria that induce nodule formation on plant roots and differentiate into nitrogen-fixing bacteroids. A detailed understanding of this complex symbiosis is essential for advancing ongoing efforts to engineer novel symbioses with cereal crops for sustainable agriculture. Here, we reconstruct and validate a genome-scale metabolic model for Rhizobium leguminosarum bv. viciae 3841. By integrating the model with various experimental datasets specific to different stages of symbiosis formation, we elucidate the metabolic characteristics of rhizosphere bacteria, undifferentiated bacteria inside root nodules, and nitrogen-fixing bacteroids. Our model predicts metabolic flux patterns for these three distinct lifestyles, thus providing a framework for the interpretation of genome-scale experimental datasets and identifying targets for future experimental studies.

Assessment of plant-associative bacterial nitrogen (N) fixation is crucial for selection and development of elite diazotrophic inoculants that could be used to supply cereal crops with nitrogen in a sustainable manner. Although diazotrophic bacteria possess diverse oxygen tolerance mechanisms, most require a sub 21% oxygen environment to achieve optimal stability and function of the N-fixing catalyst nitrogenase. Consequently, assessment of N fixation is routinely carried out on “free-living” bacteria grown in the absence of a host plant and such experiments may not accurately divulge activity in the rhizosphere where the availability and forms of nutrients such as carbon and N, which are key regulators of N fixation, may vary widely. Here, we present a modified in situ acetylene reduction assay (ARA), utilizing the model cereal barley as a host to comparatively assess nitrogenase activity in diazotrophic bacteria. The assay is rapid, highly reproducible, applicable to a broad range of diazotrophs, and can be performed with simple equipment commonly found in most laboratories that investigate plant-microbe interactions. Thus, the assay could serve as a first point of order for high-throughput identification of elite plant-associative diazotrophs.

Assessment of plant-associative bacterial nitrogen (N) fixation is crucial for selection and development of elite diazotrophic inoculants that could be used to supply cereal crops with nitrogen in a sustainable manner. Although diazotrophic bacteria possess diverse oxygen tolerance mechanisms, most require a sub 21% oxygen environment to achieve optimal stability and function of the N-fixing catalyst nitrogenase. Consequently, assessment of N fixation is routinely carried out on “free-living” bacteria grown in the absence of a host plant and such experiments may not accurately divulge activity in the rhizosphere where the availability and forms of nutrients such as carbon and N, which are key regulators of N fixation, may vary widely. Here, we present a modified in situ acetylene reduction assay (ARA), utilizing the model cereal barley as a host to comparatively assess nitrogenase activity in diazotrophic bacteria. The assay is rapid, highly reproducible, applicable to a broad range of diazotrophs, and can be performed with simple equipment commonly found in most laboratories that investigate plant-microbe interactions. Thus, the assay could serve as a first point of order for high-throughput identification of elite plant-associative diazotrophs.

Summary Bacterial colonization of the rhizosphere is critical for the establishment of plant–bacteria interactions that represent a key determinant of plant health and productivity. Plants influence bacterial colonization primarily through modulating the composition of their root exudates and mounting an innate immune response. The outcome is a horizontal filtering of bacteria from the surrounding soil, resulting in a gradient of reduced bacterial diversity coupled with a higher degree of bacterial specialization towards the root. Bacteria–bacteria interactions (BBIs) are also prevalent in the rhizosphere, influencing bacterial persistence and root colonization through metabolic exchanges, secretion of antimicrobial compounds and other processes. Traditionally, bacterial colonization has been examined under sterile laboratory conditions that mitigate the influence of BBIs. Using simplified synthetic bacterial communities combined with microfluidic imaging platforms and transposon mutagenesis screening approaches, we are now able to begin unravelling the molecular mechanisms at play during the early stages of root colonization. This review explores the current state of knowledge regarding bacterial root colonization and identifies key tools for future exploration.

Exploitation of plant growth promoting (PGP) rhizobacteria (PGPR) as crop inoculants could propel sustainable intensification of agriculture to feed our rapidly growing population. However, field performance of PGPR is typically inconsistent due to suboptimal rhizosphere colonisation and persistence in foreign soils, promiscuous host-specificity, and in some cases, the existence of undesirable genetic regulation that has evolved to repress PGP traits. While the genetics underlying these problems remain largely unresolved, molecular mechanisms of PGP have been elucidated in rigorous detail. Engineering and subsequent transfer of PGP traits into selected efficacious rhizobacterial isolates or entire bacterial rhizosphere communities now offers a powerful strategy to generate improved PGPR that are tailored for agricultural use. Through harnessing of synthetic plant-to-bacteria signalling, attempts are currently underway to establish exclusive coupling of plant-bacteria interactions in the field, which will be crucial to optimise efficacy and establish biocontainment of engineered PGPR. This review explores the many ecological and biotechnical facets of this research.

The influence of wheat (modern wheat, both bread and pasta, their wild ancestors and synthetic hybrids) on the microbiota of their roots and surrounding soil is characterized. We isolated lines of bread wheat by hybridizing diploid (Aegilops tauschii) with tetraploid Triticum durum and crossed it with a modern cultivar of Triticum aestivum. The newly created, synthetic hybrid wheat, which recapitulate the breeding history of wheat through artificial selection, is found to support a microbiome enriched in beneficial Glomeromycetes fungi, but also in, potentially detrimental, Nematoda. We hypothesize that during wheat domestication this plant-microbe interaction diminished, suggesting an evolutionary tradeoff; sacrificing advantageous nutrient acquisition through fungal interactions to minimize interaction with pathogenic fungi. Increased plant selection for Glomeromycetes and Nematoda is correlated with the D genome derived from A. tauschii. Despite differences in their soil microbiota communities, overall wheat plants consistently show a low ratio of eukaryotes to prokaryotes. We propose that this is a mechanism for protection against soil-borne fungal disease and appears to be deeply rooted in the wheat genome. We suggest that the influence of plants on the composition of their associated microbiota is an integral factor, hitherto overlooked, but intrinsic to selection during wheat domestication.

Global microbial kingdom analysis conducted simultaneously on multiple plants shows that cereals, legumes, and Brassicaceae establish similar prokaryotic and similar eukaryotic communities inside and on the root surface. While the bacterial microbiota is recruited from the surrounding soil, its profile is influenced by the root fraction more so than by soil or plant species. However, in contrast, the fungal microbiota is most strongly influenced by soil. This was observed in two different soils and for all plant species examined, indicating conserved adaptation of microbial communities to plants. Microbiota structure is established within 2 weeks of plant growth in soil and remains stable thereafter. We observed a remarkable similarity in the structure of a plant’s phyllosphere and root microbiotas and show by reciprocal soil swap experiments that both fractions are colonized from the soil in which the plant is grown. Thus, the phyllosphere is continuously colonized by the soil microbiota., Plant roots influence the soil microbiota via physical interaction, secretion, and plant immunity. However, it is unclear whether the root fraction or soil is more important in determining the structure of the prokaryotic or eukaryotic community and whether this varies between plant species. Furthermore, the leaf (phyllosphere) and root microbiotas have a large overlap; however, it is unclear whether this results from colonization of the phyllosphere by the root microbiota. Soil, rhizosphere, rhizoplane, and root endosphere prokaryote-, eukaryote-, and fungus-specific microbiotas of four plant species were analyzed with high-throughput sequencing. The strengths of factors controlling microbiota structure were determined using permutational multivariate analysis of variance (PERMANOVA) statistics. The origin of the phyllosphere microbiota was investigated using a soil swap experiment. Global microbial kingdom analysis conducted simultaneously on multiple plants shows that cereals, legumes, and Brassicaceae establish similar prokaryotic and similar eukaryotic communities inside and on the root surface. While the bacterial microbiota is recruited from the surrounding soil, its profile is influenced by the root itself more so than by soil or plant species. However, in contrast, the fungal microbiota is most strongly influenced by soil. This was observed in two different soils and for all plant species examined. Microbiota structure is established within 2 weeks of plant growth in soil and remains stable thereafter. A reciprocal soil swap experiment shows that the phyllosphere is colonized from the soil in which the plant is grown.

The casing material required in mushroom cultivation presents a very rich ecological niche, which is inhabited by a diverse population of bacteria and fungi. In this work three different casing materials, blonde peat, black peat and a 50 : 50 mixture of both, were compared for their capacity to show a natural suppressive response against dry bubble, Lecanicillium fungicola (Preuss) Zare and Gams, and wet bubble, Mycogone perniciosa (Magnus) Delacroix. The highest mushroom production was collected from crops cultivated using the mixed casing and black peat, which were not significantly different in yield. However, artificial infection with mycoparasites resulted in similar yield losses irrespective of the material used, indicating that the casing materials do not confer advantages in disease suppression. The composition of the microbiome of the 50 : 50 casing mixture along the crop cycle and the compost and basidiomes was evaluated through next-generation sequencing (NGS) of the V3–V4 region of the bacterial 16S rRNA gene and the fungal ITS2 region. Once colonized by Agaricus bisporus, the bacterial diversity of the casing microbiome increased and the fungal diversity drastically decreased. From then on, the composition of the casing microbiome remained relatively stable. Analysis of the composition of the bacterial microbiome in basidiomes indicated that it is highly influenced by the casing microbiota. Notably, L. fungicola was consistently detected in uninoculated control samples of compost and casing using NGS, even in asymptomatic crops. This suggests that the naturally established casing microbiota was able to help to suppress disease development when inoculum levels were low, but was not effective in suppressing high pressure from artificially introduced fungal inoculum. Determination of the composition of the casing microbiome paves the way for the development of synthetic casing communities that can be used to investigate the role of specific components of the casing microbiota in mushroom production and disease control.

Legumes obtain nitrogen from air through rhizobia residing in root nodules. Some species of rhizobia can colonize cereals but do not fix nitrogen on them. Disabling native regulation can turn on nitrogenase expression, even in the presence of nitrogenous fertilizer and low oxygen, but continuous nitrogenase production confers an energy burden. Here, we engineer inducible nitrogenase activity in two cereal endophytes (Azorhizobium caulinodans ORS571 and Rhizobium sp. IRBG74) and the well-characterized plant epiphyte Pseudomonas protegens Pf-5, a maize seed inoculant. For each organism, different strategies were taken to eliminate ammonium repression and place nitrogenase expression under the control of agriculturally relevant signals, including root exudates, biocontrol agents and phytohormones. We demonstrate that R. sp. IRBG74 can be engineered to result in nitrogenase activity under free-living conditions by transferring a nif cluster from either Rhodobacter sphaeroides or Klebsiella oxytoca. For P. protegens Pf-5, the transfer of an inducible cluster from Pseudomonas stutzeri and Azotobacter vinelandii yields ammonium tolerance and higher oxygen tolerance of nitrogenase activity than that from K. oxytoca. Collectively, the data from the transfer of 12 nif gene clusters between 15 diverse species (including Escherichia coli and 12 rhizobia) help identify the barriers that must be overcome to engineer a bacterium to deliver a high nitrogen flux to a cereal crop.

We present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I, and have demonstrated its use for Golden Gate cloning in Escherichia coli. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. Using the principles outlined here, new modules (e.g., origin of replication for plasmids in other bacteria) can easily be designed for specific applications. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach. To demonstrate the potential of the system, three example vectors were constructed and tested. The Golden Gate level 1 vector pOGG024 (pBBR1-based broad-host range and medium copy number) was used for gene expression in laboratory-cultured Rhizobium leguminosarum. The Golden Gate level 1 vector pOGG026 (RK2-based broad-host range, lower copy number and stable in the absence of antibiotic selection) was used to demonstrate bacterial gene expression in nitrogen-fixing nodules on pea plant roots formed by R. leguminosarum. Finally, the level 2 cloning vector pOGG216 (RK2-based broad-host range, medium copy number) was used to construct a dual reporter plasmid expressing green and red fluorescent proteins.

The attachment of bacteria to roots constitutes the first physical step in many plant–microbe interactions. These interactions exert both positive and negative influences on agricultural systems depending on whether a growth-promoting, symbiotic or pathogenic relationship transpires. A common biphasic mechanism of root attachment exists across agriculturally important microbial species, including Rhizobium, Agrobacterium, Pseudomonas, Azospirillum and Salmonella. Attachment studies have revealed how plant–microbe interactions develop, and how to manipulate these relationships for agricultural benefit. Here, we review our current understanding of the molecular mechanisms governing plant–microbe root attachment and draw together a common biphasic model.

The holobiont is composed by the plant and its microbiome. In a similar way to ecological systems of higher organisms, the holobiont shows interdependent and complex dynamics [1,2]. While plants originate from seeds, the microbiome has a multitude of sources. The assemblage of these communities depends on the interaction between the emerging seedling and its surrounding environment, with soil being the main source. These microbial communities are controlled by the plant through different strategies, such as the specific profile of root exudates and its immune system. Despite this control, the microbiome is still able to adapt and thrive. The molecular knowledge behind these interactions and microbial '-omic' technologies are developing to the point of enabling holobiont engineering. For a long time microorganisms were in the background of plant biology but new multidisciplinary approaches have led to an appreciation of the importance of the holobiont, where plants and microbes are interdependent.

Plants engineer the rhizosphere to their advantage by secreting various nutrients and secondary metabolites. Coupling transcriptomic and metabolomic analyses of the pea (Pisum sativum) rhizosphere, a suite of bioreporters has been developed in Rhizobium leguminosarum bv viciae strain 3841, and these detect metabolites secreted by roots in space and time. Fourteen bacterial lux fusion bioreporters, specific for sugars, polyols, amino acids, organic acids, or flavonoids, have been validated in vitro and in vivo. Using different bacterial mutants (nodC and nifH), the process of colonization and symbiosis has been analyzed, revealing compounds important in the different steps of the rhizobium-legume association. Dicarboxylates and sucrose are the main carbon sources within the nodules; in ineffective (nifH) nodules, particularly low levels of sucrose were observed, suggesting that plant sanctions affect carbon supply to nodules. In contrast, high myo-inositol levels were observed prior to nodule formation and also in nifH senescent nodules. Amino acid biosensors showed different patterns: a γ-aminobutyrate biosensor was active only inside nodules, whereas the phenylalanine bioreporter showed a high signal also in the rhizosphere. The bioreporters were further validated in vetch (Vicia hirsuta), producing similar results. In addition, vetch exhibited a local increase of nod gene-inducing flavonoids at sites where nodules developed subsequently. These bioreporters will be particularly helpful in understanding the dynamics of root exudation and the role of different molecules secreted into the rhizosphere.

The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus with the PA M285K mutation completely phenocopied the adapted virus. Adaptation of an avian virus-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the gene for viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel mutation and a well-known adaptive mutation in the viral polymerase to support replication. IMPORTANCE Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigated the replication of human influenza A virus in bat cell lines and the barriers that the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and evolutionarily diverse New and Old World bats. Viruses mutated during infections in bat cells, resulting in increased replication and cytopathic effects. These mutations were mapped to the viral polymerase and shown to be solely responsible for adaptation to bat cells. Our data suggest that replication of human influenza A viruses in a nonnative host drives the evolution of new variants and may be an important source of genetic diversity.

In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots.

Plant–microbe interactions in the rhizosphere have important roles in biogeochemical cycling, and maintenance of plant health and productivity, yet remain poorly understood. Using RNA-based metatranscriptomics, the global active microbiomes were analysed in soil and rhizospheres of wheat, oat, pea and an oat mutant (sad1) deficient in production of anti-fungal avenacins. Rhizosphere microbiomes differed from bulk soil and between plant species. Pea (a legume) had a much stronger effect on the rhizosphere than wheat and oat (cereals), resulting in a dramatically different rhizosphere community. The relative abundance of eukaryotes in the oat and pea rhizospheres was more than fivefold higher than in the wheat rhizosphere or bulk soil. Nematodes and bacterivorous protozoa were enriched in all rhizospheres, whereas the pea rhizosphere was highly enriched for fungi. Metabolic capabilities for rhizosphere colonisation were selected, including cellulose degradation (cereals), H2 oxidation (pea) and methylotrophy (all plants). Avenacins had little effect on the prokaryotic community of oat, but the eukaryotic community was strongly altered in the sad1 mutant, suggesting that avenacins have a broader role than protecting from fungal pathogens. Profiling microbial communities with metatranscriptomics allows comparison of relative abundance, from multiple samples, across all domains of life, without polymerase chain reaction bias. This revealed profound differences in the rhizosphere microbiome, particularly at the kingdom level between plants.

Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the tricarboxylic acid (TCA) cycle to generate NAD(P)H for reduction of N 2 . Metabolic flux analysis of laboratory-grown Rhizobium leguminosarum showed that the flux from [ 13 C]succinate was consistent with respiration of an obligate aerobe growing on a TCA cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady-state labeling under N 2 -fixing conditions. Therefore, comparative metabolomic profiling was used to compare free-living R. leguminosarum with pea bacteroids. While the TCA cycle was shown to be essential for maximal rates of N 2 fixation, levels of pyruvate (5.5-fold reduced), acetyl coenzyme A (acetyl-CoA; 50-fold reduced), free coenzyme A (33-fold reduced), and citrate (4.5-fold reduced) were much lower in bacteroids. Instead of completely oxidizing acetyl-CoA, pea bacteroids channel it into both lipid and the lipid-like polymer poly-β-hydroxybutyrate (PHB), the latter via a type III PHB synthase that is active only in bacteroids. Lipogenesis may be a fundamental requirement of the redox poise of electron donation to N 2 in all legume nodules. Direct reduction by NAD(P)H of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance the production of NAD(P)H from oxidation of acetyl-CoA in the TCA cycle with its storage in PHB and lipids. IMPORTANCE Biological nitrogen fixation by symbiotic bacteria (rhizobia) in legume root nodules is an energy-expensive process. Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the TCA cycle to generate NAD(P)H for reduction of N 2 . However, direct reduction of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance oxidation of plant-derived dicarboxylates in the TCA cycle with lipid synthesis. Pea bacteroids channel acetyl-CoA into both lipid and the lipid-like polymer poly-β-hydroxybutyrate, the latter via a type II PHB synthase. Lipogenesis is likely to be a fundamental requirement of the redox poise of electron donation to N 2 in all legume nodules.

Background Förster resonance energy transfer (FRET) biosensors are powerful tools to detect biologically important ligands in real time. Currently FRET bisosensors are available for twenty-two compounds distributed in eight classes of chemicals (two pentoses, two hexoses, two disaccharides, four amino acids, one nucleobase, two nucleotides, six ions and three phytoestrogens). To expand the number of available FRET biosensors we used the induction profile of the Sinorhizobium meliloti transportome to systematically screen for new FRET biosensors. Methodology/Principal Findings Two new vectors were developed for cloning genes for solute-binding proteins (SBPs) between those encoding FRET partner fluorescent proteins. In addition to a vector with the widely used cyan and yellow fluorescent protein FRET partners, we developed a vector using orange (mOrange2) and red fluorescent protein (mKate2) FRET partners. From the sixty-nine SBPs tested, seven gave a detectable FRET signal change on binding substrate, resulting in biosensors for D-quinic acid, myo-inositol, L-rhamnose, L-fucose, β-diglucosides (cellobiose and gentiobiose), D-galactose and C4-dicarboxylates (malate, succinate, oxaloacetate and fumarate). To our knowledge, we describe the first two FRET biosensor constructs based on SBPs from Tripartite ATP-independent periplasmic (TRAP) transport systems. Conclusions/Significance FRET based on orange (mOrange2) and red fluorescent protein (mKate2) partners allows the use of longer wavelength light, enabling deeper penetration of samples at lower energy and increased resolution with reduced back-ground auto-fluorescence. The FRET biosensors described in this paper for four new classes of compounds; (i) cyclic polyols, (ii) L-deoxy sugars, (iii) β-linked disaccharides and (iv) C4-dicarboxylates could be developed to study metabolism in vivo.

Rhizobium leguminosarum bv. viciae 3841 contains six putative quaternary ammonium transporters (Qat), of the ABC family. Qat6 was strongly induced by hyperosmosis although the solute transported was not identified. All six systems were induced by the quaternary amines choline and glycine betaine. It was confirmed by microarray analysis of the genome that pRL100079-83 (qat6) is the most strongly upregulated transport system under osmotic stress, although other transporters and 104 genes are more than threefold upregulated. A range of quaternary ammonium compounds were tested but all failed to improve growth of strain 3841 under hyperosmotic stress. One Qat system (gbcXWV) was induced 20-fold by glycine betaine and choline and a Tn5::gbcW mutant was severely impaired for both transport and growth on these compounds, demonstrating that it is the principal system for their use as carbon and nitrogen sources. It transports glycine betaine and choline with a high affinity (apparent K(m), 168 and 294 nM, respectively).

Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O 2 was used to understand how O 2 concentration alters metabolism. Two transcriptional regulators were required for growth on glucose (pRL120207 [ eryD ] and RL0547 [ phoB ]), five were required on succinate (pRL100388, RL1641, RL1642, RL3427, and RL4524 [ ecfL ]), and three were required on 1% O 2 (pRL110072, RL0545 [ phoU ], and RL4042). A novel toxin-antitoxin system was identified that could be important for generation of new plasmidless rhizobial strains. Rlv3841 appears to use the methylglyoxal pathway alongside the Entner-Doudoroff (ED) pathway and tricarboxylic acid (TCA) cycle for optimal growth on glucose. Surprisingly, the ED pathway was required for growth on succinate, suggesting that sugars made by gluconeogenesis must undergo recycling. Altered amino acid metabolism was specifically needed for growth on glucose, including RL2082 ( gatB ) and pRL120419 ( opaA , encoding omega-amino acid:pyruvate transaminase). Growth on succinate specifically required enzymes of nucleobase synthesis, including ribose-phosphate pyrophosphokinase (RL3468 [ prs ]) and a cytosine deaminase (pRL90208 [ codA ]). Succinate growth was particularly dependent on cell surface factors, including the PrsD-PrsE type I secretion system and UDP-galactose production. Only RL2393 ( glnB , encoding nitrogen regulatory protein PII) was specifically essential for growth on succinate at 1% O 2 , conditions similar to those experienced by N 2 -fixing bacteroids. Glutamate synthesis is constitutively activated in glnB mutants, suggesting that consumption of 2-ketoglutarate may increase flux through the TCA cycle, leading to excess reductant that cannot be reoxidized at 1% O 2 and cell death. IMPORTANCE Rhizobium leguminosarum , a soil bacterium that forms N 2 -fixing symbioses with several agriculturally important leguminous plants (including pea, vetch, and lentil), has been widely utilized as a model to study Rhizobium -legume symbioses. Insertion sequencing (INSeq) has been used to identify factors needed for its growth on different carbon sources and O 2 levels. Identification of these factors is fundamental to a better understanding of the cell physiology and core metabolism of this bacterium, which adapts to a variety of different carbon sources and O 2 tensions during growth in soil and N 2 fixation in symbiosis with legumes.

Ntr system in Rhizobium leguminosarum strain Rlv3841 caused a pleiotropic phenotype as observed with many bacte- ria. The mutant formed dry colonies and grew poorly on organic nitrogen or dicarboxylates. Most strikingly the ptsP mutant had low activity of a broad range of ATP-dependent ABC transporters. This lack of activa- tion, which occurred post-translationally, may explain many of the pleiotropic effects. In contrast proton- coupledtransportsystemswerenotinhibitedinaptsP mutant.RegulationbyPtsPalsoinvolvestwocopiesof ptsN that code for EIIA Ntr , resulting in a phosphoryla- tioncascade.AsinEscherichiacoli,theRlv3841PTS Ntr

Mutation of gltB (encoding glutamate oxoglutarate amidotransferase or GOGAT) in RU2307 increased the intracellular Gln:Glu ratio and inhibited amino acid transport via Aap and Bra. The mechanism probably involves global post-translational inhibition independent of Ntr. Transport was separately restored by increased gene expression of Aap or heterologous transporters. Likewise, second site suppressor mutations in the RNA chaperone Hfq elevated transport by Aap and Bra by increasing mRNA levels. Microarrays showed Hfq regulates 34 ABC transporter genes, including aap, bra and opp. The genes coding for integral membrane proteins and ABC subunits aapQMP braDEFGC were more strongly elevated in the hfq mutants than solute-binding proteins (aapJ braC). aapQMP and braDEFG are immediately downstream of stem-loops, indicating Hfq attenuates downstream translation and stability of mRNA, explaining differential expression of ABC genes. RU2307 nodulated peas and bacteria grew down infection threads, but bacteroid development was arrested and N(2) was not fixed. This probably results from an inability to synthesize or transport amino acids. However, GOGAT and GOGAT/AldA double mutants carrying suppressor mutations that increased amino acid uptake fixed N(2) on pea plants. Thus de novo ammonium assimilation into amino acids is unnecessary in bacteroids demonstrating sufficient amino acids are supplied by plants.

IVET was used to identify genes that are specifically expressed in the rhizosphere of the pea-nodulating bacterium Rhizobium leguminosarum A34. A library of R. leguminosarum A34 cloned in the integration vector pIE1, with inserts upstream of a promoter-less purN:gfp:gusA, was conjugated into purN host RU2249 and recombined into the genome. After removal of colonies that expressed the reporter genes of the vector under laboratory conditions, the library was inoculated into a nonsterile pea rhizosphere. The key result is that 29 rhizosphere-induced loci were identified. Sequence analysis of these clones showed that a wide variety of R. leguminosarum A34 genes are expressed specifically in the rhizosphere including those encoding proteins involved in environmental sensing, control of gene expression, metabolic reactions and membrane transport. These genes are likely to be important for survival and colonization of the pea rhizosphere.

Mutations in a Rhizobium leguminosarum gene, rirA (rhizobial iron regulator), caused high-level, constitutive expression of at least eight operons whose transcription is normally Fe-responsive and whose products are involved in the synthesis or uptake of siderophores, or in the uptake of haem or of other iron sources. Close homologues of RirA exist in other rhizobia and in the pathogen Brucella; many other bacteria have deduced proteins with more limited sequence similarity. None of these homologues had been implicated in Fe-mediated gene regulation. Transcription of rirA itself is about twofold higher in cells grown in Fe-replete than in Fe-deficient growth media. Mutations in rirA reduced growth rates in Fe-replete and -depleted medium, but did not appear to affect symbiotic N2 fixation.

A Rhizobium leguminosarum bv. viciae VF39 gene (gabT) encoding a γ-aminobutyrate (GABA) aminotransferase was identified, cloned and characterized. This gene is thought to be involved in GABA metabolism via the GABA shunt pathway, a theoretical bypass of the 2-oxoglutarate dehydrogenase complex. Mutants in gabT are still able to grow on GABA as a sole carbon and nitrogen source. 2-Oxoglutarate-dependent GABA aminotransferase activity is absent in these mutants, while pyruvate-dependent activity remains unaffected. This indicates that at least two enzymes with different substrate specifities are involved in the GABA metabolism of R. leguminosarum bv. viciae VF39. The gabT promoter was cloned into a newly constructed, stable promoter-probe vector pJP2, suitable for the study of transcriptional GUS fusions in free-living bacteria and during symbiosis. Under free-living conditions the gabT promoter is induced by GABA and repressed by succinate. Transcriptional regulation is mediated by GabR in a repressor-like manner. During symbiosis with the pea host plant gabT is induced and highly expressed in the symbiotic zone. Nodules induced by gabT mutants, however, are still effective in nitrogen fixation.

We examined succession of the rhizosphere microbiota of three model plants (Arabidopsis, Medicago and Brachypodium) in compost and sand and three crops (Brassica, Pisum and Triticum) in compost alone. We used serial inoculation of 24 independent replicate microcosms over three plant generations for each plant/soil combination. Stochastic variation between replicates was surprisingly weak and by the third generation, replicate microcosms for each plant had communities that were very similar to each other but different to those of other plants or unplanted soil. Microbiota diversity remained high in compost, but declined drastically in sand, with bacterial opportunists and putative autotrophs becoming dominant. These dramatic differences indicate that many microbes cannot thrive on plant exudates alone and presumably also require carbon sources and/or nutrients from soil. Arabidopsis had the weakest influence on its microbiota and in compost replicate microcosms converged on three alternative community compositions rather than a single distinctive community. Organisms selected in rhizospheres can have positive or negative effects. Two abundant bacteria are shown to promote plant growth, but in Brassica the pathogen Olpidium brassicae came to dominate the fungal community. So plants exert strong selection on the rhizosphere microbiota but soil composition is critical to its stability. microbial succession/ plant-microbe interactions/rhizosphere microbiota/selection.

Successful replication of influenza virus requires the coordinated expression of viral genes and replication of the genome by the viral polymerase, composed of the subunits PA, PB1, and PB2. Polymerase activity is regulated by both viral and host factors, yet the mechanisms of regulation and how they contribute to viral pathogenicity and tropism are poorly understood. To characterize these processes, we created a series of mutants in the 627 domain of the PB2 subunit. This domain contains a conserved “P[F/P]AAAPP” sequence motif and the well-described amino acid 627, whose identity regulates host range. A lysine present at position 627 in most mammalian viral isolates creates a basic face on the domain surface and confers high-level activity in humans compared to the glutamic acid found at this position in avian isolates. Mutation of the basic face or the P[F/P]AAAPP motif impaired polymerase activity, assembly of replication complexes, and viral replication. Most of these residues are required for general polymerase activity, whereas PB2 K586 and R589 were preferentially required for function in human versus avian cells. Thus, these data identify residues in the 627 domain and other viral proteins that regulate polymerase activity, highlighting the importance of the surface charge and structure of this domain for virus replication and host adaptation. IMPORTANCE Influenza virus faces barriers to transmission across species as it emerges from its natural reservoir in birds to infect mammals. The viral polymerase is an important regulator of this process and undergoes discrete changes to adapt to replication in mammals. Many of these changes occur in the polymerase subunit PB2. Here we describe the systematic analysis of a key region in PB2 that controls species-specific polymerase activity. We report the importance of conserved residues that contribute to the overall charge of the protein as well as those that likely affect protein structure. These findings provide further insight into the molecular events dictating species-specific polymerase function and viral replication.

A cysE gene encoding a serine acetyltransferase (SAT) potentially involved in the biosynthesis of cysteine was identified C 4 kb upstream of the previously described aapJQMP gene cluster that encodes an amino acid permease in Rhizobium leguminosarum strain 3841. The gene exhibitsS 40% identity to the family of SATs containing N-terminal extensions that have been described for other bacteria and plants. The ORF has three possible translation initiation sites which potentially encode polypeptides of 311, 277 and/or 259 amino acid residues, respectively. All three ORFs complemented the cysE mutation in an Escherichia coli cysteine auxotroph, strain JM39. Insertion of Tn5-lacZ into cysE in the genome of R. leguminosarum (strain RU632) lowered SAT activity in crude extracts byS 95%. However, RU632 was not a cysteine auxotroph, which suggests that R. leguminosarum possesses some redundancy in cysteine biosynthesis. Additional copies of cysE could not be detected in the genome when the R. leguminosarum cysE gene was used as a hybridization probe. Therefore it is possible that R. leguminosarum possesses an alternative pathway for cysteine biosynthesis which avoids O-acetylserine. Strain RU632 was unaffected in its ability to nodulate Pisum sativum, and the nodules were effective for N2 fixation (measured by C2H2 reduction). Transcriptional activity of cysE was determined by measuring the b-galactosidase arising from cysE::Tn5-lacZ fusions. Maximal levels of expression were observed during early exponential growth and were not influenced by the level of sulphur (supplied as sulphate). However, transcription was repressed by approximately twofold in ammonium-grown, as opposed to glutamate-grown, cultures. Repression by ammonium was not seen in a strain defective for ntrC.

The techniques of differential fluorescence induction (DFI) and optical trapping (OT) have been combined to allow the identification of environmentally induced genes in single bacterial cells. Designated DFI-OT, this technique allows the in situ isolation of genes driving the expression of green fluorescent protein (Gfp) using temporal and spatial criteria. A series of plasmid-based promoter probe vectors (pOT) was developed for the construction of random genomic libraries that are linked to gfpUV or egfp. Bacteria that do not express Gfp on laboratory medium (i.e. non-fluorescent) were inoculated into the environment, and induced genes were detected with a combined fluorescence/optical trapping microscope. Using this selection strategy, rhizosphere-induced genes with homology to thiamine pyrophosphorylase (thiE) and cyclic glucan synthase (ndvB) were isolated. Other genes were expressed late in the stationary phase or as a consequence of surface-dependent growth, including fixND and metX, and a putative ABC transporter of putrescine. This strategy provides a unique ability to combine spatial, temporal and physical information to identify environmental regulation of bacterial gene expression.

There are two subfamilies of ABC uptake systems for amino acids in bacteria, the polar amino acid transport family and the hydrophobic amino acid transport family. We consider the general properties of these families and we examine the specific transporters. Focusing on some of the best-studied ATP binding cassette transporters we also examine the mechanism of amino acid uptake, paying particular attention to the question of bidirectionality of solute movement.

The ability of sera to recognise secreted macromolecules of Staphylococcus aureus was examined by ELISA and Western immunoblotting. Individual secreted proteins were also studied using both human sera and sera from rabbits immunised with secreted macromolecules. Patients sera showed a wide range of IgG antibody titres to secreted macromolecules and whole bacteria. Controls showed a significantly lower IgG response. Western immunoblotting revealed that a significant number of secreted proteins were recognised by circulating IgG antibodies. Surprisingly, both the sera from controls and from patients recognised similar macromolecules including a number of potential virulence factors. The major difference was in the IgG binding to a 16-kDa component, which was recognised by the majority of the sera from infected individuals, but only by a small number of sera from healthy controls. The higher incidence of antibodies recognising the 16 kDa component may be related to our earlier finding that the major bone resorbing component of S. aureus is a heterodimeric protein containing a 16-kDa subunit, the activity of which could be blocked by sera.

A gene, cpaA, with similarity to calcium proton antiporters has been identified adjacent to lpcAB in Rhizobium leguminosarum. LpcA is a galactosyl transferase while LpcB is a 2-keto-3-deoxyoctonate transferase, both of which are required to form the lipopolysaccharide (LPS) core in R. leguminosarum. Mutations in lpcAB result in a rough LPS phenotype with a requirement for elevated calcium concentrations to allow growth, suggesting that truncation of the LPS core exposes a highly negatively charged molecule. This is consistent with the LPS core being one of the main sites for binding calcium in the Gram-negative outer membrane. Strain RU1109 (cpaA::Tn5-lacZ) has a normal LPS layer, as measured by silver staining and Western blotting. This indicates that cpaA mutants are not grossly affected in their LPS layer. LacZ fusion analysis indicates that cpaA is constitutively expressed and is not directly regulated by the calcium concentration. Over-expression of cpaA increased the concentration of calcium required for growth, consistent with CpaA mediating calcium export from the cytosol. The location of lpcA, lpcB and cpaA as well as the phenotype of lpcB mutants suggests that CpaA might provide a specific export pathway for calcium to the LPS core.