Rhizobium determinants of rhizosphere persistence and root colonisation.

Bacterial persistence in the rhizosphere and colonisation of root niches are critical for the establishment of many beneficial plant-bacteria interactions including those between Rhizobium leguminosarum and its host legumes. Despite this, most studies on R. leguminosarum have focused on its symbiotic lifestyle as an endosymbiont in root nodules. Here, we use random barcode transposon sequencing (RB-TnSeq) to assay gene contributions of R. leguminosarum during competitive growth in the rhizosphere and colonisation of various plant species. This facilitated the identification of 189 genes commonly required for growth in diverse plant rhizospheres, mutation of 111 of which also affected subsequent root colonisation (rhizosphere progressive), and a further 119 genes necessary for colonisation. Common determinants reveal a need to synthesise essential compounds (amino acids, ribonucleotides, and cofactors), adapt metabolic function, respond to external stimuli, and withstand various stresses (such as changes in osmolarity). Additionally, chemotaxis and flagella-mediated motility are prerequisites for root colonisation. Many genes showed plant-specific dependencies highlighting significant adaptation to different plant species. This work provides a greater understanding of factors promoting rhizosphere fitness and root colonisation in plant-beneficial bacteria, facilitating their exploitation for agricultural benefit.

The genotype of barley cultivars influences multiple aspects of their associated microbiota via differential root exudate secretion.

Plant-associated microbes play vital roles in promoting plant growth and health, with plants secreting root exudates into the rhizosphere to attract beneficial microbes. Exudate composition defines the nature of microbial recruitment, with different plant species attracting distinct microbiota to enable optimal adaptation to the soil environment. To more closely examine the relationship between plant genotype and microbial recruitment, we analysed the rhizosphere microbiomes of landrace (Chevallier) and modern (NFC Tipple) barley (Hordeum vulgare) cultivars. Distinct differences were observed between the plant-associated microbiomes of the 2 cultivars, with the plant-growth promoting rhizobacterial genus Pseudomonas substantially more abundant in the Tipple rhizosphere. Striking differences were also observed between the phenotypes of recruited Pseudomonas populations, alongside distinct genotypic clustering by cultivar. Cultivar-driven Pseudomonas selection was driven by root exudate composition, with the greater abundance of hexose sugars secreted from Tipple roots attracting microbes better adapted to growth on these metabolites and vice versa. Cultivar-driven selection also operates at the molecular level, with both gene expression and the abundance of ecologically relevant loci differing between Tipple and Chevallier Pseudomonas isolates. Finally, cultivar-driven selection is important for plant health, with both cultivars showing a distinct preference for microbes selected by their genetic siblings in rhizosphere transplantation assays.

Evaluation of variant calling algorithms for wastewater-based epidemiology using mixed populations of SARS-CoV-2 variants in synthetic and wastewater samples.

Wastewater-based epidemiology has been used extensively throughout the COVID-19 (coronavirus disease 19) pandemic to detect and monitor the spread and prevalence of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and its variants. It has proven an excellent, complementary tool to clinical sequencing, supporting the insights gained and helping to make informed public-health decisions. Consequently, many groups globally have developed bioinformatics pipelines to analyse sequencing data from wastewater. Accurate calling of mutations is critical in this process and in the assignment of circulating variants; yet, to date, the performance of variant-calling algorithms in wastewater samples has not been investigated. To address this, we compared the performance of six variant callers (VarScan, iVar, GATK, FreeBayes, LoFreq and BCFtools), used widely in bioinformatics pipelines, on 19 synthetic samples with known ratios of three different SARS-CoV-2 variants of concern (VOCs) (Alpha, Beta and Delta), as well as 13 wastewater samples collected in London between the 15th and 18th December 2021. We used the fundamental parameters of recall (sensitivity) and precision (specificity) to confirm the presence of mutational profiles defining specific variants across the six variant callers. Our results show that BCFtools, FreeBayes and VarScan found the expected variants with higher precision and recall than GATK or iVar, although the latter identified more expected defining mutations than other callers. LoFreq gave the least reliable results due to the high number of false-positive mutations detected, resulting in lower precision. Similar results were obtained for both the synthetic and wastewater samples.

Genome-Scale Metabolic Modelling of Lifestyle Changes in Rhizobium leguminosarum.

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. IMPORTANCE Rhizobia 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 data sets 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 data sets and identifying targets for future experimental studies.

Genomic Diversity of Pigeon Pea (Cajanus cajan L. Millsp.) Endosymbionts in India and Selection of Potential Strains for Use as Agricultural Inoculants.

Pigeon pea (Cajanus cajan L. Millsp. ) is a legume crop resilient to climate change due to its tolerance to drought. It is grown by millions of resource-poor farmers in semiarid and tropical subregions of Asia and Africa and is a major contributor to their nutritional food security. Pigeon pea is the sixth most important legume in the world, with India contributing more than 70% of the total production and harbouring a wide variety of cultivars. Nevertheless, the low yield of pigeon pea grown under dry land conditions and its yield instability need to be improved. This may be done by enhancing crop nodulation and, hence, biological nitrogen fixation (BNF) by supplying effective symbiotic rhizobia through the application of "elite" inoculants. Therefore, the main aim in this study was the isolation and genomic analysis of effective rhizobial strains potentially adapted to drought conditions. Accordingly, pigeon pea endosymbionts were isolated from different soil types in Southern, Central, and Northern India. After functional characterisation of the isolated strains in terms of their ability to nodulate and promote the growth of pigeon pea, 19 were selected for full genome sequencing, along with eight commercial inoculant strains obtained from the ICRISAT culture collection. The phylogenomic analysis [Average nucleotide identity MUMmer (ANIm)] revealed that the pigeon pea endosymbionts were members of the genera Bradyrhizobium and Ensifer. Based on nodC phylogeny and nod cluster synteny, Bradyrhizobium yuanmingense was revealed as the most common endosymbiont, harbouring nod genes similar to those of Bradyrhizobium cajani and Bradyrhizobium zhanjiangense. This symbiont type (e.g., strain BRP05 from Madhya Pradesh) also outperformed all other strains tested on pigeon pea, with the notable exception of an Ensifer alkalisoli strain from North India (NBAIM29). The results provide the basis for the development of pigeon pea inoculants to increase the yield of this legume through the use of effective nitrogen-fixing rhizobia, tailored for the different agroclimatic regions of India.

Regulation and Characterization of Mutants of fixABCX in Rhizobium leguminosarum.

Symbiosis between Rhizobium leguminosarum and Pisum sativum requires tight control of redox balance in order to maintain respiration under the microaerobic conditions required for nitrogenase while still producing the eight electrons and sixteen molecules of ATP needed for nitrogen fixation. FixABCX, a cluster of electron transfer flavoproteins essential for nitrogen fixation, is encoded on the Sym plasmid (pRL10), immediately upstream of nifA, which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically regulated NifA-dependent promoter upstream of fixA (PnifA1), as well as an additional basal constitutive promoter driving background expression of nifA (PnifA2). These were confirmed by 5'-end mapping of transcription start sites using differential RNA-seq. Complementation of polar fixAB and fixX mutants (Fix- strains) confirmed expression of nifA from PnifA1 in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of fixAB mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant fixAB bacteroids were observed. One was larger than wild-type bacteroids and contained high levels of polyhydroxy-3-butyrate, a complex energy/reductant storage product. A second bacteroid phenotype was morphologically and compositionally different and resembled wild-type infection thread cells. From these two characteristic fixAB mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Multiple sensors provide spatiotemporal oxygen regulation of gene expression in a Rhizobium-legume symbiosis.

Regulation by oxygen (O2) in rhizobia is essential for their symbioses with plants and involves multiple O2 sensing proteins. Three sensors exist in the pea microsymbiont Rhizobium leguminosarum Rlv3841: hFixL, FnrN and NifA. At low O2 concentrations (1%) hFixL signals via FxkR to induce expression of the FixK transcription factor, which activates transcription of downstream genes. These include fixNOQP, encoding the high-affinity cbb3-type terminal oxidase used in symbiosis. In free-living Rlv3841, the hFixL-FxkR-FixK pathway was active at 1% O2, and confocal microscopy showed hFixL-FxkR-FixK activity in the earliest stages of Rlv3841 differentiation in nodules (zones I and II). Work on Rlv3841 inside and outside nodules showed that the hFixL-FxkR-FixK pathway also induces transcription of fnrN at 1% O2 and in the earliest stages of Rlv3841 differentiation in nodules. We confirmed past findings suggesting a role for FnrN in fixNOQP expression. However, unlike hFixL-FxkR-FixK, Rlv3841 FnrN was only active in the near-anaerobic zones III and IV of pea nodules. Quantification of fixNOQP expression in nodules showed this was driven primarily by FnrN, with minimal direct hFixL-FxkR-FixK induction. Thus, FnrN is key for full symbiotic expression of fixNOQP. Without FnrN, nitrogen fixation was reduced by 85% in Rlv3841, while eliminating hFixL only reduced fixation by 25%. The hFixL-FxkR-FixK pathway effectively primes the O2 response by increasing fnrN expression in early differentiation (zones I-II). In zone III of mature nodules, near-anaerobic conditions activate FnrN, which induces fixNOQP transcription to the level required for wild-type nitrogen fixation activity. Modelling and transcriptional analysis indicates that the different O2 sensitivities of hFixL and FnrN lead to a nuanced spatiotemporal pattern of gene regulation in different nodule zones in response to changing O2 concentration. Multi-sensor O2 regulation is prevalent in rhizobia, suggesting the fine-tuned control this enables is common and maximizes the effectiveness of the symbioses.

Lifestyle adaptations of Rhizobium from rhizosphere to symbiosis.

By analyzing successive lifestyle stages of a model Rhizobium-legume symbiosis using mariner-based transposon insertion sequencing (INSeq), we have defined the genes required for rhizosphere growth, root colonization, bacterial infection, N2-fixing bacteroids, and release from legume (pea) nodules. While only 27 genes are annotated as nif and fix in Rhizobium leguminosarum, we show 603 genetic regions (593 genes, 5 transfer RNAs, and 5 RNA features) are required for the competitive ability to nodulate pea and fix N2 Of these, 146 are common to rhizosphere growth through to bacteroids. This large number of genes, defined as rhizosphere-progressive, highlights how critical successful competition in the rhizosphere is to subsequent infection and nodulation. As expected, there is also a large group (211) specific for nodule bacteria and bacteroid function. Nodule infection and bacteroid formation require genes for motility, cell envelope restructuring, nodulation signaling, N2 fixation, and metabolic adaptation. Metabolic adaptation includes urea, erythritol and aldehyde metabolism, glycogen synthesis, dicarboxylate metabolism, and glutamine synthesis (GlnII). There are 17 separate lifestyle adaptations specific to rhizosphere growth and 23 to root colonization, distinct from infection and nodule formation. These results dramatically highlight the importance of competition at multiple stages of a Rhizobium-legume symbiosis.

A novel biosensor to monitor proline in pea root exudates and nodules under osmotic stress and recovery.

BACKGROUND AND AIMS: Plant and bacteria are able to synthesise proline, which acts as a compound to counteract the negative effects of osmotic stresses. Most methodologies rely on the extraction of compounds using destructive methods. This work describes a new proline biosensor that allows the monitoring of proline levels in a non-invasive manner in root exudates and nodules of legume plants. METHODS: The proline biosensor was constructed by cloning the promoter region of pRL120553, a gene with high levels of induction in the presence of proline, in front of the lux cassette in Rhizobium leguminosarum bv. viciae. RESULTS: Free-living assays show that the proline biosensor is sensitive and specific for proline. Proline was detected in both root exudates and nodules of pea plants. The luminescence detected in bacteroids did not show variations during osmotic stress treatments, but significantly increased during recovery. CONCLUSIONS: This biosensor is a useful tool for the in vivo monitoring of proline levels in root exudates and bacteroids of symbiotic root nodules, and it contributes to our understanding of the metabolic exchange occurring in nodules under abiotic stress conditions.

Sequential induction of three recombination directionality factors directs assembly of tripartite integrative and conjugative elements.

Tripartite integrative and conjugative elements (ICE3) are a novel form of ICE that exist as three separate DNA regions integrated within the genomes of Mesorhizobium spp. Prior to conjugative transfer the three ICE3 regions of M. ciceri WSM1271 ICEMcSym1271 combine and excise to form a single circular element. This assembly requires three coordinated recombination events involving three site-specific recombinases IntS, IntG and IntM. Here, we demonstrate that three excisionases-or recombination directionality factors-RdfS, RdfG and RdfM are required for ICE3 excision. Transcriptome sequencing revealed that expression of ICE3 transfer and conjugation genes was induced by quorum sensing. Quorum sensing activated expression of rdfS, and in turn RdfS stimulated transcription of both rdfG and rdfM. Therefore, RdfS acts as a "master controller" of ICE3 assembly and excision. The dependence of all three excisive reactions on RdfS ensures that ICE3 excision occurs via a stepwise sequence of recombination events that avoids splitting the chromosome into a non-viable configuration. These discoveries expose a surprisingly simple control system guiding molecular assembly of these novel and complex mobile genetic elements and highlight the diverse and critical functions of excisionase proteins in control of horizontal gene transfer.

Rhizobia: from saprophytes to endosymbionts.

Rhizobia are some of the best-studied plant microbiota. These oligotrophic Alphaproteobacteria or Betaproteobacteria form symbioses with their legume hosts. Rhizobia must exist in soil and compete with other members of the microbiota before infecting legumes and forming N2-fixing bacteroids. These dramatic lifestyle and developmental changes are underpinned by large genomes and even more complex pan-genomes, which encompass the whole population and are subject to rapid genetic exchange. The ability to respond to plant signals and chemoattractants and to colonize nutrient-rich roots are crucial for the competitive success of these bacteria. The availability of a large body of genomic, physiological, biochemical and ecological studies makes rhizobia unique models for investigating community interactions and plant colonization.

Translation initiation events on structured eukaryotic mRNAs generate gene expression noise.

Gene expression stochasticity plays a major role in biology, creating non-genetic cellular individuality and influencing multiple processes, including differentiation and stress responses. We have addressed the lack of knowledge about posttranscriptional contributions to noise by determining cell-to-cell variations in the abundance of mRNA and reporter protein in yeast. Two types of structural element, a stem-loop and a poly(G) motif, not only inhibit translation initiation when inserted into an mRNA 5΄ untranslated region, but also generate noise. The noise-enhancing effect of the stem-loop structure also remains operational when combined with an upstream open reading frame. This has broad significance, since these elements are known to modulate the expression of a diversity of eukaryotic genes. Our findings suggest a mechanism for posttranscriptional noise generation that will contribute to understanding of the generally poor correlation between protein-level stochasticity and transcriptional bursting. We propose that posttranscriptional stochasticity can be linked to cycles of folding/unfolding of a stem-loop structure, or to interconversion between higher-order structural conformations of a G-rich motif, and have created a correspondingly configured computational model that generates fits to the experimental data. Stochastic events occurring during the ribosomal scanning process can therefore feature alongside transcriptional bursting as a source of noise.

Manganese transport is essential for N2 -fixation by Rhizobium leguminosarum in bacteroids from galegoid but not phaseoloid nodules.

Rhizobium leguminosarum has two high-affinity Mn2+ transport systems encoded by sitABCD and mntH. In symbiosis, sitABCD and mntH were expressed throughout nodules and also strongly induced in Mn2+ -limited cultures of free-living cells. Growth of a sitA mntH double mutant was severely reduced under Mn2+ limitation and sitA and mntH single mutants were more sensitive to oxidative stress. The double sitA mntH mutant of R. leguminosarum was unable to fix nitrogen (Fix- ) with legumes belonging to the galegoid clade (Pisum sativum, Vicia faba and Vicia hirsuta). The presence of infection thread-like structures and sparsely-packed plant cells in nodules suggest that bacteroid development was blocked, either at a late stage of infection thread progression or during bacteroid-release. In contrast, a double sitA mntH mutant was Fix+ on common bean (Phaseoli vulgaris), a member of the phaseoloid clade of legumes, indicating a host-specific symbiotic requirement for Mn2+ transport.

Role of O2 in the Growth of Rhizobium leguminosarum bv. viciae 3841 on Glucose and Succinate.

Insertion sequencing (INSeq) analysis of Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) grown on glucose or succinate at both 21% and 1% O2 was used to understand how O2 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% O2 (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% O2, conditions similar to those experienced by N2-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% O2 and cell death. IMPORTANCE: Rhizobium leguminosarum, a soil bacterium that forms N2-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 O2 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 O2 tensions during growth in soil and N2 fixation in symbiosis with legumes.

Transcriptional and Post-Transcriptional Modulation of SPI1 and SPI2 Expression by ppGpp, RpoS and DksA in Salmonella enterica sv Typhimurium.

The expression of genes within Salmonella Pathogenicity Islands 1 and 2 (SPI1, SPI2) is required to facilitate invasion and intracellular replication respectively of S. Typhimurium in host cell lines. Control of their expression is complex and occurs via a variety of factors operating at transcriptional and post-transcriptional levels in response to the environmental stimuli found within the host. Several of the factors that modulate SPI1 and SPI2 expression are involved in the redistribution or modification of RNA polymerase (RNAP) specificity. These factors include the bacterial alarmone, ppGpp, the alternative sigma factor, RpoS, and the RNAP accessory protein, DksA. In this report we show not only how these three factors modulate SPI1 and SPI2 expression but also how they contribute to the 'phased' expression of SPI1 and SPI2 during progress through late-log and stationary phase in aerobic rich broth culture conditions. In addition, we demonstrate that the expression of at least one SPI1-encoded protein, SipC is subject to DksA-dependent post-transcriptional control.

PerR controls oxidative stress defence and aerotolerance but not motility-associated phenotypes of Campylobacter jejuni.

The foodborne bacterial pathogen Campylobacter jejuni is an obligate microaerophile that is exposed to atmospheric oxygen during transmission through the food chain. Survival under aerobic conditions requires the concerted control of oxidative stress systems, which in C. jejuni are intimately connected with iron metabolism via the PerR and Fur regulatory proteins. Here, we have characterized the roles of C. jejuni PerR in oxidative stress and motility phenotypes, and its regulon at the level of transcription, protein expression and promoter interactions. Insertional inactivation of perR in the C. jejuni reference strains NCTC 11168, 81-176 and 81116 did not result in any growth deficiencies, but strongly increased survival in atmospheric oxygen conditions, and allowed growth around filter discs infused with up to 30 % H2O2 (8.8 M). Expression of catalase, alkyl hydroperoxide reductase, thioredoxin reductase and the Rrc desulforubrerythrin was increased in the perR mutant, and this was mediated at the transcriptional level as shown by electrophoretic mobility shift assays of the katA, ahpC and trxB promoters using purified PerR. Differential RNA-sequencing analysis of a fur perR mutant allowed the identification of eight previously unknown transcription start sites of genes controlled by Fur and/or PerR. Finally, inactivation of perR in C. jejuni did not result in reduced motility, and did not reduce killing of Galleria melonella wax moth larvae. In conclusion, PerR plays an important role in controlling oxidative stress resistance and aerobic survival of C. jejuni, but this role does not extend into control of motility and associated phenotypes.

Nutritional and metabolic requirements for the infection of HeLa cells by Salmonella enterica serovar Typhimurium.

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food--and water--borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.

The primary transcriptome of Salmonella enterica Serovar Typhimurium and its dependence on ppGpp during late stationary phase.

We have used differential RNA-seq (dRNA-seq) to characterise the transcriptomic architecture of S. Typhimurium SL1344, and its dependence on the bacterial alarmone, guanosine tetraphosphate (ppGpp) during late stationary phase, (LSP). Under LSP conditions we were able to identify the transcriptional start sites (TSSs) for 53% of the S. Typhimurium open reading frames (ORFs) and discovered 282 candidate non-coding RNAs (ncRNAs). The mapping of LSP TSSs enabled a detailed comparison with a previous dRNA-seq study of the early stationary phase (ESP) transcriptional architecture of S. Typhimurium SL1344 and its dependence on ppGpp. For the purposes of this study, LSP was defined as an aerobic LB culture grown to a later optical density reading (OD600 = 3.6) compared to ESP (OD600 = 2.3). The precise nucleotide positions of the majority of S. Typhimurium TSSs at LSP agreed closely with those identified at ESP. However, the identification of TSSs at different positions, or where additional or fewer TSSs were found at LSP compared to ESP enabled the genome-wide categorisation of growth phase dependent changes in promoter structure, the first time such an analysis has been done on this scale. Comparison of the ppGpp-dependency LSP and ESP TSSs for mRNAs and ncRNAs revealed a similar breadth of ppGpp-activation and repression. However, we note several ncRNAs previously shown to be involved in virulence were highly ppGpp-dependent at LSP. Finally, although SPI1 was expressed at ESP, we found SPI1 was not as highly expressed at LSP, instead we observed elevated expression of SPI2 encoded genes. We therefore also report an analysis of SPI2 transcriptional architecture at LSP resulting in localisation of SsrB binding sites and identification of a previously unreported SPI2 TSS. We also show that ppGpp is required for nearly all of SPI2 expression at LSP as well as for expression of SPI1 at ESP.

The architecture and ppGpp-dependent expression of the primary transcriptome of Salmonella Typhimurium during invasion gene expression.

BACKGROUND: Invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium (S. Typhimurium) requires expression of the extracellular virulence gene expression programme (ST(EX)), activation of which is dependent on the signalling molecule guanosine tetraphosphate (ppGpp). Recently, next-generation transcriptomics (RNA-seq) has revealed the unexpected complexity of bacterial transcriptomes and in this report we use differential RNA sequencing (dRNA-seq) to define the high-resolution transcriptomic architecture of wild-type S. Typhimurium and a ppGpp null strain under growth conditions which model ST(EX). In doing so we show that ppGpp plays a much wider role in regulating the S. Typhimurium ST(EX) primary transcriptome than previously recognised. RESULTS: Here we report the precise mapping of transcriptional start sites (TSSs) for 78% of the S. Typhimurium open reading frames (ORFs). The TSS mapping enabled a genome-wide promoter analysis resulting in the prediction of 169 alternative sigma factor binding sites, and the prediction of the structure of 625 operons. We also report the discovery of 55 new candidate small RNAs (sRNAs) and 302 candidate antisense RNAs (asRNAs). We discovered 32 ppGpp-dependent alternative TSSs and determined the extent and level of ppGpp-dependent coding and non-coding transcription. We found that 34% and 20% of coding and non-coding RNA transcription respectively was ppGpp-dependent under these growth conditions, adding a further dimension to the role of this remarkable small regulatory molecule in enabling rapid adaptation to the infective environment. CONCLUSIONS: The transcriptional architecture of S. Typhimurium and finer definition of the key role ppGpp plays in regulating Salmonella coding and non-coding transcription should promote the understanding of gene regulation in this important food borne pathogen and act as a resource for future research.

Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics.

BACKGROUND: The rhizosphere is the microbe-rich zone around plant roots and is a key determinant of the biosphere's productivity. Comparative transcriptomics was used to investigate general and plant-specific adaptations during rhizosphere colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet (non-legume). Gene expression data were compared to metabolic and transportome maps to understand adaptation to the rhizosphere. RESULTS: Carbon metabolism was dominated by organic acids, with a strong bias towards aromatic amino acids, C1 and C2 compounds. This was confirmed by induction of the glyoxylate cycle required for C2 metabolism and gluconeogenesis in all rhizospheres. Gluconeogenesis is repressed in R. leguminosarum by sugars, suggesting that although numerous sugar and putative complex carbohydrate transport systems are induced in the rhizosphere, they are less important carbon sources than organic acids. A common core of rhizosphere-induced genes was identified, of which 66% are of unknown function. Many genes were induced in the rhizosphere of the legumes, but not sugar beet, and several were plant specific. The plasmid pRL8 can be considered pea rhizosphere specific, enabling adaptation of R. leguminosarum to its host. Mutation of many of the up-regulated genes reduced competitiveness for pea rhizosphere colonization, while two genes specifically up-regulated in the pea rhizosphere reduced colonization of the pea but not alfalfa rhizosphere. CONCLUSIONS: Comparative transcriptome analysis has enabled differentiation between factors conserved across plants for rhizosphere colonization as well as identification of exquisite specific adaptation to host plants.