Resource Allocation During the Transition to Diazotrophy in Klebsiella oxytoca.

Free-living nitrogen-fixing bacteria can improve growth yields of some non-leguminous plants and, if enhanced through bioengineering approaches, have the potential to address major nutrient imbalances in global crop production by supplementing inorganic nitrogen fertilisers. However, nitrogen fixation is a highly resource-costly adaptation and is de-repressed only in environments in which sources of reduced nitrogen are scarce. Here we investigate nitrogen fixation (nif) gene expression and nitrogen starvation response signaling in the model diazotroph Klebsiella oxytoca (Ko) M5a1 during ammonium depletion and the transition to growth on atmospheric N2. Exploratory RNA-sequencing revealed that over 50% of genes were differentially expressed under diazotrophic conditions, among which the nif genes are among the most highly expressed and highly upregulated. Isotopically labelled QconCAT standards were designed for multiplexed, absolute quantification of Nif and nitrogen-stress proteins via multiple reaction monitoring mass spectrometry (MRM-MS). Time-resolved Nif protein concentrations were indicative of bifurcation in the accumulation rates of nitrogenase subunits (NifHDK) and accessory proteins. We estimate that the nitrogenase may account for more than 40% of cell protein during diazotrophic growth and occupy approximately half the active ribosome complement. The concentrations of free amino acids in nitrogen-starved cells were insufficient to support the observed rates of Nif protein expression. Total Nif protein accumulation was reduced 10-fold when the NifK protein was truncated and nitrogenase catalysis lost (nifK 1 - 1 203), implying that reinvestment of de novo fixed nitrogen is essential for further nif expression and a complete diazotrophy transition. Several amino acids accumulated in non-fixing ΔnifLA and nifK 1 - 1203 mutants, while the rest remained highly stable despite prolonged N starvation. Monitoring post-translational uridylylation of the PII-type signaling proteins GlnB and GlnK revealed distinct nitrogen regulatory roles in Ko M5a1. GlnK uridylylation was persistent throughout the diazotrophy transition while a ΔglnK mutant exhibited significantly reduced Nif expression and nitrogen fixation activity. Altogether, these findings highlight quantitatively the scale of resource allocation required to enable the nitrogen fixation adaptation to take place once underlying signaling processes are fulfilled. Our work also provides an omics-level framework with which to model nitrogen fixation in free-living diazotrophs and inform rational engineering strategies.

Transcriptome and phytohormone changes associated with ethylene-induced onion bulb dormancy.

Control of dormancy and sprouting in onion bulbs is commercially important for postharvest management. Although ethylene application is sometimes used to extend dormancy, the underlying mechanisms regulating dormancy transition remain unclear. Since the sprout leaves emerge from the bulb baseplate, we used this tissue to assess the impact of ethylene treatment and storage time on the hormone profile and the transcriptome. Reads from 30 libraries were assembled and annotated, with 94,840 unigenes retained after filtering. The de novo transcriptome assembly was of high quality and continuity (N50: 1809 bp, GC content: 36.21 %), and was used to analyse differential expression and Gene Onotologies. Across two years, applied ethylene resulted in delayed dormancy break and reduced post-dormancy sprout vigour. Ethylene supplementation enhanced endogenous ethylene production and caused a transient climacteric-like increase in respiration. Significant changes in hormone and associated transcript profiles occurred through storage and in response to ethylene. In particular, abscisic acid (ABA) and its metabolite phaseic acid (PA) increased under ethylene during the longer dormancy period; however, cytokinin increases observed during storage appeared largely independent of ethylene treatment. Several hormone-related transcripts showed differential expression over time and/or in response to ethylene. Expression of ethylene biosynthesis (ACO), receptor (EIN4) and transcription factor (EIL3) genes were modified by ethylene, as were ABA biosynthesis genes such NCED, and cytokinin biosynthesis genes such as LOG and CKX. We conclude that ethylene substantially modifies expression of genes in several phytohormone pathways, and some of these changes may underlie the dormancy-extending effects of exogenous ethylene.

The induction of the fibroblast extracellular senescence metabolome is a dynamic process.

Cellular senescence is often associated with irreparable DNA double strand breaks (IrrDSBs) which accumulate with chronological age (IrrDSBsen). The removal of senescent cells ameliorates several age-related diseases in mice but the translation of these findings into a clinical setting would be aided by the characterisation of non-invasive biomarkers of senescent cells. Several serum metabolites are independent indicators of chronological age and some of these accumulate outside senescent fibroblasts independently of cell cycle arrest, repairable DNA breaks and cell size (the extracellular senescence metabolome, or ESM). The post-mitotic phase of senescence is dynamic, making the detection of senescent cells in vivo difficult. An unbiased metabolomic screen of the IrrDSBsen fibroblast ESM also showed differences in the times of initiation and maintenance of different metabolites but generally the ESM altered progressively over the 20 day study period unlike the reported transcriptional profiles. This more detailed analysis of IrrDSBsen identified several new ESM metabolites that are associated with chronological ageing. Targeted analysis of citrate confirmed the dynamic nature of this metabolite in two cell lines and revealed its independence from the senescence effector p16INK4A. These data will aid our understanding of metabolic signatures of ageing and their relationship to cellular senescence and IrrDSBs.

GlnK Facilitates the Dynamic Regulation of Bacterial Nitrogen Assimilation.

Ammonium assimilation in Escherichia coli is regulated by two paralogous proteins (GlnB and GlnK), which orchestrate interactions with regulators of gene expression, transport proteins, and metabolic pathways. Yet how they conjointly modulate the activity of glutamine synthetase, the key enzyme for nitrogen assimilation, is poorly understood. We combine experiments and theory to study the dynamic roles of GlnB and GlnK during nitrogen starvation and upshift. We measure time-resolved in vivo concentrations of metabolites, total and posttranslationally modified proteins, and develop a concise biochemical model of GlnB and GlnK that incorporates competition for active and allosteric sites, as well as functional sequestration of GlnK. The model predicts the responses of glutamine synthetase, GlnB, and GlnK under time-varying external ammonium level in the wild-type and two genetic knock-outs. Our results show that GlnK is tightly regulated under nitrogen-rich conditions, yet it is expressed during ammonium run-out and starvation. This suggests a role for GlnK as a buffer of nitrogen shock after starvation, and provides a further functional link between nitrogen and carbon metabolisms.

Comparative proteomics of cucurbit phloem indicates both unique and shared sets of proteins.

Cucurbits are well-studied models for phloem biology but unusually possess both fascicular phloem (FP) within vascular bundles and additional extrafascicular phloem (EFP). Although the functional differences between the two systems are not yet clear, sugar analysis and limited protein profiling have established that FP and EFP have divergent compositions. Here we report a detailed comparative proteomics study of FP and EFP in two cucurbits, pumpkin and cucumber. We re-examined the sites of exudation by video microscopy, and confirmed that in both species, the spontaneous exudate following tissue cutting derives almost exclusively from EFP. Comparative gel electrophoresis and mass spectrometry-based proteomics of exudates, sieve element contents and microdissected stem tissues established that EFP and FP profiles are highly dissimilar, and that there are also species differences. Searches against cucurbit databases enabled identification of more than 300 FP proteins from each species. Few of the detected proteins (about 10%) were shared between the sieve element contents of FP and EFP, and enriched Gene Ontology categories also differed. To explore quantitative differences in the proteomes, we developed multiple reaction monitoring methods for cucumber proteins that are representative markers for FP or EFP and assessed exudate composition at different times after tissue cutting. Based on failure to detect FP markers in exudate samples, we conclude that FP is blocked very rapidly and therefore makes a minimal contribution to the exudates. Overall, the highly divergent contents of FP and EFP indicate that they are substantially independent vascular compartments.

Glucosinolate and Desulfo-glucosinolate Metabolism by a Selection of Human Gut Bacteria.

Glucosinolate (GSL) hydrolysis is mediated by the enzyme myrosinase which together with specifier proteins can give rise to isothiocyanates (ITCs), thiocyanates, and nitriles (NITs) in cruciferous plants. However, little is known about the metabolism of GSLs by the human gut flora. The aim of the work was to investigate the metabolic fates of sinigrin (SNG), glucotropaeolin (GTP), gluconasturtiin (GNT), and their corresponding desulfo-GSLs (DS-GSLs). Three human gut bacterial strains, Enterococcus casseliflavus CP1, Lactobacillus agilis R16, and Escherichia coli VL8, were chosen for this study. GNT was metabolized to completion within 24 h to phenethyl ITC and phenethyl NIT (PNIT) by all bacteria, except for L. agilis R16 which produced only PNIT. At least 80 % of GTP and SNG were metabolized by all bacteria within 24 h to the corresponding ITCs and NITs. The pH of media over time gradually became acidic for both L. agilis R16 and E. coli VL8, while for E. casseliflavus CP1 the media became slightly alkaline with NIT and ITC production occurring between pH 3.0 and 7.5. ITC production peaked between 4 and 10 h in most cases and gradually declined while NIT production increased and remained relatively constant over time. The total percentage products accounted for 3-53 % of the initial GSL. NITs were produced from DS-GSLs suggesting an alternative metabolism via desulfation for the food based GSLs. The metal ion dependency for NIT production for GNT and its DS form was investigated where it was shown that Fe(2+) increased NIT production, while Mg(2+) stimulated the formation of ITC.

Discovery of a Bacterial Glycoside Hydrolase Family 3 (GH3) ß-Glucosidase with Myrosinase Activity from a Citrobacter Strain Isolated from Soil.

A Citrobacter strain (WYE1) was isolated from a UK soil by enrichment using the glucosinolate sinigrin as sole carbon source. The enzyme myrosinase was purified using a combination of ion exchange and gel filtration to give a pure protein of approximately 66 kDa. The N-terminal amino acid and internal peptide sequence of the purified protein were determined and used to identify the gene, which, based on InterPro sequence analysis, belongs to the family GH3, contains a signal peptide, and is a periplasmic protein with a predicted molecular mass of 71.8 kDa. A preliminary characterization was carried out using protein extracts from cell-free preparations. The apparent KM and Vmax were 0.46 mM and 4.91 mmol dm(-3) min(-1) mg(-1), respectively, with sinigrin as substrate. The optimum temperature and pH for enzyme activity were 25 °C and 6.0, respectively. The enzyme was marginally activated with ascorbate by a factor of 1.67.

An in vivo crosslinking system for identifying mycobacterial protein-protein interactions.

The analysis of protein-protein interactions in Mycobacterium tuberculosis has the potential to shed light on the functions of the large number of predicted open-reading frames annotated as conserved hypothetical proteins. We have developed a formaldehyde crosslinking system to detect in vivo interactions in mycobacteria. Our Gateway-adapted vector system uses three promoter strengths, including constitutive and regulatable versions, for the expression of target proteins with either an N- or C-terminal His-Strep-Strep tag. Tandem affinity purification using the His- and Strep-tags is well-suited to the isolation of protein complexes with a high purity and no detectable background. We have validated this approach using the well-described pyruvate dehydrogenase complex.

Differential secretome analysis of Pseudomonas syringae pv tomato using gel-free MS proteomics.

The plant pathogen Pseudomonas syringae pv.tomato (DC3000) causes virulence by delivering effector proteins into host plant cells through its type three secretion system (T3SS). In response to the plant environment DC3000 expresses hypersensitive response and pathogenicity genes (hrp). Pathogenesis depends on the ability of the pathogen to manipulate the plant metabolism and to inhibit plant immunity, which depends to a large degree on the plant's capacity to recognize both pathogen and microbial determinants (PAMP/MAMP-triggered immunity). We have developed and employed MS-based shotgun and targeted proteomics to (i) elucidate the extracellular and secretome composition of DC3000 and (ii) evaluate temporal features of the assembly of the T3SS and the secretion process together with its dependence of pH. The proteomic screen, under hrp inducing in vitro conditions, of extracellular and cytoplasmatic fractions indicated the segregated presence of not only T3SS implicated proteins such as HopP1, HrpK1, HrpA1 and AvrPto1, but also of proteins not usually associated with the T3SS or with pathogenicity. Using multiple reaction monitoring MS (MRM-MS) to quantify HrpA1 and AvrPto1, we found that HrpA1 is rapidly expressed, at a strict pH-dependent rate and is post-translationally processed extracellularly. These features appear to not interfere with rapid AvrPto1 expression and secretion but may suggest some temporal post-translational regulatory mechanism of the T3SS assembly. The high specificity and sensitivity of the MRM-MS approach should provide a powerful tool to measure secretion and translocation in infected tissues.

Conditional Auxin Response and Differential Cytokinin Profiles in Shoot Branching Mutants.

Strigolactone (SL), auxin, and cytokinin (CK) are hormones that interact to regulate shoot branching. For example, several ramosus (rms) branching mutants in pea (Pisum sativum) have SL defects, perturbed xylem CK levels, and diminished responses to auxin in shoot decapitation assays. In contrast with the last of these characteristics, we discovered that buds on isolated nodes (explants) of rms plants instead respond normally to auxin. We hypothesized that the presence or absence of attached roots would result in transcriptional and hormonal differences in buds and subtending stem tissues, and might underlie the differential auxin response. However, decapitated plants and explants both showed similar up-regulation of CK biosynthesis genes, increased CK levels, and down-regulation of auxin transport genes. Moreover, auxin application counteracted these trends, regardless of the effectiveness of auxin at inhibiting bud growth. Multivariate analysis revealed that stem transcript and CK changes were largely associated with decapitation and/or root removal and auxin response, whereas bud transcript profiles related more to SL defects. CK clustering profiles were indicative of additional zeatin-type CKs in decapitated stems being supplied by roots and thus promoting bud growth in SL-deficient genotypes even in the presence of added auxin. This difference in CK content may explain why rms buds on explants respond better to auxin than those on decapitated plants. We further conclude that rapid changes in CK status in stems are auxin dependent but largely SL independent, suggesting a model in which auxin and CK are dominant regulators of decapitation-induced branching, whereas SLs are more important in intact plants.

Chp8, a diguanylate cyclase from Pseudomonas syringae pv. Tomato DC3000, suppresses the pathogen-associated molecular pattern flagellin, increases extracellular polysaccharides, and promotes plant immune evasion.

UNLABELLED: The bacterial plant pathogen Pseudomonas syringae causes disease in a wide range of plants. The associated decrease in crop yields results in economic losses and threatens global food security. Competition exists between the plant immune system and the pathogen, the basic principles of which can be applied to animal infection pathways. P. syringae uses a type III secretion system (T3SS) to deliver virulence factors into the plant that promote survival of the bacterium. The P. syringae T3SS is a product of the hypersensitive response and pathogenicity (hrp) and hypersensitive response and conserved (hrc) gene cluster, which is strictly controlled by the codependent enhancer-binding proteins HrpR and HrpS. Through a combination of bacterial gene regulation and phenotypic studies, plant infection assays, and plant hormone quantifications, we now report that Chp8 (i) is embedded in the Hrp regulon and expressed in response to plant signals and HrpRS, (ii) is a functional diguanylate cyclase, (iii) decreases the expression of the major pathogen-associated molecular pattern (PAMP) flagellin and increases extracellular polysaccharides (EPS), and (iv) impacts the salicylic acid/jasmonic acid hormonal immune response and disease progression. We propose that Chp8 expression dampens PAMP-triggered immunity during early plant infection. IMPORTANCE: The global demand for food is projected to rise by 50% by 2030 and, as such, represents one of the major challenges of the 21st century, requiring improved crop management. Diseases caused by plant pathogens decrease crop yields, result in significant economic losses, and threaten global food security. Gaining mechanistic insights into the events at the plant-pathogen interface and employing this knowledge to make crops more resilient is one important strategy for improving crop management. Plant-pathogen interactions are characterized by the sophisticated interplay between plant immunity elicited upon pathogen recognition and immune evasion by the pathogen. Here, we identify Chp8 as a contributor to the major effort of the plant pathogen Pseudomonas syringae pv. tomato DC3000 to evade immune responses of the plant.

Analysis of Chinese hamster ovary cell metabolism through a combined computational and experimental approach.

Optimization of cell culture processes can benefit from the systematic analysis of experimental data and their organization in mathematical models, which can be used to decipher the effect of individual process variables on multiple outputs of interest. Towards this goal, a kinetic model of cytosolic glucose metabolism coupled with a population-level model of Chinese hamster ovary cells was used to analyse metabolic behavior under batch and fed-batch cell culture conditions. The model was parameterized using experimental data for cell growth dynamics, extracellular and intracellular metabolite profiles. The results highlight significant differences between the two culture conditions in terms of metabolic efficiency and motivate the exploration of lactate as a secondary carbon source. Finally, the application of global sensitivity analysis to the model parameters highlights the need for additional experimental information on cell cycle distribution to complement metabolomic analyses with a view to parameterize kinetic models.

Nitrogen and carbon status are integrated at the transcriptional level by the nitrogen regulator NtrC in vivo.

UNLABELLED: Nitrogen regulation in Escherichia coli is a model system for gene regulation in bacteria. Growth on glutamine as a sole nitrogen source is assumed to be nitrogen limiting, inferred from slow growth and strong NtrB/NtrC-dependent gene activation. However, we show that under these conditions, the intracellular glutamine concentration is not limiting but 5.6-fold higher than in ammonium-replete conditions; in addition, α-ketoglutarate concentrations are elevated. We address this glutamine paradox from a systems perspective. We show that the dominant role of NtrC is to regulate glnA transcription and its own expression, indicating that the glutamine paradox is not due to NtrC-independent gene regulation. The absolute intracellular NtrC and GS concentrations reveal molecular control parameters, where NtrC-specific activities were highest in nitrogen-starved cells, while under glutamine growth, NtrC showed intermediate specific activity. We propose an in vivo model in which α-ketoglutarate can derepress nitrogen regulation despite nitrogen sufficiency. IMPORTANCE: Nitrogen is the most important nutrient for cell growth after carbon, and its metabolism is coordinated at the metabolic, transcriptional, and protein levels. We show that growth on glutamine as a sole nitrogen source, commonly assumed to be nitrogen limiting and used as such as a model system for nitrogen limitation, is in fact nitrogen replete. Our integrative quantitative analysis of key molecules involved in nitrogen assimilation and regulation reveal that glutamine is not necessarily the dominant molecule signaling nitrogen sufficiency and that α-ketoglutarate may play a more important role in signaling nitrogen status. NtrB/NtrC integrates α-ketoglutarate and glutamine signaling--sensed by the UTase (glnD) and PII (glnB), respectively--and regulates the nitrogen response through self-regulated expression and phosphorylation-dependent activation of the nitrogen (ntr) regulon. Our findings support α-ketoglutarate acting as a global regulatory metabolite.

Earthworms produce phytochelatins in response to arsenic.

Phytochelatins are small cysteine-rich non-ribosomal peptides that chelate soft metal and metalloid ions, such as cadmium and arsenic. They are widely produced by plants and microbes; phytochelatin synthase genes are also present in animal species from several different phyla, but there is still little known about whether these genes are functional in animals, and if so, whether they are metal-responsive. We analysed phytochelatin production by direct chemical analysis in Lumbricus rubellus earthworms exposed to arsenic for a 28 day period, and found that arsenic clearly induced phytochelatin production in a dose-dependent manner. It was necessary to measure the phytochelatin metabolite concentrations directly, as there was no upregulation of phytochelatin synthase gene expression after 28 days: phytochelatin synthesis appears not to be transcriptionally regulated in animals. A further untargetted metabolomic analysis also found changes in metabolites associated with the transsulfuration pathway, which channels sulfur flux from methionine for phytochelatin synthesis. There was no evidence of biological transformation of arsenic (e.g. into methylated species) as a result of laboratory arsenic exposure. Finally, we compared wild populations of earthworms sampled from the field, and found that both arsenic-contaminated and cadmium-contaminated mine site worms had elevated phytochelatin concentrations.

Uncoupling of reactive oxygen species accumulation and defence signalling in the metal hyperaccumulator plant Noccaea caerulescens.

The metal hyperaccumulator plant Noccaea caerulescens is protected from disease by the accumulation of high concentrations of metals in its aerial tissues, which are toxic to many pathogens. As these metals can lead to the production of damaging reactive oxygen species (ROS), metal hyperaccumulator plants have developed highly effective ROS tolerance mechanisms, which might quench ROS-based signals. We therefore investigated whether metal accumulation alters defence signalling via ROS in this plant. We studied the effect of zinc (Zn) accumulation by N. caerulescens on pathogen-induced ROS production, salicylic acid accumulation and downstream defence responses, such as callose deposition and pathogenesis-related (PR) gene expression, to the bacterial pathogen Pseudomonas syringae pv. maculicola. The accumulation of Zn caused increased superoxide production in N. caerulescens, but inoculation with P. syringae did not elicit the defensive oxidative burst typical of most plants. Defences dependent on signalling through ROS (callose and PR gene expression) were also modified or absent in N. caerulescens, whereas salicylic acid production in response to infection was retained. These observations suggest that metal hyperaccumulation is incompatible with defence signalling through ROS and that, as metal hyperaccumulation became effective as a form of elemental defence, normal defence responses became progressively uncoupled from ROS signalling in N. caerulescens.

Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation.

PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation.

A viral RNA silencing suppressor interferes with abscisic acid-mediated signalling and induces drought tolerance in Arabidopsis thaliana.

Cucumber mosaic virus (CMV) encodes the 2b protein, which plays a role in local and systemic virus movement, symptom induction and suppression of RNA silencing. It also disrupts signalling regulated by salicylic acid and jasmonic acid. CMV induced an increase in tolerance to drought in Arabidopsis thaliana. This was caused by the 2b protein, as transgenic plants expressing this viral factor showed increased drought tolerance, but plants infected with CMVΔ2b, a viral mutant lacking the 2b gene, did not. The silencing effector ARGONAUTE1 (AGO1) controls a microRNA-mediated drought tolerance mechanism and, in this study, we noted that plants (dcl2/3/4 triple mutants) lacking functional short-interfering RNA-mediated silencing were also drought tolerant. However, drought tolerance engendered by CMV may be independent of the silencing suppressor activity of the 2b protein. Although CMV infection did not alter the accumulation of the drought response hormone abscisic acid (ABA), 2b-transgenic and ago1-mutant seeds were hypersensitive to ABA-mediated inhibition of germination. However, the induction of ABA-regulated genes in 2b-transgenic and CMV-infected plants was inhibited more strongly than in ago1-mutant plants. The virus engenders drought tolerance by altering the characteristics of the roots and not of the aerial tissues as, compared with the leaves of silencing mutants, leaves excised from CMV-infected or 2b-transgenic plants showed greater stomatal permeability and lost water more rapidly. This further indicates that CMV-induced drought tolerance is not mediated via a change in the silencing-regulated drought response mechanism. Under natural conditions, virus-induced drought tolerance may serve viruses by aiding susceptible hosts to survive periods of environmental stress.

The dormancy regulator DosR controls ribosome stability in hypoxic mycobacteria.

It is thought that during latent infection, Mycobacterium tuberculosis bacilli are retained within granulomas in a low-oxygen environment. The dormancy survival (Dos) regulon, regulated by the response regulator DosR, appears to be essential for hypoxic survival in M. tuberculosis, but it is not known how the regulon promotes survival. Here we report that mycobacteria, in contrast to enteric bacteria, do not form higher-order structures (e.g. ribosomal dimers) upon entry into stasis. Instead, ribosomes are stabilized in the associated form (70S). Using a strategy incorporating microfluidic, proteomic, and ribosomal profiling techniques to elucidate the fate of mycobacterial ribosomes during hypoxic stasis, we show that the dormancy regulator DosR is required for optimal ribosome stabilization. We present evidence that the majority of this effect is mediated by the DosR-regulated protein MSMEG_3935 (a S30AE domain protein), which is associated with the ribosome under hypoxic conditions. A Δ3935 mutant phenocopies the ΔdosR mutant during hypoxia, and complementation of ΔdosR with the MSMEG_3935 gene leads to complete recovery of dosR mutant phenotypes during hypoxia. We suggest that this protein is named ribosome-associated factor under hypoxia (RafH) and that it is the major factor responsible for DosR-mediated hypoxic survival in mycobacteria.

Arabidopsis histidine kinase 5 regulates salt sensitivity and resistance against bacterial and fungal infection.

• The ability of plants to adapt to multiple stresses imposed by the natural environment requires cross-talk and fine-tuning of stress signalling pathways. The hybrid histidine kinase Arabidopsis histidine kinase 5 (AHK5) is known to mediate stomatal responses to exogenous and endogenous signals in Arabidopsis thaliana. The purpose of this study was to determine whether the function of AHK5 in stress signalling extends beyond stomatal responses. • Plant growth responses to abiotic stresses, tissue susceptibility to bacterial and fungal pathogens, and hormone production and metabolism of reactive oxygen species were monitored in a T-DNA insertion mutant of AHK5. • The findings of this study indicate that AHK5 positively regulates salt sensitivity and contributes to resistance to the bacterium Pseudomonas syringae pv. tomato DC3000 and the fungal pathogen Botrytis cinerea. • This is the first report of a role for AHK5 in the regulation of survival following challenge by a hemi-biotrophic bacterium and a necrotrophic fungus, as well as in the growth response to salt stress. The function of AHK5 in regulating the production of hormones and redox homeostasis is discussed.

Isolation of bacterial ribosomes with monolith chromatography.

We report the development of a rapid chromatographic method for the isolation of bacterial ribosomes from crude cell lysates in less than ten minutes. Our separation is based on the use of strong anion exchange monolithic columns. Using a simple stepwise elution program we were able to purify ribosomes whose composition is comparable to those isolated by sucrose gradient ultracentrifugation, as confirmed by quantitative proteomic analysis (iTRAQ). The speed and simplicity of this approach could accelerate the study of many different aspects of ribosomal biology.