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International audience; Phosphate, as a constituent of the high energy molecules, ATP/GTP and polyphosphate, plays a crucial role in most of the metabolic processes of living organisms. Therefore, the adaptation to low Pi availability is a major challenge for bacteria. In Streptomyces, this adaptation is tightly controlled by the two component PhoR/PhoP system. In this study, the free intracellular Pi, ATP, ADP and polyP content of the wild type and the phoP mutant strain of S. lividans TK24 were analyzed at discrete time points throughout growth in Pi replete and limited media. PolyP length and content was shown to be directly related to the Pi content of the growth medium. In Pi repletion, ATP and high molecular weight (HMW) polyP contents were higher in the phoP mutant than in the WT strain. This supports the recently proposed repressive effect of PhoP on oxidative phosphorylation. High oxidative phosphor-ylation activity might also have a direct or indirect positive impact on HMW polyP synthesis. In Pi sufficiency as in Pi limitation, the degradation of these polymers was shown to be clearly delayed in the phoP mutant, indicating PhoP dependent expression of the enzymes involved in this degradation. The efficient storage of Pi as polyphosphate and/or its inefficient degradation in Pi in the phoP mutant resulted in low levels of free Pi and ATP that are likely to be, at least in part, responsible for the very poor growth of this mutant in Pi limitation. Furthermore , short polyP was shown to be present outside the cell, tightly bound to the myceli-um via electrostatic interactions involving divalent cations. Less short polyP was found to be associated with the mycelium of the phoP mutant than with that of the WT strain, indicating that generation and externalization of these short polyP molecules was directly or indirectly dependent on PhoP.

The haloarchaeon Haloferax volcanii contains nearly 2800 small non-coding RNAs (sRNAs). One intergenic sRNA, sRNA132, was chosen for a detailed characterization. A deletion mutant had a growth defect and thus underscored the importance of sRNA132. A microarray analysis identified the transcript of an operon for a phosphate-specific ABC transporter as a putative target of sRNA132. Both the sRNA132 and the operon transcript accumulated under low phosphate concentrations, indicating a positive regulatory role of sRNA132. A kinetic analysis revealed that sRNA132 is essential shortly after the onset of phosphate starvation, while other regulatory processes take over after several hours. Comparison of the transcriptomes of wild-type and the sRNA132 gene deletion mutant 30 min after the onset of phosphate starvation revealed that sRNA132 controls a regulon of about 40 genes. Remarkably, the regulon included a second operon for a phosphate-specific ABC transporter, which also depended on sRNA132 for rapid induction in the absence of phosphate. Competitive growth experiments of the wild-type and ABC transporter operon deletion mutants underscored the importance of both transporters for growth at low phosphate concentrations. Northern blot analyses of four additional members of the sRNA132 regulon verified that all four transcripts depended on sRNA132 for rapid regulation after the onset of phosphate starvation. Importantly, this is the first example for the transient importance of a sRNA for any archaeal and bacterial species. In addition, this study unraveled the first sRNA regulon for haloarchaea.

A healthy gastrointestinal (GI) tract with a properly established microbiota is necessary for a foal to develop into a healthy weanling. A foal's health can be critically impacted by aberrations in the microbiome such as with diarrhea which can cause great morbidity and mortality in foals. In this study, we hypothesized that gut establishment in the foal transitioning from a diet of milk to a diet of grain, forage, and pasture would be detectable through analyses of the fecal microbiotas. Fecal samples from 37 sets of foals and mares were collected at multiple time points ranging from birth to weaning. Bacterial DNA was isolated from the samples, and the V4 domain of bacterial 16S rRNA genes were amplified via polymerase chain reaction. Next generation sequencing was then performed on the resulting amplicons, and analyses were performed to characterize the microbiome as well as the relative abundance of microbiota present. We found that bacterial population compositions followed a pattern throughout the early life of the foal in an age-dependent manner. As foals transitioned from milk consumption to a forage and grain diet, there were recognizable changes in fecal microbial compositions from initial populations predominant in the ability to metabolize milk to populations capable of utilizing fibrous plant material. We were also able to recognize differences in microbial populations amongst diarrheic foals as well as microbial population differences associated with differences in management styles between facilities. Future efforts will gauge the effects of lesser abundant bacterial populations that could also be essential to GI health, as well as to determine how associations between microbial population profiles and animal management practices can be used to inform strategies for improving upon the health and growth of horses overall.

Regulators of the WhiB-like (wbl) family are playing important role in the complex regulation of metabolic and morphological differentiation in Streptomyces. In this study, we investigated the role of wblI, a member of this family, in the regulation of secondary metabolite production in Streptomyces lividans. The over-expression of wblI was correlated with an enhanced biosynthesis of undecylprodigiosin and actinorhodin and with a reduction of the biosynthesis of yCPK and of the grey spore pigment encoded by the whiE locus. Five regulatory targets of WblI were identified using in vitro formaldehyde crosslinking and confirmed by EMSA and qRT-PCR. These included the promoter regions of wblI itself, two genes of the ACT cluster (actVA3 and the intergenic region between the divergently orientated genes actII-1 and actII-2) and that of wblA, another member of the Wbl family. Quantitative RT-PCR analysis indicated that the expression of actVA3 encoding a protein of unknown function as well as that of actII-1, a TetR regulator repressing the expression of actII-2, encoding the ACT transporter, were down regulated in the WblI over-expressing strain. Consistently the expression of the transporter actII-2 was up-regulated. The expression of WblA, that is known to have a negative impact on ACT biosynthesis, was strongly down regulated in the WblI over-expressing strain. These data are consistent with the positive impact that WblI over-expression has on ACT biosynthesis. The latter might result from direct activation of ACT biosynthesis and export and from repression of the expression of WblA, a likely indirect, repressor of ACT biosynthesis.

Over 100 genome-scale metabolic networks (GSMNs) have been published in recent years and widely used for phenotype prediction and pathway design. However, GSMNs for a specific organism reconstructed by different research groups usually produce inconsistent simulation results, which makes it difficult to use the GSMNs for precise optimal pathway design. Therefore, it is necessary to compare and identify the discrepancies among networks and build a consensus metabolic network for an organism. Here we proposed a process for systematic comparison of metabolic networks at pathway level. We compared four published GSMNs of Pseudomonas putida KT2440 and identified the discrepancies leading to inconsistent pathway calculation results. The mistakes in the models were corrected based on information from literature so that all the calculated synthesis and uptake pathways were the same. Subsequently we built a pathway-consensus model and then further updated it with the latest genome annotation information to obtain modelPpuQY1140 for P. putida KT2440, which includes 1140 genes, 1171 reactions and 1104 metabolites. We found that even small errors in a GSMN could have great impacts on the calculated optimal pathways and thus may lead to incorrect pathway design strategies. Careful investigation of the calculated pathways during the metabolic network reconstruction process is essential for building proper GSMNs for pathway design.

The permanent draft genome sequence of Actinotignum schaalii DSM 15541T is presented. The annotated genome includes 2,130,987 bp, with 1777 protein-coding and 58 rRNA-coding genes. Genome sequence analysis revealed absence of genes encoding for: components of the PTS systems, enzymes of the TCA cycle, glyoxylate shunt and gluconeogensis. Genomic data revealed that A. schaalii is able to oxidize carbohydrates via glycolysis, the nonoxidative pentose phosphate and the Entner-Doudoroff pathways. Besides, the genome harbors genes encoding for enzymes involved in the conversion of pyruvate to lactate, acetate and ethanol, which are found to be the end products of carbohydrate fermentation. The genome contained the gene encoding Type I fatty acid synthase required for de novo FAS biosynthesis. The plsY and plsX genes encoding the acyltransferases necessary for phosphatidic acid biosynthesis were absent from the genome. The genome harbors genes encoding enzymes responsible for isoprene biosynthesis via the mevalonate (MVA) pathway. Genes encoding enzymes that confer resistance to reactive oxygen species (ROS) were identified. In addition, A. schaalii harbors genes that protect the genome against viral infections. These include restriction-modification (RM) systems, type II toxin-antitoxin (TA), CRISPR-Cas and abortive infection system. A. schaalii genome also encodes several virulence factors that contribute to adhesion and internalization of this pathogen such as the tad genes encoding proteins required for pili assembly, the nanI gene encoding exo-alpha-sialidase, genes encoding heat shock proteins and genes encoding type VII secretion system. These features are consistent with anaerobic and pathogenic lifestyles. Finally, resistance to ciprofloxacin occurs by mutation in chromosomal genes that encode the subunits of DNA-gyrase (GyrA) and topisomerase IV (ParC) enzymes, while resistant to metronidazole was due to the frxA gene, which encodes NADPH-flavin oxidoreductase.

Cropping soils vary in extent of natural suppression of soil-borne plant diseases. However, it is unknown whether similar variation occurs across pastoral agricultural systems. We examined soil microbial community properties known to be associated with disease suppression across 50 pastoral fields varying in management intensity. The composition and abundance of the disease-suppressive community were assessed from both taxonomic and functional perspectives. Pseudomonas bacteria were selected as a general taxonomic indicator of disease suppressive potential, while genes associated with the biosynthesis of a suite of secondary metabolites provided functional markers (GeoChip 5.0 microarray analysis). The composition of both the Pseudomonas communities and disease suppressive functional genes were responsive to land use. Underlying soil properties explained 37% of the variation in Pseudomonas community structure and up to 61% of the variation in the abundance of disease suppressive functional genes. Notably, measures of soil organic matter quality, C:P ratio, and aromaticity of the dissolved organic matter content (carbon recalcitrance), influenced both the taxonomic and functional disease suppressive potential of the pasture soils. Our results suggest that key components of the soil microbial community may be managed on-farm to enhance disease suppression and plant productivity.

The prevalence of lipids devoid of phosphorus suggests that the availability of phosphorus limits microbial growth and activity in many anoxic, stratified environments. To better understand the response of anaerobic bacteria to phosphate limitation and starvation, this study combines microscopic and lipid analyses with the measurements of fitness of pooled barcoded transposon mutants of the model sulfate reducing bacterium Desulfovibrio alaskensis G20. Phosphate-limited G20 has lower growth rates and replaces more than 90% of its membrane phospholipids by a mixture of monoglycosyl diacylglycerol (MGDG), glycuronic acid diacylglycerol (GADG) and ornithine lipids, lacks polyphosphate granules, and synthesizes other cellular inclusions. Analyses of pooled and individual mutants reveal the importance of the high-affinity phosphate transport system (the Pst system), PhoR, and glycolipid and ornithine lipid synthases during phosphate limitation. The phosphate-dependent synthesis of MGDG in G20 and the widespread occurrence of the MGDG/GADG synthase among sulfate reducing ∂-Proteobacteria implicate these microbes in the production of abundant MGDG in anaerobic environments where the concentrations of phosphate are lower than 10 μM. Numerous predicted changes in the composition of the cell envelope and systems involved in transport, maintenance of cytoplasmic redox potential, central metabolism and regulatory pathways also suggest an impact of phosphate limitation on the susceptibility of sulfate reducing bacteria to other anthropogenic or environmental stresses.

Previous experiments revealed that DHH1, a RNA helicase involved in the regulation of mRNA stability and translation, complemented the phenotype of a Saccharomyces cerevisiae mutant affected in the expression of genes coding for monocarboxylic-acids transporters, JEN1 and ADY2 (Paiva S, Althoff S, Casal M, Leao C. FEMS Microbiol Lett, 1999, 170:301-306). In wild type cells, JEN1 expression had been shown to be undetectable in the presence of glucose or formic acid, and induced in the presence of lactate. In this work, we show that JEN1 mRNA accumulates in a dhh1 mutant, when formic acid was used as sole carbon source. Dhh1 interacts with the decapping activator Dcp1 and with the deadenylase complex. This led to the hypothesis that JEN1 expression is post-transcriptionally regulated by Dhh1 in formic acid. Analyses of JEN1 mRNAs decay in wild-type and dhh1 mutant strains confirmed this hypothesis. In these conditions, the stabilized JEN1 mRNA was associated to polysomes but no Jen1 protein could be detected, either by measurable lactate carrier activity, Jen1-GFP fluorescence detection or western blots. These results revealed the complexity of the expression regulation of JEN1 in S. cerevisiae and evidenced the importance of DHH1 in this process. Additionally, microarray analyses of dhh1 mutant indicated that Dhh1 plays a large role in metabolic adaptation, suggesting that carbon source changes triggers a complex interplay between transcriptional and post-transcriptional effects., This study was supported by the Portuguese grant POCI/BIA-BCM/57812/2004 (Eixo 2, Medida 2.3, QCAIII - FEDER). N.V. received a FCT PhD fellowship (SFRH/BD/23503/2005). S.M. received a FCT PhD fellowship (SFRH/BD/74790/2010). F.D.'s work is supported by a grant from the Agence pour la Recherche contre le Cancer (ARC). Support to C.B.M.A. by FEDER through POFC-COMPETE and by Portuguese funds from FCT through the project PEst-OE/BIA/UI4050/2014 is also acknowledged. The authors thank Josette Banroques and Kyle Tanner for their advice regarding polysome gradients. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.