Search for novel targets of the PII signal transduction protein in Bacteria identifies the BCCP component of acetyl‐CoA carboxylase as a PII binding partner

The P_II family comprises a group of widely distributed signal transduction proteins. The archetypal function of P_II is to regulate nitrogen metabolism in bacteria. As P_II can sense a range of metabolic signals, it has been suggested that the number of metabolic pathways regulated by P_II may be much greater than described in the literature. In order to provide experimental evidence for this hypothesis a P_II protein affinity column was used to identify P_II targets in Azospirillum brasilense. One of the P_II partners identified was the biotin carboxyl carrier protein (BCCP), a component of the acetyl‐CoA carboxylase which catalyses the committed step in fatty acid biosynthesis. As BCCP had been previously identified as a P_II target in Arabidopsis thaliana we hypothesized that the P_II–BCCP interaction would be conserved throughout Bacteria. In vitro experiments using purified proteins confirmed that the P_II–BCCP interaction is conserved in Escherichia coli. The BCCP–P_II interaction required MgATP and was dissociated by increasing 2‐oxoglutarate. The interaction was modestly affected by the post‐translational uridylylation status of P_II; however, it was completely dependent on the post‐translational biotinylation of BCCP.     

Exploring a Pool‐seq‐only approach for gaining population genomic insights in nonmodel species

Developing genomic insights is challenging in nonmodel species for which resources are often scarce and prohibitively costly. Here, we explore the potential of a recently established approach using Pool‐seq data to generate a de novo genome assembly for mining exons, upon which Pool‐seq data are used to estimate population divergence and diversity. We do this for two pairs of sympatric populations of brown trout (Salmo trutta): one naturally sympatric set of populations and another pair of populations introduced to a common environment. We validate our approach by comparing the results to those from markers previously used to describe the populations (allozymes and individual‐based single nucleotide polymorphisms [SNPs]) and from mapping the Pool‐seq data to a reference genome of the closely related Atlantic salmon (Salmo salar). We find that genomic differentiation (F_ST) between the two introduced populations exceeds that of the naturally sympatric populations (F_ST = 0.13 and 0.03 between the introduced and the naturally sympatric populations, respectively), in concordance with estimates from the previously used SNPs. The same level of population divergence is found for the two genome assemblies, but estimates of average nucleotide diversity differ ( ≈ 0.002 and  ≈ 0.001 when mapping to S. trutta and S. salar, respectively), although the relationships between population values are largely consistent. This discrepancy might be attributed to biases when mapping to a haploid condensed assembly made of highly fragmented read data compared to using a high‐quality reference assembly from a divergent species. We conclude that the Pool‐seq‐only approach can be suitable for detecting and quantifying genome‐wide population differentiation, and for comparing genomic diversity in populations of nonmodel species where reference genomes are lacking.     

The type II secretion system (Xcp) of Pseudomonas putida is active and involved in the secretion of phosphatases

The genome of the Gram‐negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these genes is induced under inorganic phosphate (P_i) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low P_i conditions and transported across the cell envelope in an Xcp‐dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin‐arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P. putida genome, of which one, named tat‐1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat‐1 genes was strongly induced by low P_i levels, indicating a function of this system in survival during P_i starvation.     

Inhibition of photosynthesis by high temperature in oak (Quercus pubescens L.) leaves grown under natural conditions closely correlates with a reversible heat-dependent reduction of the activation state of ribulose-1,5-bisphosphate carboxylase/oxygenase

Inhibition of the net photosynthetic CO₂ assimilation rate (P_n) by high temperature was examined in oak (Quercus pubescens L.) leaves grown under natural conditions. Combined measurements of gas exchange and chlorophyll (Chl) a fluorescence were employed to differentiate between inhibition originating from heat effects on components of the thylakoid membranes and that resulting from effects on photosynthetic carbon metabolism. Regardless of whether temperature was increased rapidly or gradually, P_n decreased with increasing leaf temperature and was more than 90% reduced at 45 °C as compared to 25 °C. Inhibition of P_n by heat stress did not result from reduced stomatal conductance (g_s), as heat‐induced reduction of g_s was accompanied by an increase of the intercellular CO₂ concentration (C_i). Chl a fluorescence measurements revealed that between 25 and 45 °C heat‐dependent alterations of thylakoid‐associated processes contributed only marginally, if at all, to the inhibition of P_n by heat stress, with photosystem II being remarkably well protected against thermal inactivation. The activation state of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) decreased from about 90% at 25 °C to less than 30% at 45 °C. Heat stress did not affect Rubisco per se, since full activity could be restored by incubation with CO₂ and Mg²⁺. Western‐blot analysis of leaf extracts disclosed the presence of two Rubisco activase polypeptides, but heat stress did not alter the profile of the activase bands. Inhibition of P_n at high leaf temperature could be markedly reduced by artificially increasing C_i. A high C_i also stimulated photosynthetic electron transport and resulted in reduced non‐photochemical fluorescence quenching. Recovery experiments showed that heat‐dependent inhibition of P_n was largely, if not fully, reversible. The present results demonstrate that in Q. pubescens leaves the thylakoid membranes in general and photosynthetic electron transport in particular were well protected against heat‐induced perturbations and that inhibition of P_n by high temperature closely correlated with a reversible heat‐dependent reduction of the Rubisco activation state.     

The CarD/CarG regulatory complex is required for the action of several members of the large set of Myxococcus xanthus extracytoplasmic function σ factors

Extracytoplasmic function (ECF) σ factors are critical players in signal transduction networks involved in bacterial response to environmental changes. The Myxococcus xanthus genome reveals ～45 putative ECF‐σ factors, but for the overwhelming majority, the specific signals or mechanisms for selective activation and regulation remain unknown. One well‐studied ECF‐σ, CarQ, binds to its anti‐σ, CarR, and is inactive in the dark but drives its own expression from promoter P_QRS on illumination. This requires the CarD/CarG complex, the integration host factor (IHF) and a specific CarD‐binding site upstream of P_QRS. Here, we show that DdvS, a previously uncharacterized ECF‐σ, activates its own expression in a CarD/CarG‐dependent manner but is inhibited when specifically bound to the N‐terminal zinc‐binding anti‐σ domain of its cognate anti‐σ, DdvA. Interestingly, we find that the autoregulatory action of 11 other ECF‐σ factors studied here depends totally or partially on CarD/CarG but not IHF. In silico analysis revealed possible CarD‐binding sites that may be involved in direct regulation by CarD/CarG of target promoter activity. CarD/CarG‐linked ECF‐σ regulation likely recurs in other myxobacteria with CarD/CarG orthologous pairs and could underlie, at least in part, the global regulatory effect of the complex on M. xanthus gene expression.     

Tolerance to oxidative stress is required for maximal xylem colonization by the xylem‐limited bacterial phytopathogen,Xylella fastidiosa

Bacterial plant pathogens often encounter reactive oxygen species (ROS) during host invasion. In foliar bacterial pathogens, multiple regulatory proteins are involved in the sensing of oxidative stress and the activation of the expression of antioxidant genes. However, it is unclear whether xylem‐limited bacteria, such as Xylella fastidiosa, experience oxidative stress during the colonization of plants. Examination of the X. fastidiosa genome uncovered only one homologue of oxidative stress regulatory proteins, OxyR. Here, a knockout mutation in the X. fastidiosa oxyR gene was constructed; the resulting strain was significantly more sensitive to hydrogen peroxide (H₂O₂) relative to the wild‐type. In addition, during early stages of grapevine infection, the survival rate was 1000‐fold lower for the oxyR mutant than for the wild‐type. This supports the hypothesis that grapevine xylem represents an oxidative environment and that X. fastidiosa must overcome this challenge to achieve maximal xylem colonization. Finally, the oxyR mutant exhibited reduced surface attachment and cell–cell aggregation and was defective in biofilm maturation, suggesting that ROS could be a potential environmental cue stimulating biofilm development during the early stages of host colonization.     

Co‐ordinated functions of Mms proteins define the surface structure of cubo‐octahedral magnetite crystals in magnetotactic bacteria

Magnetotactic bacteria synthesize magnetosomes comprised of membrane‐enveloped single crystalline magnetite (Fe₃O₄). The size and morphology of the nano‐sized magnetite crystals (< 100 nm) are highly regulated and bacterial species dependent. However, the control mechanisms of magnetite crystal morphology remain largely unknown. The group of proteins, called Mms (Mms5, Mms6, Mms7, and Mms13), was previously isolated from the surface of cubo‐octahedral magnetite crystals in Magnetospirillum magneticum strain AMB‐1. Analysis of an mms6 gene deletion mutant suggested that the Mms6 protein plays a major role in the regulation of magnetite crystal size and morphology. In this study, we constructed various mms gene deletion mutants and characterized the magnetite crystals formed by the mutant strains. Comparative analysis showed that all mms genes were involved in the promotion of crystal growth in different manners. The phenotypic characterization of magnetites also suggested that these proteins are involved in controlling the geometries of the crystal surface structures. Thus, the co‐ordinated functions of Mms proteins regulate the morphology of the cubo‐octahedral magnetite crystals in magnetotactic bacteria.     

Genome‐wide patterns of latitudinal differentiation among populations of Drosophila melanogaster from North America

Understanding the genetic underpinnings of adaptive change is a fundamental but largely unresolved problem in evolutionary biology. Drosophila melanogaster, an ancestrally tropical insect that has spread to temperate regions and become cosmopolitan, offers a powerful opportunity for identifying the molecular polymorphisms underlying clinal adaptation. Here, we use genome‐wide next‐generation sequencing of DNA pools (‘pool‐seq’) from three populations collected along the North American east coast to examine patterns of latitudinal differentiation. Comparing the genomes of these populations is particularly interesting since they exhibit clinal variation in a number of important life history traits. We find extensive latitudinal differentiation, with many of the most strongly differentiated genes involved in major functional pathways such as the insulin/TOR, ecdysone, torso, EGFR, TGFβ/BMP, JAK/STAT, immunity and circadian rhythm pathways. We observe particularly strong differentiation on chromosome 3R, especially within the cosmopolitan inversion In(3R)Payne, which contains a large number of clinally varying genes. While much of the differentiation might be driven by clinal differences in the frequency of In(3R)P, we also identify genes that are likely independent of this inversion. Our results provide genome‐wide evidence consistent with pervasive spatially variable selection acting on numerous loci and pathways along the well‐known North American cline, with many candidates implicated in life history regulation and exhibiting parallel differentiation along the previously investigated Australian cline.