Geographically structured genetic variation in the Medicago lupulina–Ensifer mutualism

Gene flow between genetically differentiated populations can maintain variation in species interactions, especially when population structure is congruent between interacting species. However, large‐scale empirical comparisons of the population structure of interacting species are rare, particularly in positive interspecific interactions (mutualisms). One agriculturally and ecologically important mutualism is the partnership between legume plants and rhizobia. Through characterizing and comparing the population genomic structure of the legume Medicago lupulina and two rhizobial species (Ensifer medicae and E. meliloti), we explored the spatial scale of population differentiation between interacting partners in their introduced range in North America. We found high proportions of E. meliloti in southeastern populations and high proportions of E. medicae in northwestern populations. Medicago lupulina and the Ensifer genus showed similar patterns of spatial genetic structure (isolation by distance). However, we detected no evidence of isolation by distance or population structure within either species of bacteria. Genome‐wide nucleotide diversity within each of the two Ensifer species was low, suggesting limited introduction of strains, founder events, or severe bottlenecks. Our results suggest that there is potential for geographically structured coevolution between M. lupulina and the Ensifer genus, but not between M. lupulina and either Ensifer species.     

A nodule endophytic Bacillus megaterium strain isolated from Medicago polymorpha enhances growth,promotes nodulation by Ensifer medicae and alleviates salt stress in alfalfa plants

A Gram‐positive, fast‐growing, endophytic bacterium was isolated from root nodules of Medicago polymorpha and identified as Bacillus megaterium. The isolate, named NMp082, co‐inhabited nodules with the symbiotic rhizobium Ensifer medicae. B. megaterium NMp082 contained nifH and nodD genes that were 100% identical to those of Ensifer meliloti, an unusual event that suggested previous lateral gene transfer from a different rhizobial species. Despite the presence of nodulation and nitrogen fixation genes, the endophyte was not able to form effective nodules; however, it induced nodule‐like unorganised structures in alfalfa roots. Axenic inoculation promoted plant growth in M. polymorpha, Medicago lupulina, Medicago truncatula and Medicago sativa, and co‐inoculation with E. medicae enhanced growth and nodulation of Medicago spp. plants compared with inoculation with either bacterium alone. B. megaterium NMp082 also induced tolerance to salt stress in alfalfa and Arabidopsis plants. The ability to produce indole acetic acid (IAA) and the 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase activity displayed by the endophyte in vitro might explain the observed plant growth promotion and salt stress alleviation. The isolate was also highly tolerant to salt stress, water deficit and to the presence of different heavy metals. The newly characterised endophytic bacterium possessed specific characteristics that point at potential applications to sustain plant growth and nodulation under abiotic stress.     

Virulence of Agrobacterium tumefaciens requires lipid homeostasis mediated by the lysyl‐phosphatidylglycerol hydrolase AcvB

Agrobacterium tumefaciens transfers oncogenic T‐DNA via the type IV secretion system (T4SS) into plants causing tumor formation. The acvB gene encodes a virulence factor of unknown function required for plant transformation. Here we specify AcvB as a periplasmic lysyl‐phosphatidylglycerol (L‐PG) hydrolase, which modulates L‐PG homeostasis. Through functional characterization of recombinant AcvB variants, we showed that the C‐terminal domain of AcvB (residues 232–456) is sufficient for full enzymatic activity and defined key residues for catalysis. Absence of the hydrolase resulted in ~10‐fold increase in L‐PG in Agrobacterium membranes and abolished T‐DNA transfer and tumor formation. Overproduction of the L‐PG synthase gene (lpiA) in wild‐type A. tumefaciens resulted in a similar increase in the L‐PG content (~7‐fold) and a virulence defect even in the presence of intact AcvB. These results suggest that elevated L‐PG amounts (either by overproduction of the synthase or absence of the hydrolase) are responsible for the virulence phenotype. Gradually increasing the L‐PG content by complementation with different acvB variants revealed that cellular L‐PG levels above 3% of total phospholipids interfere with T‐DNA transfer. Cumulatively, this study identified AcvB as a novel virulence factor required for membrane lipid homeostasis and T‐DNA transfer.     

Characterization of root-nodulating bacteria associated to <Emphasis Type="Italic">Prosopis farcta</Emphasis> growing in the arid regions of Tunisia

Diversity of 50 bacterial isolates recovered from root nodules of Prosopis farcta grown in different arid soils in Tunisia, was investigated. Characterization of isolates was assessed using a polyphasic approach including phenotypic characteristics, 16S rRNA gene PCR–RFLP and sequencing, nodA gene sequencing and MLSA. It was found that most of isolates are tolerant to high temperature (40°C) and salinity (3%). Genetic characterization emphasizes that isolates were assigned to the genus Ensifer (80%), Mesorhizobium (4%) and non-nodulating endophytic bacteria (16%). Forty isolates belonging to the genus Ensifer were affiliated to Ensifer meliloti, Ensifer xinjiangense/Ensifer fredii and Ensifer numidicus species. Two isolates belonged to the genus Mesorhizobium. Eight isolates failing to renodulate their host plant were endophytic bacteria and belonged to Bacillus, Paenibacillus and Acinetobacter genera. Symbiotic properties of nodulating isolates showed a diversity in their capacity to infect their host plant and fix atmospheric nitrogen. Isolate PG29 identified as Ensifer meliloti was the most effective one. Ability of Prosopis farcta to establish symbiosis with rhizobial species confers an important advantage for this species to be used in reforestation programs. This study offered the first systematic information about the diversity of microsymbionts nodulating Prosopis farcta in the arid regions of Tunisia.     

Novel putative Mesorhizobium and Ensifer genomospecies together with a novel symbiovar psoraleae nodulate legumes of agronomic interest grown in Tunisia

Forty rhizobial strains were isolated from Lotus creticus, L. pusillus and Bituminaria bituminosa endemic to Tunisia, and they belonged to the Mesorhizobium and Ensifer genera based on 16S rDNA sequence phylogeny. According to the concatenated recA and glnII sequence-based phylogeny, four Bituminaria isolates Pb5, Pb12, Pb8 and Pb17 formed a monophyletic group with Mesorhizobium chacoense ICMP14587^T, whereas four other strains Pb1, Pb6, Pb13 and Pb15 formed two separate lineages within the Ensifer genus. Among the L. pusillus strains, Lpus9 and Lpus10 showed a 96% identical nucleotide with Ensifer meliloti CCBAU83493^T; whereas six other strains could belong to previously undescribed Mesorhizobium and Ensifer species. For L. creticus strains, Lcus37, Lcus39 and Lcus44 showed 98% sequence identity with Ensifer aridi JNVU TP6, and Lcus42 shared a 96% identical nucleotide with Ensifer meliloti CCBAU83493^T; whereas another four strains were divergent from all the described Ensifer and Mesorhizobium species. The analysis of the nodC gene-based phylogeny identified four symbiovar groups; Mesorhizobium sp. sv. anthyllidis (Lpus3 and Lpus11 from L. pusillus, Lcus43 from L. creticus), Ensifer medicae sv. meliloti (four strains from L. creticus and two strains from L. pusillus), E. meliloti sv. meliloti (four from L. creticus, four from L. pusillus and four from B. bituminosa). In addition, four B. bituminosa strains (Pb5, Pb8, Pb12, and Pb17) displayed a distinctive nodC sequence distant from those of other symbiovars described to date. According to their symbiotic gene sequences and host range, the B. bituminosa symbionts (Pb5, Pb8, Pb12 and Pb17) would represent a new symbiovar of M. chacoense for which sv. psoraleae is proposed.     

Abrogation of ospAB constitutively activates the Rrp2‐RpoN‐RpoS pathway (sigmaN‐sigmaS cascade) in Borrelia burgdorferi

Molecular mechanisms underlying the reciprocal regulation of the two major surface lipoproteins and virulence factors of Borrelia burgdorferi, OspA and OspC, are not fully understood. Herein, we report that inactivation of the ospAB operon resulted in overproduction of OspC and many other lipoproteins via the constitutive activation of the Rrp2‐RpoN‐RpoS pathway. Complementing the ospAB mutant with a wild‐type copy of ospA, but not an ospA variant that lacks the lipoprotein signal sequence, restored normal regulation of the Rrp2‐RpoN‐RpoS pathway; these results indicate that the phenotype was not caused by spurious mutations. Interestingly, while most of the ospAB mutant clones displayed a constitutive ospC expression phenotype, some ospAB mutant clones showed little or no ospC expression. Further analyses revealed that this OspC‐negative phenotype was independent of abrogation of ospAB. While activation of the Rrp2‐RpoN‐RpoS pathway was recently shown to downregulate ospA, our findings suggest that reduction of OspA can also activate this pathway. We postulate that the activation of the Rrp2‐RpoN‐RpoS pathway and downregulation of OspA form a positive feedback loop that allows spirochaetes to produce and maintain a constant high level of OspC and other lipoproteins during tick feeding, a strategy that is critical for spirochaetal transmission and mammalian infection.     

Structure and expression of the alternative sigma factor, RpoN, in Rhodobacter capsulatus; physiological relevance of an autoactivated nifU2-rpoN superoperon

The alternative sigma factor, RpoN (σ⁵⁴) is responsible for recruiting core RNA polymerase to the promoters of genes required for diverse physiological functions In a variety of eubacterial species. The RpoN protein In Rhodobacter capsulatus is a putative sigma factor specific for nitrogen fixation (nif) genes. Insertional mutagenesis was used to define regions important for the function of the R. capsulatus RpoN protein. Insertions of four amino acids in the predicted helix-turn-helix or in the highly conserved C-terminal eight amino acid residues (previously termed the RpoN box), and an in-frame deletion of the glutamine-rich M-terminus completely inactivated the R. capsulatus RpoN protein. Two separate insertions in the second hydrophobic heptad repeat, a putative leucine zipper, resulted in a partially functional RpoN protein. Eight other linkers in the rpoN open reading frame (ORF) resulted in a completeiy or partially functional RpoN protein. The rpoN gene in R capsulatus is downstream from the nifHDKU2 genes, in a nifU2-rpoN operon. Results of genetic experiments on the nifU2-rpoN locus show that the rpoN gene is organized in a nifU2-rpoN superoperon. A primary promoter directly upstream of the rpoN ORF is responsible for the initial expression of rpoN. Deletion analysis and insertional mutagenesis were used to define the primary promoter to 50 bp, between 37 and 87 nucleotides upstream of the predicted rpoN translational start site. This primary promoter is expressed constitutively with respect to nitrogen, and it is necessary and sufficient for growth under nitrogen-limiting conditions typically used in the laboratory. A secondary promoter upstream of nifU2 is autoactivated by RpoN and NifA to increase the expression of rpoN, which ultimately results in higher expression of RpoN dependent genes. Moreover. rpoN expression from this secondary promoter is physiologically beneficial under certain stressful conditions, such as nitrogen-limiting environments that contain high salt (>50mM NaCl) or low iron (<400nM FeS0₄).     

Establishing microbial co‐cultures for 3‐hydroxybenzoic acid biosynthesis on glycerol

Converting renewable feedstocks to aromatic compounds using engineered microbes offers a robust approach for sustainable, environment‐friendly, and cost‐effective production of these value‐added products without the reliance on petroleum. In this study, rationally designed E. coli–E. coli co‐culture systems were established for converting glycerol to 3‐hydroxybenzoic acid (3HB). Specifically, the 3HB pathway was modularized and accommodated by two metabolically engineered E. coli strains. The co‐culture biosynthesis was optimized by using different cultivation temperatures, varying the inoculum ratio between the co‐culture strains, recruitment of a key pathway intermediate transporter, strengthening the critical pathway enzyme expression, and adjusting the timing for inducing pathway gene expression. Compared with the E. coli mono‐culture, the optimized co‐culture showed 5.3‐fold improvement for 3HB biosynthesis. This study demonstrated the applicability of modular co‐culture engineering for addressing the challenges of aromatic compound biosynthesis.     

RPW8 promoter is involved in pathogen‐And stress‐inducible expression from Vitis pseudoreticulata

Based on previous cloning of VpRPW8‐e, we obtained a 1,126 bp VpRPW8‐e promoter sequence in this study. A large number of TATA‐boxes, CAAT‐boxes, and other cis‐acting elements were predicted including light‐responsive elements, hormone‐responsive elements, stress‐responsive elements, and growth‐ and development‐associated elements within the promoter sequence. To further investigate the function of this promoter, we examined its activity in response to biotic and abiotic stress. The VpRPW8‐e promoter was strongly activated by Plasmopara viticola infection, and activation also occurred when the orientation of the promoter was reversed, although to a lesser extent. Deletion analysis showed that the ?1,126 to ?475 bp region of VpRPW8‐e promoter had high activity. A promoter fragment 5′ deleted to ?475 bp (P_?475) was activated in response to heat and cold stress, and even more strongly in response to Phytophthora capsici and salicylic acid (SA). Furthermore, Transgenic Nicotiana benthamiana were generated, VpRPW8‐e driven by P_?475 enhanced resistance to Ph. capsici in N. benthamiana. Based on these results, the ?475 bp region was deduced to be an indispensable part of the VpRPW8‐e promoter. VpRPW8‐e promoter is involved in pathogen‐ and stress‐inducible expression.     

Borrelia burgdorferi genes,bb0639‐0642, encode a putative putrescine/spermidine transport system,PotABCD, that is spermidine specific and essential for cell survival

Polyamines are an essential class of metabolites found throughout all kingdoms in life. Borrelia burgdorferi harbors no enzymes to synthesize or degrade polyamines yet does contain a polyamine uptake system, potABCD. In this report, we describe the initial characterization of this putative transport system. After several unsuccessful attempts to inactivate potABCD, we placed the operon under the control of an inducible LacI promoter expression system. Analyses of this construct confirmed that potABCD was required for in vitro survival. Additionally, we demonstrated that the potABCD operon were upregulated in vitro by low osmolarity. Previously, we had shown that low osmolarity triggers the activation of the Rrp2/RpoN/RpoS regulatory cascade, which regulates genes essential for the transmission of spirochetes from ticks to mammalian hosts. Interestingly, induction of the pot operon was only affected in an rpoS mutant but not in a rpoN mutant, suggesting that the genes were RpoS dependent and RpoN independent. Furthermore, potABCD was upregulated during tick feeding concomitant with the initiation of spirochete replication. Finally, uptake experiments determined the specificity of B. burgdorferi's PotABCD for spermidine.