A broadly distributed predicted helicase/nuclease confers phage resistance via abortive infection.

There is strong selection for the evolution of systems that protect bacterial populations from viral attack. We report a single phage defense protein, Hna, that provides protection against diverse phages in Sinorhizobium meliloti, a nitrogen-fixing alpha-proteobacterium. Homologs of Hna are distributed widely across bacterial lineages, and a homologous protein from Escherichia coli also confers phage defense. Hna contains superfamily II helicase motifs at its N terminus and a nuclease motif at its C terminus, with mutagenesis of these motifs inactivating viral defense. Hna variably impacts phage DNA replication but consistently triggers an abortive infection response in which infected cells carrying the system die but do not release phage progeny. A similar host cell response is triggered in cells containing Hna upon expression of a phage-encoded single-stranded DNA binding protein (SSB), independent of phage infection. Thus, we conclude that Hna limits phage spread by initiating abortive infection in response to a phage protein.

Minimal gene set from Sinorhizobium (Ensifer) meliloti pSymA required for efficient symbiosis with Medicago.

Reduction of N2 gas to ammonia in legume root nodules is a key component of sustainable agricultural systems. Root nodules are the result of a symbiosis between leguminous plants and bacteria called rhizobia. Both symbiotic partners play active roles in establishing successful symbiosis and nitrogen fixation: while root nodule development is mostly controlled by the plant, the rhizobia induce nodule formation, invade, and perform N2 fixation once inside the plant cells. Many bacterial genes involved in the rhizobia-legume symbiosis are known, and there is much interest in engineering the symbiosis to include major nonlegume crops such as corn, wheat, and rice. We sought to identify and combine a minimal bacterial gene complement necessary and sufficient for symbiosis. We analyzed a model rhizobium, Sinorhizobium (Ensifer) meliloti, using a background strain in which the 1.35-Mb symbiotic megaplasmid pSymA was removed. Three regions representing 162 kb of pSymA were sufficient to recover a complete N2-fixing symbiosis with alfalfa, and a targeted assembly of this gene complement achieved high levels of symbiotic N2 fixation. The resulting gene set contained just 58 of 1,290 pSymA protein-coding genes. To generate a platform for future synthetic manipulation, the minimal symbiotic genes were reorganized into three discrete nod, nif, and fix modules. These constructs will facilitate directed studies toward expanding the symbiosis to other plant partners. They also enable forward-type approaches to identifying genetic components that may not be essential for symbiosis, but which modulate the rhizobium's competitiveness for nodulation and the effectiveness of particular rhizobia-plant symbioses.

A Key Regulator of the Glycolytic and Gluconeogenic Central Metabolic Pathways in Sinorhizobium meliloti.

The order Rhizobiales contains numerous agriculturally, biotechnologically, and medically important bacteria, including the rhizobia, and the genera Agrobacterium, Brucella, and Methylobacterium, among others. These organisms tend to be metabolically versatile, but there has been relatively little investigation into the regulation of their central carbon metabolic pathways. Here, RNA-sequencing and promoter fusion data are presented to show that the PckR protein is a key regulator of central carbon metabolism in Sinorhizobium meliloti; during growth with gluconeogenic substrates, PckR represses expression of the complete Entner-Doudoroff glycolytic pathway and induces expression of the pckA and fbaB gluconeogenic genes. Electrophoretic mobility shift assays indicate that PckR binds an imperfect palindromic sequence that overlaps the promoter or transcriptional start site in the negatively regulated promoters, or is present in tandem upstream the promoter motifs in the positively regulated promoters. Genetic and in vitro electrophoretic mobility shift assay experiments suggest that elevated concentrations of a PckR effector ligand results in the dissociation of PckR from its target binding site, and evidence is presented that suggests phosphoenolpyruvate may function as the effector. Characterization of missense pckR alleles identified three conserved residues important for increasing the affinity of PckR for its cognate effector molecule. Bioinformatics analyses illustrates that PckR is limited to a narrow phylogenetic range consisting of the Rhizobiaceae, Phyllobacteriaceae, Brucellaceae, and Bartonellaceae families. These data provide novel insights into the regulation of the core carbon metabolic pathways of this pertinent group of α-proteobacteria.

Calculating the Na⁺ translocating V-ATPase catalytic site affinity for substrate binding by homology modeled NtpA monomer using molecular dynamics/free energy calculation.

Vacuolar ATPase (V-ATPase) of Enterococcus hirae is composed of a soluble catalytic domain (V1; NtpA3-B3-D-G) and an integral membrane domain (V0; NtpI-K10) connected by a central and two peripheral stalks (NtpC, NtpD-G and NtpE-F). Recently nucleotide binding of catalytic NtpA monomer has been reported (Arai et al.). In the present study, we calculated the nucleotide binding affinity of NtpA by molecular dynamics (MD) simulation/free energy calculation using MM-GBSA approach based on homology modeled structure of NtpA monomer docked with ATP analogue, adenosine 5'-[ß, γ-imido] triphosphate (AMP-PNP). The calculated binding free energies showed qualitatively good agreement with experimental data. The calculation was cross-validated further by the rigorous method, thermodynamic integration (TI) simulation. Finally, the interaction between NtpA and nucleotides at the atomic level was investigated by the analyses of components of free energy and the optimized model structures obtained from MD simulations, suggesting that electrostatic contribution is responsible for the difference in nucleotide binding to NtpA monomer. This is the first observation and suggestion to explain the difference of nucleotide binding properties in V-ATPase NtpA subunit, and our method can be a valuable primary step to predict nucleotide binding affinity to other subunits (NtpAB, NtpA3B3) and to explore subunit interactions and eventually may help to understand energy transduction mechanism of E. hirae V-ATPase.