Juan Carlos Alonso

Visualization of DNA double‐strand break repair in live bacteria reveals dynamic recruitment of Bacillus subtilis RecF, RecO and RecN proteins to distinct sites on the nucleoids

We have found that SMC‐like RecN protein, RecF and RecO proteins that are involved in DNA recombination play an important role in DNA double‐strand break (DSB) repair in Bacillus subtilis. Upon induction of DNA DSBs, RecN, RecO and RecF localized as a discrete focus on the nucleoids in a majority of cells, whereas two or three foci were rarely observed. RecN, RecO and RecF co‐localized to the induced foci, with RecN localizing first, while RecO localized later, followed by RecF. Thus, three repair proteins were differentially recruited to distinct sites on the nucleoids, potentially constituting active DSB repair centres (RCs). RecF did not form regular foci in the absence of RecN and failed to form any foci in recO cells, demonstrating a central role for RecN and RecO in initializing the formation of RCs. RecN/O/F foci were detected in recA, recG or recU mutant cells, indicating that the proteins act upstream of proteins involved in synapsis or post‐synapsis. In the absence of exogenous DNA damage, RCs were rare, but they accumulated in recA and recU cells, suggesting that DSBs occur frequently in the absence of RecA or RecU. The results suggest a model in which RecN that forms multimers in solution and high‐molecular‐weight complexes in cells containing DSBs initiates the formation of RCs that mediate DSB repair with the homologous sister chromosome, which presents a novel concept for DSB repair in prokaryotes.

Crystal structure of the plasmid maintenance system epsilon/zeta: functional mechanism of toxin zeta and inactivation by epsilon 2 zeta 2 complex formation.

Programmed cell death in prokaryotes is frequently found as postsegregational killing. It relies on antitoxin/toxin systems that secure stable inheritance of low and medium copy number plasmids during cell division and kill cells that have lost the plasmid. The broad-host-range, low-copy-number plasmid pSM19035 from Streptococcus pyogenes carries the genes encoding the antitoxin/toxin system ɛ/ζ and antibiotic resistance proteins, among others. The crystal structure of the biologically nontoxic ɛ2ζ2 protein complex at a 1.95-Å resolution and site-directed mutagenesis showed that free ζ acts as phosphotransferase by using ATP/GTP. In ɛ2ζ2, the toxin ζ is inactivated because the N-terminal helix of the antitoxin ɛ blocks the ATP/GTP-binding site. To our knowledge, this is the first prokaryotic postsegregational killing system that has been entirely structurally characterized.

Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family

The Lrp/AsnC family of transcriptional regulatory proteins is found in both archaea and bacteria. Members of the family influence cellular metabolism in both a global (Lrp) and specific (AsnC) manner, often in response to exogenous amino acid effectors. In the present study we have determined both the first bacterial and the highest resolution structures for members of the family. Escherichia coli AsnC is a specific gene regulator whose activity is triggered by asparagine binding. Bacillus subtilis LrpC is a global regulator involved in chromosome condensation. Our AsnC-asparagine structure is the first for a regulator–effector complex and is revealed as an octameric disc. Key ligand recognition residues are identified together with a route for ligand access. The LrpC structure reveals a stable octamer supportive of a topological role in dynamic DNA packaging. The structures yield significant clues to the functionality of Lrp/AsnC-type regulators with respect to ligand binding and oligomerization states as well as to their role in specific and global DNA regulation.

A field test of ideal free distribution in flock-feeding common cranes

This is a contribution to Project PB91-0081 of the Direccion General de Investigacion Cientifica y Tecnica.

Functional Analysis of the Terminase Large Subunit, G2P, of Bacillus subtilis Bacteriophage SPP1

The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities. H6-G2P introduces a cut with preference at the 5′-RCGG↓CW-3′ sequence. Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at bothbona fide 5′-CTATTGCGG↓C-3′ sequences withinpacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and Pi. H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1PΔN62 (lacking domain I) stimulate 3.5- and 1.9-fold, respectively, the ATPase activity of H6-G2P. The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC. In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated. Based on these results, we propose a model that can account for the role of terminase in headful packaging.

In vitro and in vivo stability of the epsilon2zeta2 protein complex of the broad host-range Streptococcus pyogenes pSM19035 addiction system

Abstract Streptococcus pyogenes pSM19035-encoded (10.7 kDa) and ζ (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with ζ acting as toxin that kills plasmiddeprived cells and as an antitoxin that neutralises the activity of ζ. The and ζ proteins copurify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as 2ζ2 heterotetramer in solution. Cocrystals of the 2ζ2 complex contain and ζ in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the ζ protein has a significantly lower thermodynamic stability than the protein both in free state and in the complex. Proteolytic studies indicate that ζ protein is more stable in the the 2ζ2 complex than in the free state. In vivo studies reveal a short halflife of the antitoxin (~18min) and a long lifetime of the ζ toxin (>60min). When transcriptiontranslation of a plasmid containing the and ζ genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the protein from the background.

Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination

ABSTRACT The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (β-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly orientedsix sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding β-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the β-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation.

Patch use in cranes: a field test of optimal foraging predictions

Abstract The marginal value theorem states that foragers should leave patches when the instantaneous capture rate has fallen to the average capture rate for the habitat. This predicts that patch residence time should increase with increasing patch quality and decrease with increasing habitat quality. These and other predictions from prescient, Bayesian and fixed-time models were tested using observations on 14 radio-tagged free-living common cranes,Grus grus, foraging in cereal farmland. Cranes behaved as Bayesian foragers. Their intake rates on leaving patches changed with patch and habitat quality. The behaviour of cranes was consistent with marginal value theorem predictions only in patches where energy return was lower than required to meet daily food requirements. In contrast, birds left richer patches earlier than expected and at higher intake rates than poor patches. In addition, cranes stayed longer in larger flocks. These results suggest that cranes changed their foraging rules according to their expected energy balance.

Mitigation of bird collisions with transmission lines through groundwire marking

Abstract A study was conducted to evaluate the effectiveness of groundwire marking in reducing bird mortality through collision at a power transmission line in southwestern Spain. Monthly flight intensity observations and weekly searches for dead birds were carried out at four sectors of the line comprising 28·2 km, during two consecutive winters, 1989–1990 and 1990–1991, respectively before and after groundwire marking with coloured PVC spirals. Flight intensity and collision frequency decreased respectively by 61% and 60% at marked spans compared to the same spans prior to marking, while there was no significant change in collision frequency at spans left unmarked. After marking, the percentage of birds flying between the cables decreased and that flying above them increased. Our results suggest that many birds avoided flying across the marked spans of the line or climbed while approaching them, and therefore collided less frequently. The percentage decrease in mortality observed in our study falls within the range of results of other groundwire marking or removal studies.

Bacillus subtilis Bacteriophage SPP1 DNA Packaging Motor Requires Terminase and Portal Proteins

Initiation of headful packaging of SPP1 DNA concatemers involves the interaction of the terminase, G1P and G2P, and the portal protein, G6P. G1P, which specifically recognizes the non-adjacent pacL and pacR subsites and directs loading of G2P to pacC, interacts with G6P. G2P, which has endonuclease, DNA binding, and ATPase activities, interacts with G1P and does it transiently with G6P. The stoichiometry of G1P on the G1P·G2P complex promotes the transition from a G2P endonuclease to an ATPase. G6P does not alter the endonuclease activity of G2P. Both G1P and G6P, which do not have endogenous ATPase activity, synergistically enhance and modulate the ATPase activity of G2P. Based on these results, we propose a model in which the modulation of the ATPase and endonuclease activities of G2P accounts for the role of the terminase in headful packaging.