Natural bacterial transformation involves the internalization and chromosomal integration of DNA and has now been documented in ~80 species. Recent advances have established that phylogenetically distant species share conserved uptake and processing proteins but differ in the inducing cues and regulatory mechanisms that are involved. In this Review, we highlight divergent and common principles that govern the transformation process in different bacteria. We discuss how this cumulative knowledge enables the prediction of new transformable species and supports the idea that the main role of internalized DNA is in the generation of genetic diversity or in chromosome repair rather than in nutrition.

Bacterial transformation: distribution, shared mechanisms and divergent control

Lactic acid bacteria (LAB) are important starter, commensal, or pathogenic microorganisms. The stress physiology of LAB has been studied in depth for over 2 decades, fueled mostly by the technological implications of LAB robustness in the food industry. Survival of probiotic LAB in the host and the potential relatedness of LAB virulence to their stress resilience have intensified interest in the field. Thus, a wealth of information concerning stress responses exists today for strains as diverse as starter (e.g., Lactococcus lactis), probiotic (e.g., several Lactobacillus spp.), and pathogenic (e.g., Enterococcus and Streptococcus spp.) LAB. Here we present the state of the art for LAB stress behavior. We describe the multitude of stresses that LAB are confronted with, and we present the experimental context used to study the stress responses of LAB, focusing on adaptation, habituation, and cross-protection as well as on self-induced multistress resistance in stationary phase, biofilms, and dormancy. We also consider stress responses at the population and single-cell levels. Subsequently, we concentrate on the stress defense mechanisms that have been reported to date, grouping them according to their direct participation in preserving cell energy, defending macromolecules, and protecting the cell envelope. Stress-induced responses of probiotic LAB and commensal/pathogenic LAB are highlighted separately due to the complexity of the peculiar multistress conditions to which these bacteria are subjected in their hosts. Induction of prophages under environmental stresses is then discussed. Finally, we present systems-based strategies to characterize the "stressome" of LAB and to engineer new food-related and probiotic LAB with improved stress tolerance.

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Stress Physiology of Lactic Acid Bacteria

Structural Biochemistry of a Bacterial Checkpoint Protein Reveals Diadenylate Cyclase Activity Regulated by DNA Recombination Intermediates

The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes.

Regulation of bacterial RecA protein function.

In the ancient anaerobic environment, ferrous iron (Fe2+) was one of the first metal cofactors. Oxygenation of the ancient world challenged bacteria to acquire the insoluble ferric iron (Fe3+) and later to defend against reactive oxygen species (ROS) generated by the Fenton chemistry. To acquire Fe3+, bacteria produce low-molecular weight compounds, known as siderophores, which have extremely high affinity for Fe3+. However, during infection the host restricts iron from pathogens by producing iron- and siderophore-chelating proteins, by exporting iron from intracellular pathogen-containing compartments, and by limiting absorption of dietary iron. Ferric Uptake Regulator (Fur) is a transcription factor which utilizes Fe2+ as a corepressor and represses siderophore synthesis in pathogens. Fur, directly or indirectly, controls expression of enzymes that protect against ROS damage. Thus, the challenges of iron homeostasis and defense against ROS are addressed via Fur. Although the role of Fur as a repressor is well documented, emerging evidence demonstrates that Fur can function as an activator. Fur activation can occur through three distinct mechanisms 1) indirectly via small RNAs, 2) binding at cis regulatory elements that enhance recruitment of the RNA polymerase holoenzyme (RNAP), and 3) functioning as an antirepressor by removing or blocking DNA binding of a repressor of transcription. In addition, Fur homologs control defense against peroxide stress (PerR) and control uptake of other metals such as zinc (Zur) and manganese (Mur) in pathogenic bacteria. Fur family members are important for virulence within bacterial pathogens since mutants of fur, perR, or zur exhibit reduced virulence within numerous animal and plant models of infection. This review focuses on the breadth of Fur regulation in pathogenic bacteria.

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Transcriptional Regulation by Ferric Uptake Regulator (Fur) in Pathogenic Bacteria

In all living organisms, the response to double-strand breaks (DSBs) is critical for the maintenance of chromosome integrity. Homologous recombination (HR), which utilizes a homologous template to prime DNA synthesis and to restore genetic information lost at the DNA break site, is a complex multistep response. In Bacillus subtilis, this response can be subdivided into five general acts: (1) recognition of the break site(s) and formation of a repair center (RC), which enables cells to commit to HR; (2) end-processing of the broken end(s) by different avenues to generate a 3'-tailed duplex and RecN-mediated DSB 'coordination'; (3) loading of RecA onto single-strand DNA at the RecN-induced RC and concomitant DNA strand exchange; (4) branch migration and resolution, or dissolution, of the recombination intermediates, and replication restart, followed by (5) disassembly of the recombination apparatus formed at the dynamic RC and segregation of sister chromosomes. When HR is impaired or an intact homologous template is not available, error-prone nonhomologous end-joining directly rejoins the two broken ends by ligation. In this review, we examine the functions that are known to contribute to DNA DSB repair in B. subtilis, and compare their properties with those of other bacterial phyla.

Double-strand break repair in bacteria: a view from Bacillus subtilis.

We have investigated the structural, biochemical and cellular roles of the two single-stranded (ss) DNA-binding proteins from Bacillus subtilis, SsbA and SsbB. During transformation, SsbB localizes at the DNA entry pole where it binds and protects internalized ssDNA. The 2.8-A resolution structure of SsbB bound to ssDNA reveals a similar overall protein architecture and ssDNA-binding surface to that of Escherichia coli SSB. SsbA, which binds ssDNA with higher affinity than SsbB, co-assembles onto SsbB-coated ssDNA and the two proteins inhibit ssDNA binding by the recombinase RecA. During chromosomal transformation, the RecA mediators RecO and DprA provide RecA access to ssDNA. Interestingly, RecO interaction with ssDNA-bound SsbA helps to dislodge both SsbA and SsbB from the DNA more efficiently than if the DNA is coated only with SsbA. Once RecA is nucleated onto the ssDNA, RecA filament elongation displaces SsbA and SsbB and enables RecA-mediated DNA strand exchange. During plasmid transformation, RecO localizes to the entry pole and catalyzes annealing of SsbA- or SsbA/SsbB-coated complementary ssDNAs to form duplex DNA with ssDNA tails. Our results provide a mechanistic framework for rationalizing the coordinated events modulated by SsbA, SsbB and RecO that are crucial for RecA-dependent chromosomal transformation and RecA-independent plasmid transformation.

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Genetic recombination in Bacillus subtilis : a division of labor between two single-strand DNA-binding proteins

Bacillus subtilis RecU protein is involved in homologous recombination, DNA repair, and chromosome segregation. Purified RecU binds preferentially to three- and four-strand junctions when compared to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) (≈10- and ≈40-fold lower efficiency, respectively). RecU cleaves mobile four-way junctions but fails to cleave a linear dsDNA with a putative cognate site, a finding consistent with a similar genetic defect observed for genes classified within the e epistatic group (namely ruvA, recD, and recU). In the presence of Mg2+, RecU also anneals a circular ssDNA and a homologous linear dsDNA with a ssDNA tail and a linear ssDNA and a homologous supercoiled dsDNA substrate. These results suggest that RecU, which cleaves recombination intermediates with high specificity, might also help in their assembly.

Bacillus subtilis RecU protein cleaves Holliday junctions and anneals single-stranded DNA

Previous studies have established that the expression of Salmonella enterica pathogenicity island 1 (SPI1), which is essential for epithelial invasion, is mainly regulated by the HilD protein. The ferric uptake regulator, Fur, in turn modulates the expression of the S. enterica hilD gene, albeit through an unknown mechanism. Here we report that S. enterica Fur, in its metal-bound form, specifically binds to an AT-rich region (BoxA), located upstream of the hilD promoter (PhilD), at position -191 to -163 relative to the hilD transcription start site. Furthermore, in a PhilD variant with mutations in BoxA, PhilD*, Fur·Mn2+ binding is impaired. In vivo experiments using S. enterica strains carrying wild-type PhilD or the mutant variant PhilD* showed that Fur activates hilD expression, while in vitro experiments revealed that the Fur·Mn2+ protein is sufficient to increase hilD transcription. Together, these results present the first evidence that Fur·Mn2+, by binding to the upstream BoxA sequence, directly stimulates the expression of hilD in S. enterica.

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Fur activates the expression of Salmonella enterica pathogenicity island 1 by directly interacting with the hilD operator in vivo and in vitro.

The Bacillus subtilis RecU protein is able to catalyze in vitro DNA strand annealing and Holliday-junction resolution. The interaction between the RecA and RecU proteins, in the presence or absence of a single-stranded binding (SSB) protein, was studied. Substoichiometric amounts of RecU enhanced RecA loading onto single-stranded DNA (ssDNA) and stimulated RecA-catalyzed D-loop formation. However, RecU inhibited the RecA-mediated three-strand exchange reaction and ssDNA-dependent dATP or rATP hydrolysis. The addition of an SSB protein did not reverse the negative effect exerted by RecU on RecA function. Annealing of circular ssDNA and homologous linear 3'-tailed double-stranded DNA by RecU was not affected by the addition of RecA both in the presence and in the absence of SSB. We propose that RecU modulates RecA activities by promoting RecA-catalyzed strand invasion and inhibiting RecA-mediated branch migration, by preventing RecA filament disassembly, and suggest a potential mechanism for the control of resolvasome assembly.

Begoña Carrasco

Papers

Double-strand break repair in bacteria: a view from Bacillus subtilis.

Genetic recombination in Bacillus subtilis : a division of labor between two single-strand DNA-binding proteins

Bacillus subtilis RecU protein cleaves Holliday junctions and anneals single-stranded DNA

Fur activates the expression of Salmonella enterica pathogenicity island 1 by directly interacting with the hilD operator in vivo and in vitro.

Bacillus subtilis RecU Holliday-junction resolvase modulates RecA activities