Specific Expansion of Protein Families in the Radioresistant Bacterium Deinococcus Radiodurans

Computer analysis of the complete genome of Deinococcus radioduransR1 reveals a number of protein families, which are over-represented in this organism, compared to most other bacteria with known genome sequences. These families include both previously characterized and uncharacterized proteins. Most of the families whose functions are known or could be predicted seem to be related to stress-response and elimination of damage products (cell-cleaning). The two most prominent family expansions are the Nudix (MutT) family of pyrophosphohydrolases and a previously unnoticed family of proteins related to Bacillus subtilisDinB that could possess a metal-dependent enzymatic activity whose exact nature remains to be determined. Several proteins of the expanded families, particularly the Nudix family, are fused to other domains and form multidomain proteins that are so far unique for Deinococcus. The domain composition of some of these proteins indicates that they could be involved in novel DNA-repair pathways. Such unique proteins are good targets for knock-out and gene expression studies, which are aimed to shed light on the unusual features of this interesting10.6pt bacterium.     

The Role of DNA Repair Genes in Recombination between Repeated Sequences in Yeast

The presence of repeated sequences in the genome represents a potential source of karyotypic instability. Genetic control of recombination is thus important to preserve the integrity of the genome. To investigate the genetic control of recombination between repeated sequences, we have created a series of isogenic strains in which we could assess the role of genes involved in DNA repair in two types of recombination: direct repeat recombination and ectopic gene conversion. Naturally occurring (Ty elements) and artificially constructed repeats could be compared in the same cell population. We have found that direct repeat recombination and gene conversion have different genetic requirements. The role of the RAD51, RAD52, RAD54, RAD55, and RAD57 genes, which are involved in recombinational repair, was investigated. Based on the phenotypes of single and double mutants, these genes can be divided into three functional subgroups: one composed of RAD52, a second one composed of RAD51 and RAD54, and a third one that includes the RAD55 and RAD57 genes. Among seven genes involved in excision repair tested, only RAD1 and RAD10 played a role in the types of recombination studied. We did not detect a differential effect of any rad mutation on Ty elements as compared to artificially constructed repeats.     

Recombination between a resident plasmid and the chromosome following irradiation of the radioresistant bacterium Deinococcus radiodurans

Interplasmidic and intrachromosomal recombination in Deinococcus radiodurans has been studied recently and has been found to occur at high frequency following exposure to ionizing radiation. In the current work, we document plasmid-chromosome recombination following exposure of D. radiodurans to 1.75 Mrad (17.5 kGy) ⁶⁰Co, when the plasmid is present in the cell at the time of irradiation. Recombination is assayed using both physical and allelic polymorphisms of homologous genes in the plasmid and chromosome. Recombination was found to be largely, but not entirely, recA-dependent. Crossovers occur frequently, and a significant fraction of these are non-reciprocal.     

Repair of Oxidized Bases in the Extremely Radiation-Resistant Bacterium Deinococcus radiodurans

Deinococcus radiodurans is able to resist and survive extreme DNA damage induced by ionizing radiation and many other DNA-damaging agents. It is believed that it possesses highly efficient DNA repair mechanisms. To characterize the repair pathway of oxidized purines in this bacteria, we have purified, from crude extracts, proteins that recognize these oxidized bases. We report here that D. radiodurans possesses two proteins excising the oxidized purines (formamidopyrimidine and 8-oxoguanine) by a DNA glycosylase–a purinic/apyrimidine lyase mechanism. Moreover, one of those proteins is endowed with a thymine glycol DNA glycosylase activity. One of these proteins could be the homolog of the Escherichia coli Fpg enzyme, which confirms the existence of a base excision repair system in this bacteria.     

The HSV-1 Exonuclease, UL12, Stimulates Recombination by a Single Strand Annealing Mechanism

Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5′-to-3′ exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection.     

Targeted Mutagenesis by Duplication Insertion in the Radioresistant Bacterium Deinococcus radiodurans: Radiation Sensitivities of Catalase (katA) and Superoxide Dismutase (sodA) Mutants

Deinococcus radiodurans R1 is extremely resistant to both oxidative stress and ionizing radiation. A simple and general targeted mutagenesis method was developed to generate catalase (katA) and superoxide dismutase (sodA) mutants. Both mutants were shown to be more sensitive to ionizing radiation than the wild type.     

Approaches to Half-Tetrad Analysis in Bacteria: Recombination between Repeated, Inverse-Order Chromosomal Sequences

In standard bacterial recombination assays, a linear fragment of DNA is transferred to a recipient cell and, at most, a single selected recombinant type is recovered from each merozygote. This contrasts with fungal systems, for which tetrads allow recovery of all meiotic products, including both ultimate recombinant products of an apparent single act of recombination. We have developed a bacterial recombination system in which two recombining sequences are placed in inverse order at widely separated sites in the circular chromosome of Salmonella typhimurium. Recombination can reassort markers between these repeated sequences (double recombination and apparent gene conversion), or can exchange flanking sequences, leading to inversion of the chromosome segment between the recombining sequences. Since two recombinant products remain in the chromosome of a recombinant with an inversion, one can, in principle, approach the capability of tetrad analysis. Using this system, the following observations have been made. (a) When long sequences (40 kb) recombine, conversion frequently accompanies exchange of flanking sequences. (b) When short sequences (5 kb) recombine, conversion rarely accompanies exchange of flanks. (c) Both recA and recB mutations eliminate inversion formation. (d) The frequency of exchanges between short repeats is more sensitive to the distance separating the recombining sequences in the chromosome. The results are presented with the assumption that inversions occur by simple interaction of two sequences in the same circular chromosome. In an appendix we discuss mechanistically more complex possibilities, some of which could also apply to standard fungal systems.     

Functional Characterization of the Role of the Chromosome I Partitioning System in Genome Segregation in Deinococcus radiodurans

Deinococcus radiodurans, a radiation-resistant bacterium, harbors a multipartite genome. Chromosome I contains three putative centromeres (segS1, segS2, and segS3), and ParA (ParA1) and ParB (ParB1) homologues. The ParB1 interaction with segS was sequence specific, and ParA1 was shown to be a DNA binding ATPase. The ATPase activity of ParA1 was stimulated when segS elements were coincubated with ParB1, but the greatest increase was observed with segS3. ParA1 incubated with the segS-ParB1 complex showed increased light scattering in the absence of ATP. In the presence of ATP, this increase was continued with segS1-ParA1B1 and segS2-ParA1B1 complexes, while it decreased rapidly after an initial increase for 30 min in the case of segS3. D. radiodurans cells expressing green fluorescent protein (GFP)-ParB1 produced foci on nucleoids, and the ΔparB1 mutant showed growth retardation and ∼13%-higher anucleation than the wild type. Unstable mini-F plasmids carrying segS1 and segS2 showed inheritance in Escherichia coli without ParA1B1, while segS3-mediated plasmid stability required the in trans expression of ParA1B1. Unlike untransformed E. coli cells, cells harboring pDAGS3, a plasmid carrying segS3 and also expressing ParB1-GFP, produced discrete GFP foci on nucleoids. These findings suggested that both segS elements and the ParA1B1 proteins of D. radiodurans are functionally active and have a role in genome segregation.     

A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in Streptomyces ambofaciens ATCC23877

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.     

Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity

The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.     

Induction of a Futile Embden-Meyerhof-Parnas Pathway in Deinococcus radiodurans by Mn: Possible Role of the Pentose Phosphate Pathway in Cell Survival

Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D. radiodurans. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO₂. Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D. radiodurans. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance.     

Membrane Chaperone SecDF Plays a Role in the Secretion of Listeria monocytogenes Major Virulence Factors

Listeria monocytogenes is a Gram-positive human intracellular pathogen that infects diverse mammalian cells. Upon invasion, L. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. It has been presumed, but was not empirically established, that the Sec translocation system is the primary mediator of this secretion. Here, we validate an important role for SecDF, a component of the Sec system, in the secretion of several critical L. monocytogenes virulence factors. A ΔsecDF mutant is demonstrated to exhibit impaired membrane translocation of listeriolysin O (LLO), PlcA, PlcB, and ActA, factors that mediate L. monocytogenes phagosomal escape and spread from cell to cell. This impaired translocation was monitored by accumulation of the factors on the bacterial membrane and by reduced activity upon secretion. This defect in secretion is shown to be associated with a severe intracellular growth defect of the ΔsecDF mutant in macrophages and a less virulent phenotype in mice, despite normal growth in laboratory medium. We further show that SecDF is upregulated when the bacteria reside in macrophage phagosomes and that it is necessary for efficient phagosomal escape. Taken together, these data support the premise that SecDF plays a role as a chaperone that facilitates the translocation of L. monocytogenes virulence factors during infection.     

Role of the Rad52 Amino-terminal DNA Binding Activity in DNA Strand Capture in Homologous Recombination

Saccharomyces cerevisiae Rad52 protein promotes homologous recombination by nucleating the Rad51 recombinase onto replication protein A-coated single-stranded DNA strands and also by directly annealing such strands. We show that the purified rad52-R70A mutant protein, with a compromised amino-terminal DNA binding domain, is capable of Rad51 delivery to DNA but is deficient in DNA annealing. Results from chromatin immunoprecipitation experiments find that rad52-R70A associates with DNA double-strand breaks and promotes recruitment of Rad51 as efficiently as wild-type Rad52. Analysis of gene conversion intermediates reveals that rad52-R70A cells can mediate DNA strand invasion but are unable to complete the recombination event. These results provide evidence that DNA binding by the evolutionarily conserved amino terminus of Rad52 is needed for the capture of the second DNA end during homologous recombination.     

Identification of PprM: a modulator of the PprI-dependent DNA damage response in Deinococcus radiodurans

Deinococcus radiodurans possesses a DNA damage response mechanism that acts via the PprI protein to induce RecA and PprA proteins, both of which are necessary in conferring extreme radioresistance. In an effort to further delineate the nature of the DNA damage response mechanism in D. radiodurans, we set out to identify novel components of the PprI-dependent signal transduction pathway in response to radiation stress. Here we demonstrate the discovery of a novel regulatory protein, PprM (a modulator of the PprI-dependent DNA damage response), which is a homolog of cold shock protein (Csp). Disruption of the pprM gene rendered D. radiodurans significantly sensitive to γ-rays. PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from D. geothermalis and Thermus thermophilus. Purified PprM is present as a homodimer under physiological conditions, as the case with Escherichia coli CspD. The pprA pprM double-disruptant strain exhibited higher sensitivity than the pprA or pprM single disruptant strains, suggesting that PprM regulates other hitherto unknown protein(s) important for radioresistance besides PprA. This study strongly suggests that PprM is involved in the radiation response mediated by PprI in D. radiodurans.     

Sexual Reproduction Plays a Major Role in the Genetic Structure of Populations of the Fungus Mycosphaerella Graminicola

The relative contributions of sexual and asexual reproduction to the genetic structure of populations can be difficult to determine for fungi that use a mixture of both types of propagation. Nuclear RFLPs and DNA fingerprints were used to make indirect and direct measures of departures from random mating in a population of the plant pathogenic fungus Mycosphaerella graminicola during the course of an epidemic cycle. DNA fingerprints resolved 617 different genotypes among 673 isolates sampled from a single field over a 3-month period. Only 7% of the isolates represented asexual clones that were found more than once in the sample. The most common clone was found four times. Genotypic diversity averaged 85% of its maximum possible value during the course of the epidemic. Analyses of multilocus structure showed that allelic distributions among RFLP loci were independent. Pairwise comparisons between individual RFLP loci showed that the majority of alleles at these loci were in gametic equilibrium. Though this fungus has the capacity for a significant level of asexual reproduction, each analysis suggested that M. graminicola populations maintain a genetic structure more consistent with random-mating over the course of an epidemic cycle.     

Hierarchy in Hfq Chaperon Occupancy of Small RNA Targets Plays a Major Role in Their Regulation

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