Hannu Myllykallio

In vivo interactions of archaeal Cdc6/Orc1 and minichromosome maintenance proteins with the replication origin

Although genome analyses have suggested parallels between archaeal and eukaryotic replication systems, little is known about the DNA replication mechanism in Archaea. By two-dimensional gel electrophoreses we positioned a replication origin (oriC) within 1 kb in the chromosomal DNA of Pyrococcus abyssi, an anaerobic hyperthermophile, and demonstrated that the oriC is physically linked to the cdc6 gene. Our chromatin immunoprecipitation assays indicated that P. abyssi Cdc6 and minichromosome maintenance (MCM) proteins bind preferentially to the oriC region in the exponentially growing cells. Whereas the oriC association of MCM was specifically inhibited by stopping DNA replication with puromycin treatment, Cdc6 protein stayed bound to the replication origin after de novo protein synthesis was inhibited. Our data suggest that archaeal and eukaryotic Cdc6 and MCM proteins function similarly in replication initiation and imply that an oriC association of MCM could be regulated by an unknown mechanism in Archaea.

Identification of putative chromosomal origins of replication in Archaea.

Recently, Novak et al. (1998, Mol Microbiol 29: 1285±1296) reported their investigation on the phenomenon of penicillin tolerance in Streptococcus pneumoniae. A library of mutants in pneumococcal surface proteins was screened for the ability to survive in the presence of 10 ́ the minimum inhibitory concentration of antibiotic. A mutant harbouring an insertion in the known gene psaA was isolated among 10 candidate tolerance mutants. Inactivation of psaA was previously shown to result in reduced virulence of S. pneumoniae (as judged by intranasal or intraperitoneal challenge of mice) and in reduced adherence to A549 cells (type II pneumocytes), leading to the suggestion that PsaA was an adhesin (Berry and Paton, 1996, Infect Immun 64: 5255±5262). This gene is part of the psa locus (Fig. 1) that encodes an ATP-binding cassette (ABC) permease belonging to cluster 9, a family of ABC metal permeases (Dintilhac et al., 1997, Mol Microbiol 25: 727±740). Novak et al. (1998, Mol Microbiol 29: 1285±1296) reported that psa mutants displayed pleiotropic phenotypes: (i) reduced sensitivity to the lytic and killing effects of penicillin; (ii) growth in chains of 40±50 (psaC ) to 200±300 (psaD ) cells; (iii) autolysis defect and loss of sensitivity to low concentrations of deoxycholate (DOC), a species characteristic trait; (iv) absence of LytA, the major autolytic amidase; (v) almost complete loss of choline-binding proteins (ChBPs) (psaC and psaD ) and absence of CbpA; (vi) loss of transformability (except psaA); and (vii) manganese (Mn) requirement for growth in a chemically de®ned medium. Because penicillin tolerance was ®rst associated with an autolysis defect (Tomasz et al., 1970, Nature 227: 138± 140), the absence of LytA (phenotype iv) could itself explain phenotypes i and iii. Dysregulation of lytA could not be investigated because, according to Novak et al. (1998, Mol Microbiol 29: 1285±1296), the dif®culty in lysing psa mutant cells prohibited Northern analysis, although lysates of the psa mutants could be obtained for immunoblot analysis of LytA and of RecA and for Southern con®rmation of the psa mutations. Nevertheless, because expression of the lytA gene has been shown to be driven by three different promoters, including Pb which is the recA basal promoter (Mortier-BarrieÁre et al., 1998, Mol Microbiol 27: 159±170), and because wild-type levels of RecA were detected in the psa mutants (Novak et al., 1998, Mol Microbiol 29: 1285±1296), it seems dif®cult to account for the complete absence of LytA on the basis of altered expression. On the other hand, phenotypes i±iv are reminiscent of alterations observed after the replacement of choline (Ch) by ethanolamine (EA) in the cell wall of pneumococcus (Tomasz, 1968, Proc Natl Acad Sci USA 59: 86±93). Similar phenotypes were also displayed by Ch-independent mutants of S. pneumoniae (Severin et al., 1997, Microb Drug Res 3: 391±400; Yother et al., 1998, J Bacteriol 180: 2093±2101). S. pneumoniae has a nutritional requirement for Ch that is incorporated by covalent bonds into the cell wall teichoic acids (TA) and in the membrane-bound lipoteichoic acid (LTA). Ch residues bound to TA (ChTA) were shown to be absolutely required for LytA activity (Holtje and Tomasz, 1975; J Biol Chem 250: 6072±6076). The action of LytA has long been thought to be restricted to pneumococcal cell walls because of this requirement. However, recent reports suggest that ChTA is required Molecular Microbiology (1999) 32(4), 881±891

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA‐primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well‐defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.

Identification of a novel gene encoding a flavin-dependent tRNA:m5U methyltransferase in bacteria--evolutionary implications.

Formation of 5-methyluridine (ribothymidine) at position 54 of the T-psi loop of tRNA is catalyzed by site-specific tRNA methyltransferases (tRNA:m5U-54 MTase). In all Eukarya and many Gram-negative Bacteria, the methyl donor for this reaction is S-adenosyl-l-methionine (S-AdoMet), while in several Gram-positive Bacteria, the source of carbon is N5, N10-methylenetetrahydrofolate (CH2H4folate). We have identified the gene for Bacillus subtilis tRNA:m5U-54 MTase. The encoded recombinant protein contains tightly bound flavin and is active in Escherichia coli mutant lacking m5U-54 in tRNAs and in vitro using T7 tRNA transcript as substrate. This gene is currently annotated gid in Genome Data Banks and it is here renamed trmFO. TrmFO (Gid) orthologs have also been identified in many other bacterial genomes and comparison of their amino acid sequences reveals that they are phylogenetically distinct from either ThyA or ThyX class of thymidylate synthases, which catalyze folate-dependent formation of deoxyribothymine monophosphate, the universal DNA precursor.

Catalytic Mechanism and Structure of Viral Flavin-Dependent Thymidylate Synthase Thyx.

By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by diethyl pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.

Mobile Cytochrome c2 and Membrane-Anchored Cytochrome cy Are Both Efficient Electron Donors to the cbb3- and aa3-Type Cytochrome c Oxidases during Respiratory Growth of Rhodobacter sphaeroides

We have recently established that the facultative phototrophic bacterium Rhodobacter sphaeroides, like the closely related Rhodobacter capsulatus species, contains both the previously characterized mobile electron carrier cytochrome c2 (cyt c2) and the more recently discovered membrane-anchored cyt cy. However, R. sphaeroides cyt cy, unlike that of R. capsulatus, is unable to function as an efficient electron carrier between the photochemical reaction center and the cyt bc1 complex during photosynthetic growth. Nonetheless, R. sphaeroides cyt cy can act at least in R. capsulatus as an electron carrier between the cyt bc1 complex and the cbb3-type cyt c oxidase (cbb3-Cox) to support respiratory growth. Since R. sphaeroides harbors both a cbb3-Cox and an aa3-type cyt c oxidase (aa3-Cox), we examined whether R. sphaeroides cyt cy can act as an electron carrier to either or both of these respiratory terminal oxidases. R. sphaeroides mutants which lacked either cyt c2 or cyt cy and either the aa3-Cox or the cbb3-Cox were obtained. These double mutants contained linear respiratory electron transport pathways between the cyt bc1 complex and the cyt c oxidases. They were characterized with respect to growth phenotypes, contents of a-, b-, and c-type cytochromes, cyt c oxidase activities, and kinetics of electron transfer mediated by cyt c2 or cyt cy. The findings demonstrated that both cyt c2 and cyt cy are able to carry electrons efficiently from the cyt bc1 complex to either the cbb3-Cox or the aa3-Cox. Thus, no dedicated electron carrier for either of the cyt c oxidases is present in R. sphaeroides. However, under semiaerobic growth conditions, a larger portion of the electron flow out of the cyt bc1 complex appears to be mediated via the cyt c2-to-cbb3-Cox and cyt cy-to-cbb3-Cox subbranches. The presence of multiple electron carriers and cyt c oxidases with different properties that can operate concurrently reveals that the respiratory electron transport pathways of R. sphaeroides are more complex than those of R. capsulatus.

Origin and Evolution of DNA and DNA Replication Machineries

The transition from the RNA to the DNA world was a major event in the history of life. The invention of DNA required the appearance of enzymatic activities for both synthesis of DNA precursors, retro-transcription of RNA templates and replication of single- and double-stranded DNA molecules. Recent data from comparative genomics, structural biology and traditional biochemistry have revealed that several of these enzymatic activities have been invented independently more than once, indicating that the transition from RNA to DNA genomes was more complex than previously thought. The distribution of the different protein families corresponding to these activities in the three domains of life (Archaea, Eukarya, and Bacteria) is puzzling. In many cases, Archaea and Eukarya contain the same version of these proteins, whereas Bacteria contain another version. However, in other cases, such as thymidylate synthases or type II DNA topoisomerases, the phylogenetic distributions of these proteins donot follow this simple pattern. Several hypotheses have been proposed to explain these observations, including independent invention of DNA and DNA replication proteins, ancient gene transfer and gene loss, and/or nonorthologous replacement. We review all of them here, with more emphasis on recent proposals suggesting that viruses have played a major role in the origin and evolution of the DNA replication proteins and possibly of DNA itself.

Structure and function of a novel endonuclease acting on branched DNA substrates

We show that Pyrococcus abyssi PAB2263 (dubbed NucS (nuclease for ss DNA) is a novel archaeal endonuclease that interacts with the replication clamp PCNA. Structural determination of P. abyssi NucS revealed a two‐domain dumbbell‐like structure that in overall does not resemble any known protein structure. Biochemical and structural studies indicate that NucS orthologues use a non‐catalytic ssDNA‐binding domain to regulate the cleavage activity at another site, thus resulting into the specific cleavage at double‐stranded DNA (dsDNA)/ssDNA junctions on branched DNA substrates. Both 3′ and 5′ extremities of the ssDNA can be cleaved at the nuclease channel that is too narrow to accommodate duplex DNA. Altogether, our data suggest that NucS proteins constitute a new family of structure‐specific DNA endonucleases that are widely distributed in archaea and in bacteria, including Mycobacterium tuberculosis.

Flavin-Dependent Thymidylate Synthase ThyX Activity: Implications for the Folate Cycle in Bacteria

Although flavin-dependent ThyX proteins show thymidylate synthase activity in vitro and functionally complement thyA defects in heterologous systems, direct proof of their cellular functions is missing. Using insertional mutagenesis of Rhodobacter capsulatus thyX, we constructed the first defined thyX inactivation mutant. Phenotypic analyses of the obtained mutant strain confirmed that R. capsulatus ThyX is required for de novo thymidylate synthesis. Full complementation of the R. capsulatus thyX::spec strain to thymidine prototrophy required not only the canonical thymidylate synthase ThyA but also the dihydrofolate reductase FolA. Strikingly, we also found that addition of exogenous methylenetetrahydrofolate transiently inhibited the growth of the different Rhodobacter strains used in this work. To rationalize these experimental results, we used a mathematical model of bacterial folate metabolism. This model suggests that a very low dihydrofolate reductase activity is enough to rescue significant thymidylate synthesis in the presence of ThyX proteins and is in agreement with the notion that intracellular accumulation of folates results in growth inhibition. In addition, our observations suggest that the presence of flavin-dependent thymidylate synthase X provides growth benefits under conditions in which the level of reduced folate derivatives is compromised.

Functional Analysis of the Mycobacterium tuberculosis FAD-Dependent Thymidylate Synthase, ThyX, Reveals New Amino Acid Residues Contributing to an Extended ThyX Motif

A novel FAD-dependent thymidylate synthase, ThyX, is present in a variety of eubacteria and archaea, including the mycobacteria. A short motif found in all thyX genes, RHRX(7-8)S, has been identified. The three-dimensional structure of the Mycobacterium tuberculosis ThyX enzyme has been solved. Building upon this information, we used directed mutagenesis to produce 67 mutants of the M. tuberculosis thyX gene. Each enzyme was assayed to determine its ability to complement the defect in thymidine biosynthesis in a delta thyA strain of Escherichia coli. Enzymes from selected strains were then tested in vitro for their ability to catalyze the oxidation of NADPH and the release of a proton from position 5 of the pyrimidine ring of dUMP. The results defined an extended motif of amino acids essential to enzyme activity in M. tuberculosis (Y44X(24)H69X(25)R95HRX(7)S105XRYX(90)R199 [with the underlined histidine acting as the catalytic residue and the underlined serine as the nucleophile]) and provided insight into the ThyX reaction mechanism. ThyX is found in a variety of bacterial pathogens but is absent in humans, which depend upon an unrelated thymidylate synthase, ThyA. Therefore, ThyX is a potential target for development of antibacterial drugs.