Structure-function analysis of the C-terminus of IcmT of Legionella pneumophila in pore formation-mediated egress from macrophages

We have recently shown an essential role of the 32 amino acids C-terminus domain of IcmT of Legionella pneumophila in bacterial egress from macrophages. Mutants expressing an IcmT protein with a truncation in the C-terminus, replicate intracellularly but are defective in pore formation-mediated egress. The C-terminus domain of IcmT is the only hydrophilic domain of IcmT that is predicted to be in the cytoplasm while the rest of the protein is in the cytoplasmic membrane. In order to characterize the structure-function of the C-terminus of IcmT in the pore-forming activity and bacterial egress, we constructed 10 icmT missense mutant alleles differing by a single amino acid in the C-terminus of icmT and introduced them into the null icmT mutant. The H58Q, W69L, R71I, R79I and R86I icmT mutant alleles showed significantly lower pore-forming activity as measured by hemolysis of sRBC. The Y59S, R68L and S77L mutant alleles showed significantly lower cytopathogenicity to U937 macrophages. All 10 mutant alleles enabled the icmT null mutant to replicate intracellularly as efficiently as icmT null mutant harboring the wild-type icmT. Seven of the icmT alleles enabled the icmT null mutant to egress from infected macrophages as efficiently as icmT null mutant harboring the wild-type icmT. The other 3 substitutions conferred a partial defect in hemolysis and two of them also conferred a defect in egress from macrophages. Thus, two amino acid residues in the C-terminus of IcmT are required for both pore formation and bacterial egress. However, certain single amino acid substitutions in the C-terminus reduce the pore-forming activity when tested in vitro, but may or may not have a detectable effect on egress of L. pneumophila from U937 macrophages.     

The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila

The ability of Legionella pneumophila to cause legionnaires' disease is dependent on its capacity to replicate within cells in the alveolar spaces. The bacteria kill mammalian cells in two phases: induction of apoptosis during the early stages of infection, followed by an independent and rapid necrosis during later stages of the infection, mediated by a pore-forming activity. In the environment, L. pneumophila is a parasite of protozoa. The molecular mechanisms by which L. pneumophila kills the protozoan cells, after their exploitation for intracellular proliferation, are not known. In an effort to decipher these mechanisms, we have examined induction of both apoptosis and necrosis in the protozoan Acanthamoeba polyphaga upon infection by L. pneumophila. Our data show that, although A. polyphaga undergoes apoptosis following treatment with actinomycin D, L. pneumophila does not induce apoptosis in these cells. Instead, intracellular L. pneumophila induces necrotic death in A. polyphaga, which is mediated by the pore-forming activity. Mutants of L. pneumophila defective in expression of the pore-forming activity are indistinguishable from the parental strain in intracellular replication within A. polyphaga. The parental strain bacteria cause necrosis-mediated lysis of all the A. polyphaga cells within 48 h after infection, and all the intracellular bacteria are released into the tissue culture medium. In contrast, all cells infected by the mutants remain intact, and the intracellular bacteria are 'trapped' within A. polyphaga after the termination of intracellular replication. Failure to exit the host cell after termination of intracellular replication results in a gradual decline in the viability of the mutant strain bacteria within A. polyphaga starting 48h after infection. Our data show that the pore-forming activity of L. pneumophila is not required for intracellular bacterial replication within A. polyphaga but is required for killing and exiting the protozoan host upon termination of intracellular replication.     

Role for the Ankyrin eukaryotic-like genes of Legionella pneumophila in parasitism of protozoan hosts and human macrophages

Legionella pneumophila is a ubiquitous organism in the aquatic environment where it is capable of invasion and intracellular proliferation within various protozoan species and is also capable of causing pneumonia in humans. In silico analysis showed that the three sequenced L. pneumophila genomes each contained a common multigene family of 11 ankyrin (ank) genes encoding proteins with approximately 30-35 amino acid tandem Ankyrin repeats that are involved in protein-protein interactions in eukaryotic cells. To examine whether the ank genes are involved in tropism of protozoan hosts, we have constructed isogenic mutants of L. pneumophila in ten of the ank genes. Among the mutants, the DeltaankH and DeltaankJ mutants exhibit significant defects in robust intracellular replication within A. polyphaga, Hartmanella vermiformis and Tetrahymena pyriformis. A similar defect is also exhibited in human macrophages. Most of the ank genes are upregulated by L. pneumophila upon growth transition into the post-exponential phase in vitro and within Acanthamoeba polyphaga, and this upregulation is mediated, at least in part, by RpoS. Single-cell analyses have shown that upon co-infection of the wild-type strain with the ankH or ankJ mutant, the replication defect of the mutant is rescued within communal phagosomes harbouring the wild-type strain, similar to dot/icm mutants. Therefore, at least two of the L. pneumophila eukaryotic-like Ank proteins play a role in intracellular replication of L. pneumophila within amoeba, ciliated protozoa and human macrophages. The Ank proteins may not be involved in host tropism in the aquatic environment. Many of the L. pneumophila eukaryotic-like ank genes are triggered upon growth transition into post-exponential phase in vitro as well as within A. polyphaga. Our data suggest a role for AnkH and AnkJ in modulation of phagosome biogenesis by L. pneumophila independent of evasion of lysosomal fusion and recruitment of the rough endoplasmic reticulum.     

Translocon-independent intracellular replication by Pseudomonas aeruginosa requires the ADP-ribosylation domain of ExoS

Pseudomonas aeruginosa, a significant cause of human morbidity and mortality, uses a type 3 secretion system (T3SS) to inject effector toxins into host cells. We previously reported that P. aeruginosa uses ADP-ribosyltransferase (ADPr) activity of the T3SS effector ExoS for intracellular replication. T3SS translocon (ΔpopB)-mutants, which can export, but not translocate effectors across host membranes, retained intracellular replication. We hypothesized that secreted effectors mediate translocon-independent intracellular replication. Translocon mutants of PAO1 lacking one or more of its three known effectors (ExoS, ExoT and ExoY) were used. All translocon mutants, irrespective of effectors expressed, localized to intracellular vacuoles. Translocon-effector null mutants and translocon-exoS mutants showed defective intracellular replication. Mutants in exoT, exoY or both replicated as efficiently as translocon mutants expressing all effectors. Complementation of translocon-effector null mutants with native exoS or a membrane localization domain mutant of exoS, but not the ADPr mutant exoS (pUCPexoSE381D), restored intracellular replication, correlating with increased bacteria per vacuole. Thus, P. aeruginosa is capable of intravacuolar replication that requires ExoS ADPr activity, but not the translocon. These data suggest that T3SS effectors can participate in pathogenesis without translocon-mediated translocation across host membranes, and that intracellular bacteria can contribute to P. aeruginosa pathogenesis within epithelial cells.     

AsgD, a new two-component regulator required for A-signalling and nutrient sensing during early development of Myxococcus xanthus

Myxococcus xanthus has a complex life cycle that includes fruiting body formation. One of the first stages in development has been called A-signalling. The asg (A-signalling) mutants have been proposed to be deficient in producing A-signal, resulting in development arresting at an early stage. In this paper, we report the identification of a new asg locus asgD. This locus appears to be involved in both environmental sensing and intercellular signalling. Expression of asgD was undetected during vegetative growth, but increased dramatically within 1 h of starvation. The AsgD protein is predicted to contain 773 amino acids and to be part of a two-component regulatory system because it has a receiver domain located at the N-terminus and a histidine protein kinase at the C-terminus. An asgD null mutant was defective in fruiting body formation and sporulation on CF medium. However, the defects of the mutant were complemented extracellularly when cells were mixed with wild-type strains or with bsgA, csgA, dsgA or esgA mutants, but were not complemented extracellularly by asgA, asgB or asgC mutants. In addition, the mutant was rescued by a subset of A-factor amino acids. Surprisingly, when the mutant was plated on stringent starvation medium rather than CF, cells were able to form fruiting bodies. Thus, it appears that AsgD is directly or indirectly involved in sensing nutritionally limiting conditions. The discovery of the asgD locus provides an important sensory transduction component of early development in M. xanthus.     

Amplification of an alternate transporter gene suppresses the avirulent phenotype of glucose transporter null mutants in Leishmania mexicana

A glucose transporter null mutant of the parasitic protozoan Leishmania mexicana , in which three linked glucose transporter genes have been deleted by targeted gene replacement, is unable to replicate as amastigote forms within phagolysomes of mammalian host macrophages and is avirulent. Spontaneous suppressors of the null mutant have been isolated that partially restore replication of parasites within macrophages. These suppressor mutants have amplified the gene for an alternative hexose transporter, the LmGT4 permease (previously called the D2 permease), on a circular extrachromosomal element, and they overexpress LmGT4 mRNA and protein. The suppressors have also regained the ability to transport hexoses, and they have reverted other phenotypes of the null mutant exhibiting enhanced resistance to oxidative killing, heat shock and starvation for nutrients, as well as augmented levels of the storage carbohydrate β-mannan, increased cell size and increased growth as insect stage promastigotes compared with the unsuppressed mutant. Complementation of the null mutant with the LmGT4 gene on a multicopy episomal expression vector also reverted these phenotypes, confirming that suppression results from amplification of the LmGT4 gene. These results underscore the importance of hexose transporters for the infectious stage of the parasite life cycle.     

Ionophore-resistant mutants of Toxoplasma gondii reveal host cell permeabilization as an early event in egress

Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. Invasion and egress by this protozoan parasite are rapid events that are dependent upon parasite motility and appear to be directed by fluctuations in intracellular [Ca(2+)]. Treatment of infected host cells with the calcium ionophore A23187 causes the parasites to undergo rapid egress in a process termed ionophore-induced egress (IIE). In contrast, when extracellular parasites are exposed to this ionophore, they quickly lose infectivity (termed ionophore-induced death [IID]). From among several Iie(-) mutants described here, two were identified that differ in several attributes, most notably in their resistance to IID. The association between the Iie(-) and Iid(-) phenotypes is supported by the observation that two-thirds of mutants selected as Iid(-) are also Iie(-). Characterization of three distinct classes of IIE and IID mutants revealed that the Iie(-) phenotype is due to a defect in a parasite-dependent activity that normally causes infected host cells to be permeabilized just prior to egress. Iie(-) parasites underwent rapid egress when infected cells were artificially permeabilized by a mild saponin treatment, confirming that this step is deficient in the Iie(-) mutants. A model is proposed that includes host cell permeabilization as a critical part of the signaling pathway leading to parasite egress. The fact that Iie(-) mutants are also defective in early stages of the lytic cycle indicates some commonality between these normal processes and IIE.     

Replication mutants of Staphylococcus aureus macrolide-lincosamide-streptogramin B resistance plasmid pT48

Copy-number mutants of Staphylococcus aureus macrolide-lincosamide-streptogramin B (MLS) resistance plasmid pT48 were isolated by their resistance to the non-inducing macrolide, tylosin. One mutant plasmid, pcopD3, showed a three- to five-fold cis-dominant increase in copy number, and nucleotide sequence analysis revealed that the mutant had a single base change within the replication region. All other pT48 mutants examined had the unusual phenotype of increased plasmid multimerization and elevated copy number. These mutants were effective in trans and DNA sequencing showed that plasmids with this phenotype were deleted in one of two ways. The deletions caused similar alterations to the C-terminus of the wild-type pT48 Rep protein. The two types of mutant Rep proteins terminate with the same pentapeptide sequence: Ala-Asn-Glu-Ile-Asp. The multimerization phenotype of these mutants can be explained by defective termination of rolling-circle type replication.     

Requirement of siderophore biosynthesis for plant colonization by Salmonella enterica

Contaminated fresh produce has become the number one vector of nontyphoidal salmonellosis to humans. However, Salmonella enterica genes essential for the life cycle of the organism outside the mammalian host are for the most part unknown. Screening deletion mutants led to the discovery that an aroA mutant had a significant root colonization defect due to a failure to replicate. AroA is part of the chorismic acid biosynthesis pathway, a central metabolic node involved in aromatic amino acid and siderophore production. Addition of tryptophan or phenylalanine to alfalfa root exudates did not restore aroA mutant replication. However, addition of ferrous sulfate restored replication of the aroA mutant, as well as alfalfa colonization. Tryptophan and phenylalanine auxotrophs had minor plant colonization defects, suggesting that suboptimal concentrations of these amino acids in root exudates were not major limiting factors for Salmonella replication. An entB mutant defective in siderophore biosynthesis had colonization and growth defects similar to those of the aroA mutant, and the defective phenotype was complemented by the addition of ferrous sulfate. Biosynthetic genes of each Salmonella siderophore, enterobactin and salmochelin, were upregulated in alfalfa root exudates, yet only enterobactin was sufficient for plant survival and persistence. Similar results in lettuce leaves indicate that siderophore biosynthesis is a widespread or perhaps universal plant colonization fitness factor for Salmonella, unlike phytobacterial pathogens, such as Pseudomonas and Xanthomonas.     

Icm/Dot-dependent upregulation of phagocytosis by Legionella pneumophila

Legionella pneumophila is the causative agent of Legionnaires' disease, a severe pneumonia. Dependent on the icm/dot loci, L. pneumophila survives and replicates in macrophages and amoebae within a specialized phagosome that does not fuse with lysosomes. Here, we report that phagocytosis of wild-type L. pneumophila is more efficient than uptake of icm/dot mutants. Compared with the wild-type strain JR32, about 10 times fewer icm/dot mutant bacteria were recovered from HL-60 macrophages in a gentamicin protection assay. The defect in phagocytosis of the mutants could be complemented by supplying the corresponding genes on a plasmid. Using fluorescence microscopy and green fluorescent protein (GFP)-expressing strains, 10-20 times fewer icm/dot mutant bacteria were found to be internalized by HL-60 cells and human monocyte-derived macrophages (HMMPhi). Compared with icm/dot mutants, wild-type L. pneumophila infected two to three times more macrophages and yielded a population of highly infected host cells (15-70 bacteria per macrophage) that was not observed with icm/dot mutant strains. Wild-type and icmT mutant bacteria were found to adhere similarly and compete for binding to HMMPhi. In addition, wild-type L. pneumophila was also phagocytosed more efficiently by Acanthamoeba castellanii, indicating that the process is independent of adherence receptor(s). Wild-type L. pneumophila enhanced phagocytosis of an icmT mutant strain in a synchronous co-infection, suggesting that increased phagocytosis results from (a) secreted effector(s) acting in trans.     

Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila

Legionella pneumophila mutants specifically defective for intracellular replication were isolated using an intracellular thymineless death enrichment strategy. Mutants belonging to two distinct phenotypic classes were unable to grow in macrophage-like cultured cells. One class of mutants was defective for both inhibition of phagosome–lysosome fusion and association of host cell organelles with bacteria-containing phagosomes (‘recruitment’). Another class of mutants was defective only for organelle recruitment, suggesting that recruitment may be necessary for intracellular growth. Recombinant clones were identified that complemented the intracellular growth defects of these mutants. A single genetic locus, designated dot (for defect in organelle trafficking), restored wild-type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome–lysosome fusion to mutants belonging to both phenotypic classes.     

Mutations in uvrD induce the SOS response in Escherichia coli.

We have isolated three new mutations in uvrD that increase expression of the Escherichia coli SOS response in the absence of DNA damage. Like other uvrD (DNA helicase II) mutants, these strains are sensitive to UV irradiation and have high spontaneous mutation frequencies. Complementation studies with uvrD+ showed that UV sensitivity and spontaneous mutator activity were recessive in these new mutants. The SOS-induction phenotype, however, was not completely complemented, which indicated that the mutant proteins were functioning in some capacity. The viability of one of the mutants in combination with rep-5 suggests that the protein is functional in DNA replication. We suggest that these mutant proteins are deficient in DNA repair activities (since UV sensitivity is complemented) but are able to participate in DNA replication. We believe that defective DNA replication in these mutants increases SOS expression.     

How does Legionella pneumophila exit the host cell?

In recent years, tremendous progress has been made in unraveling the elegant mechanisms by which intracellular pathogens invade host cells and establish intracellular infections. By contrast, our knowledge of the mechanisms of host cell cytolysis and the egress of intracellular pathogens is still in its infancy. Temporal pore-formation-mediated lysis of the host and exit by Legionella pneumophila and Leishmania could provide a new model of egress for other intracellular pathogens, many of which exhibit pore-forming or cytolysin activity     

Pore-forming activity is not sufficient for Legionella pneumophila phagosome trafficking and intracellular growth

Bacterial pathogens often subvert eukaryotic cellular processes in order to establish a replicative niche and evade host immunity. Inhibition of phagosome lysosome fusion is a strategy used by several intracellular bacteria that grow within mammalian cells. It was shown recently that Legionella pneumophila possesses a cytolytic activity that results from the insertion of pores in the macrophage membrane upon contact, and that this activity requires the dot/icm gene products, which are necessary for intracellular growth and phagosome trafficking. Other bacteria that inhibit phagosome lysosome fusion, such as Mycobacterium tuberculosis , demonstrate similar cytolytic activities, which suggests that formation of pores in the phagosome membrane may account for the defects observed in phagosome trafficking. In this study, we identify a new class of L. pneumophila mutant that retains the pore-forming activity found in virulent bacteria, but is defective in phagosome lysosome fusion inhibition and intracellular growth. These data indicate that cytolytic activity is not sufficient for L. pneumophila -induced alterations in phagosome trafficking. Rather, the pore may be a vehicle that facilitates delivery of bacterial-derived effector molecules to the host cell cytoplasm.     

A Differential Fluorescence-Based Genetic Screen Identifies Listeria monocytogenes Determinants Required for Intracellular Replication

Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen capable of causing severe invasive disease with high mortality rates in humans. While previous studies have largely elucidated the bacterial and host cell mechanisms necessary for invasion, vacuolar escape, and subsequent cell-to-cell spread, the L. monocytogenes factors required for rapid replication within the restrictive environment of the host cell cytosol are poorly understood. In this report, we describe a differential fluorescence-based genetic screen utilizing fluorescence-activated cell sorting (FACS) and high-throughput microscopy to identify L. monocytogenes mutants defective in optimal intracellular replication. Bacteria harboring deletions within the identified gene menD or pepP were defective for growth in primary murine macrophages and plaque formation in monolayers of L2 fibroblasts, thus validating the ability of the screening method to identify intracellular replication-defective mutants. Genetic complementation of the menD and pepP deletion strains rescued the in vitro intracellular infection defects. Furthermore, the menD deletion strain displayed a general extracellular replication defect that could be complemented by growth under anaerobic conditions, while the intracellular growth defect of this strain could be complemented by the addition of exogenous menaquinone. As prior studies have indicated the importance of aerobic metabolism for L. monocytogenes infection, these findings provide further evidence for the importance of menaquinone and aerobic metabolism for L. monocytogenes pathogenesis. Lastly, both the menD and pepP deletion strains were attenuated during in vivo infection of mice. These findings demonstrate that the differential fluorescence-based screening approach provides a powerful tool for the identification of intracellular replication determinants in multiple bacterial systems.     

A mycobacterial virulence gene cluster extending RD1 is required for cytolysis, bacterial spreading and ESAT-6 secretion

Initiation and maintenance of infection by mycobacteria in susceptible hosts are not well understood. A screen of Mycobacterium marinum transposon mutant library led to isolation of eight mutants that failed to cause haemolysis, all of which had transposon insertions in genes homologous to a region between Rv3866 and Rv3881c in Mycobacterium tuberculosis, which encompasses RD1 (Rv3871-Rv3879c), a known virulence gene cluster. The M. marinum mutants showed decreased virulence in vivo and failed to secrete ESAT-6, like M. tuberculosis RD1 mutants. M. marinum mutants in genes homologous to Rv3866-Rv3868 also failed to accumulate intracellular ESAT-6, suggesting a possible role for those genes in synthesis or stability of the protein. These transposon mutants and an ESAT-6/CFP-10 deletion mutant all showed reduced cytolysis and cytotoxicity to macrophages and significantly decreased intracellular growth at late stages of the infection only when the cells were infected at low multiplicity of infection, suggesting a defect in spreading. Direct evidence for cell-to-cell spread by wild-type M. marinum was obtained by microscopic detection in macrophage and epithelial monolayers, but the mutants all were defective in this assay. Expression of M. tuberculosis homologues complemented the corresponding M. marinum mutants, emphasizing the functional similarities between M. tuberculosis and M. marinum genes in this region that we designate extRD1 (extended RD1). We suggest that diminished membranolytic activity and defective spreading is a mechanism for the attenuation of the extRD1 mutants. These results extend recent findings on the genomic boundaries and functions of M. tuberculosis RD1 and establish a molecular cellular basis for the role that extRD1 plays in mycobacterial virulence. Disruption of the M. marinum homologue of Rv3881c, not previously implicated in virulence, led to a much more attenuated phenotype in macrophages and in vivo, suggesting that this gene plays additional roles in M. marinum survival in the host.     

Phosphorylation of a Myosin Motor by TgCDPK3 Facilitates Rapid Initiation of Motility during Toxoplasma gondii egress

Members of the family of calcium dependent protein kinases (CDPK’s) are abundant in certain pathogenic parasites and absent in mammalian cells making them strong drug target candidates. In the obligate intracellular parasite Toxoplasma gondii TgCDPK3 is important for calcium dependent egress from the host cell. Nonetheless, the specific substrate through which TgCDPK3 exerts its function during egress remains unknown. To close this knowledge gap we applied the proximity-based protein interaction trap BioID and identified 13 proteins that are either near neighbors or direct interactors of TgCDPK3. Among these was Myosin A (TgMyoA), the unconventional motor protein greatly responsible for driving the gliding motility of this parasite, and whose phosphorylation at serine 21 by an unknown kinase was previously shown to be important for motility and egress. Through a non-biased peptide array approach we determined that TgCDPK3 can specifically phosphorylate serines 21 and 743 of TgMyoA in vitro. Complementation of the TgmyoA null mutant, which exhibits a delay in egress, with TgMyoA in which either S21 or S743 is mutated to alanine failed to rescue the egress defect. Similarly, phosphomimetic mutations in the motor protein overcome the need for TgCDPK3. Moreover, extracellular Tgcdpk3 mutant parasites have motility defects that are complemented by expression of S21+S743 phosphomimetic of TgMyoA. Thus, our studies establish that phosphorylation of TgMyoA by TgCDPK3 is responsible for initiation of motility and parasite egress from the host-cell and provides mechanistic insight into how this unique kinase regulates the lytic cycle of Toxoplasma gondii.     

Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants.

We constructed insertion and deletion mutants with mutations within the adeno-associated virus (AAV) sequences of the infectious recombinant plasmid pSM620. Studies of these mutants revealed at least three AAV phenotypes. Mutants with mutations between 11 and 42 map units were partially or completely defective for rescue and replication of the AAV sequences from the recombinant plasmids (rep mutants). The mutants could be complemented by mutants with replication-positive phenotypes. The protein(s) that is affected in rep mutants has not been identified, but the existence of the rep mutants proves that at least one AAV-coded protein is required for viral DNA replication. Also, the fact that one of the rep mutant mutations maps within the AAV intron suggests that the intron sequences code for part of a functional AAV protein. Mutants with mutations between 63 and 91 map units synthesized normal amounts of AAV duplex DNA but could not generate single-stranded virion DNA (cap mutants). The cap phenotype could be complemented by rep mutants and is probably due to a defect in the major AAV capsid protein, VP3. This suggests that a preformed capsid or precursor is required for the accumulation of single-stranded AAV progeny DNA. Mutants with mutations between 48 and 55 map units synthesized normal amounts of AAV single-stranded and duplex DNA but produced substantially lower yields of infectious virus particles than wild-type AAV (lip mutants). The lip phenotype is probably due to a defect in the minor capsid protein, VPI, and suggests the existence of an additional (as yet undiscovered) AAV mRNA. Evidence is also presented for recombination between mutant AAV genomes during lytic growth.     

The Schizosaccharomyces pombe cdt2(+) gene,a target of G1-S phase-specific transcription factor complex DSC1, is required for mitotic and premeiotic DNA replication

We have defined five sev genes by genetic analysis of Schizosaccharomyces pombe mutants, which are defective in both proliferation and sporulation. sev1(+)/cdt2(+) was transcribed during the G1-S phase of the mitotic cell cycle, as well as during the premeiotic S phase. The mitotic expression of cdt2(+) was regulated by the MCB-DSC1 system. A mutant of a component of DSC1 affected cdt2(+) expression in vivo, and a cdt2(+) promoter fragment containing MCB motifs bound DSC1 in vitro. Cdt2 protein also accumulated in S phase and localized to the nucleus. cdt2 null mutants grew slowly at 30 degrees and were unable to grow at 19 degrees. These cdt2 mutants were also medially sensitive to hydroxyurea, camptothecin, and 4-nitroquinoline-1-oxide and were synthetically lethal in combination with DNA replication checkpoint mutations. Flow cytometry analysis and pulsed-field gel electrophoresis revealed that S-phase progression was severely retarded in cdt2 mutants, especially at low temperatures. Under sporulation conditions, premeiotic DNA replication was impaired with meiosis I blocked. Furthermore, overexpression of suc22(+), a ribonucleotide reductase gene, fully complemented the sporulation defect of cdt2 mutants and alleviated their growth defect at 19 degrees. These observations suggest that cdt2(+) plays an important role in DNA replication in both the mitotic and the meiotic life cycles of fission yeast.