These results suggest that, in human cells, the DNA-damage checkpoint involves direct inactivation of Cdc25 catalyzed by Cds1 and/or Chk1.

SummaryRhizobium bacteria produce different surface polysaccharides which are either secreted in the growth medium or contribute to a capsule surrounding the cell. Here, we describe isolation and partial characterization of a novel high molecular weight surface polysaccharide from a strain of Rhizobium leguminosarum that nodulates Pisum sativum (pea) and Vicia sativa (vetch) roots. Carbohydrate analysis showed that the polysaccharide consists for 95% of mannose and glucose, with minor amounts of galactose and rhamnose. Lectin precipitation analysis revealed high binding affinity of pea and vetch lectin for this polysaccharide, in contrast to the other known capsular and extracellular polysaccharides of this strain. Expression of the polysaccharide was independent of the presence of a Sym plasmid or the nod gene inducer naringenin. Incubation of R. leguminosarum with labelled pea lectin showed that this polysaccharide is exclusively localized on one of the poles of the bacterial cell. Vetch roots incubated with rhizobia and labelled pea lectin revealed that this bacterial pole is involved in attachment to the root surface. A mutant strain deficient in the production of this polysaccharide was impaired in attachment and root hair infection under slightly acidic conditions, in contrast to the situation at slightly alkaline conditions. Our data are consistent with the hypothesis that rhizobia can use (at least) two mechanisms for docking at the root surface, with use of a lectin-glycan mechanism under slightly acidic conditions.

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.

In the fission yeast Schizosaccharomyces pombe the rad1 ؉ gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a human homologue of the S. pombe rad1 ؉ gene, designated Hrad1, as well as a mouse homologue: Mrad1. Two Hrad1 alternative splice variants with different open reading frames have been identified; one codes for a long form, Hrad1A, and the other encodes a short form because of N-terminal truncation, Hrad1B. Hrad1A has 60% identity to the S. pombe rad1 ؉ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level. Northern blot analysis indicates elevated levels of expression in testis and cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates that Hrad1 is located on chromosome 5p13.2-13.3. This region is subject to loss of heterozygosity in several human cancers. Hrad1 also shares homology with the Saccharomyces cerevisiae RAD17 and Ustilago maydis REC1 proteins. REC1 has previously been characterized as a 3 3 5 exonuclease with a C-terminal domain essential for cell cycle checkpoint function. We have expressed and purified polyhistidine-tagged fusions of Hrad1A and Hrad1B and show that HisHrad1A has 3 3 5 exonuclease activity, whereas HisHrad1B lacks such activity. The biological functions of the two proteins remain to be determined.

In the fission yeast Schizosaccharomyces pombe the rad17 ؉ gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a human cDNA homologue of the S. pombe rad17 ؉ checkpoint gene, designated Hrad17. Hrad17 has 49% identity to the S. pombe rad17 ؉ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level. Northern blot analysis indicates elevated levels of expression in testis and in cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates that Hrad17 is located on chromosome 4q13.3-21.2. This region is subject to loss of heterozygosity in several human cancers. To begin to understand the protein-protein interactions of the human checkpoint machinery, we have used the yeast two-hybrid system to examine potential interactions between Hrad1, Hrad9, and Hrad17. We demonstrate a physical interaction between Hrad17 and Hrad1 but no interaction with Hrad9.

Analysis of two exopolysaccharide-deficient mutants of Rhizobium leguminosarum, RBL5808 and RBL5812, revealed independent Tn5 transposon integrations in a single gene, designated exo5. As judged from structural and functional homology, this gene encodes a UDP-glucose dehydrogenase responsible for the oxidation of UDP-glucose to UDP-glucuronic acid. A mutation in exo5 affects all glucuronic acid-containing polysaccharides and, consequently, all galacturonic acid-containing polysaccharides. Exo5-deficient rhizobia do not produce extracellular polysaccharide (EPS) or capsular polysaccharide (CPS), both of which contain glucuronic acid. Carbohydrate composition analysis and nuclear magnetic resonance studies demonstrated that EPS and CPS from the parent strain have very similar structures. Lipopolysaccharide (LPS) molecules produced by the mutant strains are deficient in galacturonic acid, which is normally present in the core and lipid A portions of the LPS. The sensitivity of exo5 mutant rhizobia to hydrophobic compounds shows the involvement of the galacturonic acid residues in the outer membrane structure. Nodulation studies with Vicia sativa subsp. nigra showed that exo5 mutant rhizobia are impaired in successful infection thread colonization. This is caused by strong agglutination of EPS-deficient bacteria in the root hair curl. Root infection could be restored by simultaneous inoculation with a Nod factor-defective strain which retained the ability to produce EPS and CPS. However, in this case colonization of the nodule tissue was impaired.

Rhizobial lipopolysaccharide (LPS) is required to establish an effective symbiosis with its host plant. An exo5 mutant of Rhizobium leguminosarum RBL5523, strain RBL5808, is defective in UDP-glucose (Glc) dehydrogenase that converts UDP-Glc to UDP-glucuronic acid (GlcA). This mutant is unable to synthesize either UDP-GlcA or UDP-galacturonic acid (GalA) and is unable to synthesize extracellular and capsular polysaccharides, lacks GalA in its LPS and is defective in symbiosis (Laus MC, Logman TJ, van Brussel AAN, Carlson RW, Azadi P, Gao MY, Kijne JW. 2004. Involvement of exo5 in production of surface polysaccharides in Rhizobium leguminosarum and its role in nodulation of Vicia sativa subsp. nigra. J Bacteriol. 186:6617-6625). Here, we determined and compared the structures of the RBL5523 parent and RBL5808 mutant LPSs. The parent LPS core oligosaccharide (OS), as with other R. leguminosarum and Rhizobium etli strains, is a Gal(1)Man(1)GalA(3)Kdo(3) octasaccharide in, which each of the GalA residues is terminally linked. The core OS from the mutant lacks all three GalA residues. Also, the parent lipid A consists of a fatty acylated GlcNGlcNonate or GlcNGlcN disaccharide that has a GalA residue at the 4'-position, typical of other R. leguminosarum and R. etli lipids A. The mutant lipid A lacks the 4'-GalA residue, and the proximal glycosyl residue was only present as GlcNonate. In spite of these alterations to the lipid A and core OSs, the mutant was still able to synthesize an LPS containing a normal O-chain polysaccharide (OPS), but at reduced levels. The structure of the OPS of the mutant LPS was identical to that of the parent and consists of an O-acetylated →4)-α-d-Glcp-(1→3)-α-d-QuipNAc-(1→ repeating unit.

Exopolysaccharide (EPS)-deficient strains of the root nodule symbiote Rhizobium leguminosarum induce formation of abortive infection threads in Vicia sativa subsp. nigra roots. As a result, the nodule tissue remains uninfected. Formation of an infection thread can be restored by coinoculation of the EPS-deficient mutant with a Nod factor-deficient strain, which produces a similar EPS structure. This suggests that EPS contributes to host-plant specificity of nodulation. Here, a comparison was made of i) coinoculation with heterologous strains with different EPS structures, and ii) introduction of the pRL1JI Sym plasmid or a nod gene-encoding fragment in the same heterologous strains. Most strains not complementing in coinoculation experiments were able to nodulate V. sativa roots as transconjugants. Apparently, coinoculation is a delicate approach in which differences in root colonization ability or bacterial growth rate easily affect successful infection-thread formation. Obviously, lack of infection-thread formation in coinoculation studies is not solely determined by EPS structure. Transconjugation data show that different EPS structures can allow infection-thread formation and subsequent nodulation of V. sativa roots.