Rhizobium melioti produces an acidic, Calcofluor-binding exopolysaccharide which plays a role in nodulation of alfalfa plants by this bacterium. We constructed and mapped 102 transposon insertions in a 48-kilobase (kb) region previously shown to contain several exo genes. Mutations affecting production of the Calcofluor-binding exopolysaccharide were clustered in a 22-kb region and fell into 12 complementation groups. Strains carrying mutations in seven of the complementation groups (exoA, exoB, exoF, exoL, exoM, exoP, and exoQ) produced no Calcofluor-binding exopolysaccharide and induced non-nitrogen-fixing nodules on alfalfa. Mutants in an eighth complementation group, exoH (Leigh et al., Cell 51:579-587, 1987), produce an altered exopolysaccharide and also induce the formation of non-nitrogen-fixing nodules. Mutants in the remaining four complementation groups produced less Calcofluor-binding material than the wild type. Mutants carrying mutations in two of these complementation groups (exoK and exoN) formed apparently normal, nitrogen-fixing nodules, while mutants in the other two groups (exoG and exoJ) formed normal nodules less efficiently than the wild type.Rhizobia fix nitrogen in symbiotic association with leguminous plants. In the course of this association, the bacteria induce the formation of nodules on the roots of the plant, enter these nodules through tubes called infection threads, and differentiate and begin to fix nitrogen once inside (for reviews see references 2, 20, 30, and 44). Considerable attention has been devoted to the mechanisms by which a Rhizobiqm strain and its host plant might recognize each other and by which the bacteria enter the nodules which they induce. It has been hypothesized that surface or extracellular polysaccharides produced by the bacterial symbiont are involved in these processes (2, 10). Mutants that do not produce an acidic exopolysaccharide have been described for several strains of 13,17,28,35), and in most cases such mutants form empty, non-nitrogen-fixing nodules (6-8, 13, 17, 28, 35). Exopolysaccharide-deficient mutants that fail to nodulate or that are nodulation and fixation proficient have also been described (5, 38).We have previously described TnS insertion mutants of Rhizobium meliloti RmlO21 that do not produce a Calcofluor-binding, acidic exopolysaccharide (17, 28). These exo mutants fail to invade the nodules they induce on alfalfa, which are consequently Fix-(do not fix nitrogen). All of the nod genes are required for nodule induction by these mutants (26 (18,23). Plasmids that complement these mutations have been isolated from a library of R. meliloti DNA (28). In this paper we describe the mapping of the region covered by these plasmids and the isolation of more TnS insertion mutations in this region. The number of loci in the cluster affecting exopolysaccharide biosynthesis now stands at 12. MATERIALS AND METHODSStrains, plasmids, and media. Bacterial strains and plasmids are listed in Table 1. Bacteria were grown in LB medium (32), with 2.5 mM M...
Gene 5 protein of bacteriophage T7 is a nonprocessive DNA polymerase. During infection of Escherichia col, T7 annexes the host protein thioredoxin for use as a processivity factor for 17 DNA polymerase. We describe here a genetic method to investigate the interaction between T7 gene 5 protein and E. cofl thioredoxin. The strategy is to use thioredoxin mutants that are unable to support the growth of wild-type T7 phage to select for T7 revertant phage that suppress the defect in thioredoxin. A thioredoxin mutation that replaces glycine at position 74 with aspartic acid fails to support the growth of wild-type T7. This mutation is suppressed by six different mutations within T7 gene 5, each of which results in a single amino acid substitution within gene S protein. Three of the suppressor mutations are located within the putative polymerization domain of gene 5 protein, and three are located within the putative 3'-to-5' exonucleolytic domain. Each sup pressor mutation alone is necessary and sufficient to confer the revertant phenotype.The efficient polymerization of nucleotides at a replication fork is best accomplished by a highly processive mechanism of DNA synthesis. Processive DNA synthesis can lead to the incorporation of thousands of nucleotides in a single binding event between a DNA polymerase and a primer-template. In contrast, a completely nonprocessive DNA polymerase dissociates after the incorporation of a single nucleotide. In Escherichia coli, the DNA polymerase III holoenzyme consists of 10 polypeptides, with the 13 and r subunits and the five-polypeptide 'y complex being required for processive polymerization (1,2). Processivity of the bacteriophage T4 DNA polymerase, the product of gene 43, is increased by three accessory proteins, the products ofT4 genes 44, 62, and 45 (3, 4). The DNA polymerase of herpes simplex virus type 1 requires the viral UL42 gene product for processive DNA synthesis (5). The mammalian DNA polymerase, polymerase 8, interacts with the proliferating cell nuclear antigen (PCNA) and replication factor C (RF-C) to increase its processivity (6-9). The yeast analogs of mammalian PCNA and RF-C interact with yeast DNA polymerase III to increase the processivity of DNA synthesis (10, 11).The DNA polymerase of bacteriophage T7, the subject of this communication, is the 80-kDa product of the viral gene 5 (12). The gene 5 protein has low processivity, dissociating from the primer-template after catalyzing the incorporation of 1-50 nucleotides (13). Upon infection, phage T7 annexes a host protein, thioredoxin, as a processivity factor for the gene 5 protein (14, 15). E. coli thioredoxin binds tightly (Km = 5 nM) to T7 DNA polymerase in a 1:1 stoichiometry. Thioredoxin stabilizes the binding of gene 5 protein to a primer-template by 20-to 80-fold and increases the processivity of polymerization by 1000-fold (13, 16). The characteristics of the gene 5 protein-thioredoxin interaction-a high affinity, a 1:1 stoichiometry, and the ability to form a stable complex without an energy r...
Upon infection of Escherichia coli, bacteriophage T7 annexes a host protein, thioredoxin, to serve as a processivity factor for its DNA polymerase, T7 gene 5 protein. In a previous communication (Himawan, J., and Richardson, C. C. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9774 -9778), we reported that an E. coli strain encoding a Gly-74 to Asp-74 (G74D) thioredoxin mutation could not support wild-type T7 growth and that in vivo, six mutations in T7 gene 5 could individually suppress this G74D thioredoxin defect. In the present study, we report the purification and biochemical characterization of the G74D thioredoxin mutant and two suppressor gene 5 proteins, a Glu-319 to Lys-319 (E319K) mutant of gene 5 protein and an Ala-45 to Thr-45 (A45T) mutant. The suppressor E319K mutation, positioned within the DNA polymerization domain of gene 5 protein, appears to suppress the parental thioredoxin mutation by compensating for the binding defect that was caused by the G74D alteration. We suggest that the Glu-319 residue of T7 gene 5 protein and the Gly-74 residue of E. coli thioredoxin define a contact point or site of interaction between the two proteins. In contrast, the A45T mutation in gene 5 protein, located within the 3 to 5 exonuclease domain, does not suppress the G74D thioredoxin mutation by simple restoration of binding affinity. Based upon our understanding of the mechanisms of suppression, we propose a model for the T7 gene 5 protein-E. coli thioredoxin interaction.The concept of using genetic or suppressor analysis to investigate protein-protein interaction can be described as follows. If two proteins form a complex, then there must exist a contact point, or more likely, several contact points between them. These contact points would be defined by certain amino acid residues of one protein that must be physically adjacent to certain amino acid residues of the other protein. If a contact point amino acid from one protein is structurally altered significantly by mutation, then complex formation with the second protein would be destroyed. Theoretically, a productive complex could be formed once again by an alteration in the second protein that structurally compensates for the original mutation. Therefore, by mutating one protein of a complex and selecting for extragenic suppressor mutations in the second protein, one should be able to identify the contact points between the two proteins.We (1) have used extragenic suppressor analysis to investigate the interactions between two proteins that are involved in DNA replication in Escherichia coli infected with bacteriophage T7. Similar to our studies, other workers have also used suppressor analysis to study protein-protein interactions in the E. coli DNA replication system (2) and also in the DNA replication system of the yeast Saccharomyces cerevisiae (3). Specifically, we have been investigating by suppressor analysis the interaction between T7 gene 5 protein and E. coli thioredoxin. T7 gene 5 protein, the DNA polymerase of phage T7 (4, 5), has two enzymatic act...
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