Extragenic suppressor mutations which had the ability to suppress a dnaX2016(Ts) DNA polymerization defect and which concomitantly caused cold sensitivity have been characterized within the dnaA initiation gene. When these alleles (designated Cs, Sx) were moved into dnaX ؉ strains, the new mutants became cold sensitive and phenotypically were initiation defective at 20؇C (J. R. Walker, J. A. Ramsey, and W. G. Haldenwang, Proc. Natl. Acad. Sci. USA 79:3340-3344, 1982). Detailed localization by marker rescue and DNA sequencing are reported here. One mutation changed codon 213 from Ala to Asp, the second changed Arg-432 to Leu, and the third changed codon 435 from Thr to Lys. It is striking that two of the three spontaneous mutations occurred in codons 432 and 435; these codons are within a very highly conserved, 12-residue region (K. Skarstad and E. Boye, Biochim. Biophys. Acta 1217:111-130, 1994; W. Messer and C. Weigel, submitted for publication) which must be critical for one of the DnaA activities. The dominance of wild-type and mutant alleles in both initiation and suppression activities was studied. First, in initiation function, the wild-type allele was dominant over the Cs, Sx alleles, and this dominance was independent of location. That is, the dnaA ؉ allele restored growth to dnaA(Cs, Sx) strains at 20؇C independently of which allele was present on the plasmid. The dnaA(Cs, Sx) alleles provided initiator function at 39؇C and were dominant in a dnaA(Ts) host at that temperature. On the other hand, suppression was dominant when the suppressor allele was chromosomal but recessive when it was plasmid borne. Furthermore, suppression was not observed when the suppressor allele was present on a plasmid and the chromosomal dnaA was a null allele. These data suggest that the suppressor allele must be integrated into the chromosome, perhaps at the normal dnaA location. Suppression by dnaA(Cs, Sx) did not require initiation at oriC; it was observed in strains deleted of oriC and which initiated at an integrated plasmid origin.The Escherichia coli dnaX gene encodes the and ␥ subunits of DNA polymerase III holoenzyme (20,38,51,86). , the larger protein (71.1 kDa), results from translation of the complete 643-codon message. ␥, the shorter product (47.5 kDa), results from a programmed Ϫ1 ribosomal frameshift over messenger codons 428 to 430 (10, 21, 72, 73, 75). This shift results in the translation of one unique amino acid, as residue 431, which is followed by a stop codon. The net result is that and ␥ are identical over the N-terminal 430 residues. The dnaX gene was defined by two temperature-sensitive (Ts) mutations, dnaX2016(Ts) (19) and dnaX36(Ts) (32). The dnaX2016(Ts) mutation changes codon 118 from glycine (GGT) to aspartate (GAT) and affects both and ␥ (7). Shifting the dnaX2016(Ts) mutant from the permissive temperature of 30ЊC to 42ЊC causes an immediate stop in DNA synthesis, as expected for a defect in polymerization, and growth gradually ceases (16). The dnaX36(Ts) mutation changes codon 601 from glutamat...

SummaryTemperature sensitivity of DNA polymerization and growth, resulting from mutation of the t and g subunits of Escherichia coli DNA polymerase III, are suppressed by Cs,Sx mutations of the initiator gene, dnaA. These mutations simultaneously cause defective initiation at 208C. Efficient suppression, defined as restoration of normal growth rate at 398C to essentially all the cells, depends on functional oriC. Increasing DnaA activity in a strain capable of suppression, by introducing a copy of the wild-type allele, increasing the suppressor gene dosage or introducing a seqA mutation, reversed the suppression. This suggests that the suppression mechanism depends on reduced activity of DnaACs,Sx. Models that assume that suppression results from an initiation defect or from DnaACs,Sx interaction with polymerization proteins during nascent strand synthesis are proposed.

Clostridium taeniosporum, a non-pathogenic anaerobe closely related to the C. botulinum Group II members, was isolated from Crimean lake silt about 60 years ago. Its endospores are surrounded by an encasement layer which forms a trunk at one spore pole to which about 12–14 large, ribbon-like appendages are attached. The genome consists of one 3,264,813 bp, circular chromosome (with 26.6% GC) and three plasmids. The chromosome contains 2,892 potential protein coding sequences: 2,124 have specific functions, 147 have general functions, 228 are conserved but without known function and 393 are hypothetical based on the fact that no statistically significant orthologs were found. The chromosome also contains 101 genes for stable RNAs, including 7 rRNA clusters. Over 84% of the protein coding sequences and 96% of the stable RNA coding regions are oriented in the same direction as replication. The three known appendage genes are located within a single cluster with five other genes, the protein products of which are closely related, in terms of sequence, to the known appendage proteins. The relatedness of the deduced protein products suggests that all or some of the closely related genes might code for minor appendage proteins or assembly factors. The appendage genes might be unique among the known clostridia; no statistically significant orthologs were found within other clostridial genomes for which sequence data are available. The C. taeniosporum chromosome contains two functional prophages, one Siphoviridae and one Myoviridae, and one defective prophage. Three plasmids of 5.9, 69.7 and 163.1 Kbp are present. These data are expected to contribute to future studies of developmental, structural and evolutionary biology and to potential industrial applications of this organism.

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