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In this paper, we describe a new mutation, gicD1, that gives a cold-sensitive phenotype in bacterial cell growth. Complementation analysis showed gicD1 to be allelic to infB. We identify gicD1 to be a valine to isoleucine substitution in initiation factor-2 (IF2) of a residue that seems to be well conserved in eubacterial IF2 proteins. This mutation lies in a region distant from the G-domain to which all earlier reported cold-sensitive mutations cluster. We describe a novel phenotype of the mutant that is suppression of rpsL31-mediated streptomycin resistance in cold. We provide evidence that mutant IF2 specifically interacts with rpsL31 in cold, leading to a bacteriostatic effect on host cells.

Extrachromosomal circular DNA molecules consisting of IS1-cat repeats, (IS1-cat)n, were isolated from an E. coli strain harboring nearly 30 copies of tandemly amplified transposon Tn9 located on the chromosome. The DNA 'circles' were characterized by restriction analysis followed by Southern blotting and electron microscopic examination. Their size varied from approximately 5.5 kb to 53 kb.

We have mapped the extents of two deletion sbcA mutations which result in production of DNA exonuclease VIII (ExoVIII). One mutation, sbcA8, deletes about 140 kb of DNA which includes most of the Rac prophage and the trg gene. Western blot analysis shows that the protein produced is larger than wild type ExoVIII. The nucleotide sequence shows that a translational gene fusion has occurred. The N-terminal 294 codons of recE have been deleted and the remaining C-terminal codons have been fused to the N-terminal portion of another reading frame we call sfcA. Analysis of the protein sequence encoded by sfcA shows an 83% similarity with rat and mouse NADP-linked malic enzyme. We discuss the possibility that sfcA is identical to maeA which encodes NAD-linked malic enzyme from Escherichia coli. Restriction nuclease analysis of a second deletion, sbcA81, by Southern blot technique indicates that about 105 kb of DNA have been deleted and a transcriptional gene fusion has occurred between recE and the regulatory region of an E. coli chromosomal gene. We also examined eight other sbc mutations that result in ExoVIII production. Five have no effect on restriction nucleotide fragment sizes detected by complementarity to lambda rev as probe. These are presumed point mutations. Three seem to produce additional restriction nucleotide fragments complementary to lambda rev. The possible nature of these sbc mutations is discussed.

A new pleiotropic mutation, designated cup-1 (for carbohydrate uptake), which impairs the ability of Escherichia coli cells to grow on a large number of phosphotransferase system (PTS) and non-PTS carbohydrates by blocking their entry into the cells, has been isolated, partially characterized, and mapped. The mutants grew poorly even on rich and glucose minimal media. Fast-growing revertants rapidly accumulated in cultures grown on either of the above two media and made stable maintenance of the mutation difficult. Several extragenic suppressor mutations that permitted cup cells to grow on specific single sugars or groups of sugars have been isolated. One such suppressor, which enabled cup cells to grow as well on glycerol minimal medium as their wild-type parent, has been helpful in stably maintaining these cells in this medium. cup-1 has been mapped to 97 min on the standard E. coli map. It cotransduced with a transposon Tn10 inserted clockwise to it and (very weakly) with uxuA. Surprisingly, it failed to cotransduce with pyrB, argI, or valS, three markers located nearby but counterclockwise to it. In F' merodiploids, cup-1 was dominant over its cup+ allele. Cyclic AMP permitted growth of cup-1 cells on some sugars but not all. Apparently, reduced cyclic AMP level and therefore noninduction of several sugar operons is one but not the only effect of cup.

The recombinational processes directed by the RecBC and the RecF pathways following conjugation in E. coli have been compared. The viable recombinant products of the RecF pathway show a higher incidence of mismatch correction, higher percentage of heterogeneous clones produced by single ex-conjugants and a much slower rate of integration and segregation compared to the RecBC pathway. There are reasons to suspect that the product of recB and recC genes may be necessary for conversion of the single stranded donor DNA in the zygote to double stranded DNA. Theoretical considerations suggest that an exchange involving only one strand of DNA may be a much slower process, with more stringent homology requirement for the entire exchanged segment, than a double strand exchange of a comparable length; the latter should be much faster, with stringent homology requirements for only the terminal regions of the exchanged segments. It is suggested that the RecF pathway mainly mediates replacement of relatively long stretches of single strands of recipient DNA by the corresponding strands of donor DNA while the RecBC pathway mediates exchange of mostly double stranded DNA between the donor and the recipient; in addition, the RecBC pathway may also catalyze the integration of very small segments of single strands of the donor DNA. A model based on the above basic hypothesis is described. It is further suggested that the enzymes exonucleaseV and exonucleaseI Control the relative yields of the recombinants produced by the two pathways by regulating the supply of the donor substrates required by these pathways; the former diverts the potential substrate of the RecF pathway (single stranded DNA) to the duplex substrates of the RecBC pathway while the latter destroys the substrates of the RecF pathway, especially in absence of exonucleaseV.

Conventional methods for chromosomal mapping in Escherichia coli are (i) interruption of matings to obtain minimum marker entry times, (ii) linkage analysis of recombinants, and (iii) cotransduction. Method (i) has a resolution of about 0.5 min (5 x 10(4) nucleotides) and is not useful for distances less than about 1 min; methods (ii) and (iii) are capable of better resolution but are generally not very reproducible and no general theory is available for translating crossing-over and cotransduction frequencies into physical chromosomal distances. We found that when merozygotes are irradiated (X rays or ultraviolet light) soon after marker transfer, high linkage values (0.8 to 1.0) between nearby marker pairs decrease with radiation dose to 0.5. Our results are quantitatively consistent with the idea that radiations induce crossing-over lesions proportional to dose, and the number of such lesions between two markers is proportional to the physical separation of the markers in the range that can also be measured by interruption of mating (0.5 to 4.0 min). Additivity relations among markers are also satisfied. We used this technique to measure the distances (0.1 to 1.0 min) between several pairs of closely linked markers.