The competence-related phenotypes of mutations in each of the four open reading frames associated with the comE locus of Bacillus subtilis are described. comEA and comEC are required for transformability, whereas the products of comEB and of the overlapping comER, which is transcribed in the reverse direction, are dispensable. Loss of the comEA product decreases the binding of DNA to the competent cell surface and the internalization of DNA, in addition to exhibiting a profound effect on transformability. The comEC product is required for internalization but is dispensable for DNA binding. ComEA is shown to be an integral membrane protein, as predicted from hydropathy analysis, with its C-terminal domain outside the cytoplasmic membrane. This C-terminal domain possesses a sequence with similarity to those of several proteins known to be involved in nucleic acid transactions including UvrC and a human protein that binds to the replication origin of the Epstein-Barr virus.Competent cells of Bacillus subtilis can bind, process, and internalize exogenous DNA via a process requiring several known proteins (for a review, see reference 6). Physical contact is first made with cell surface receptors of unknown identity, resulting in irreversible binding, which is followed by processing of the DNA by double-stranded cleavage and presentation to a transporter apparatus. Following internalization of singlestranded DNA, integration into the recipient chromosome occurs by homologous recombination. Our present understanding of how the competence apparatus functions relies largely on kinetic studies of the binding and uptake of radiolabeled DNA. A more detailed understanding of the specific role that each protein plays in the overall process in B. subtilis is the topic of this and other recent investigations (1,4,12,(20)(21)(22)26). comE is unique among four known competence transcription units, in that open reading frames (ORFs) within it have been shown in preliminary experiments to be essential for both the binding and uptake of DNA (11,12). In these reports we have described the identification, cloning, sequencing, and transcription mapping of the comE operon. This locus consists of four ORFs (see Fig. 1). Three are transcribed in the forward direction from a single, major apparent E A promoter, with comEB and comEC additionally transcribed from a minor promoter. The fourth ORF (comER) is transcribed in the reverse orientation from that of a promoter that is located near the end of comEA. comEA is preceded by a long untranslated leader sequence. Transcription of comER overlaps this leader and partially overlaps comEA. Insertion of Tn917-lacZ in comEA or -C has revealed that the latter is required only for DNA uptake but that insertion in comEA results in a binding defect. Since Tn917-lacZ insertions are polar and since transcription of comEA and comER overlaps, it was unclear whether inactivation of comEA, comEB, or comER was responsible for the observed binding-deficient phenotype of the Tn917 comEA insertion.In this study...

The binding and transport of DNA by competent Bacillus subtilis requires the assembly of a specialized apparatus. We present here the characterization of comE, an operon under competence control that is required for both DNA binding to the competent cell surface, and for uptake. comE contains three open reading frames (ORF1-3) read in the forward direction, preceded by a long untranslated leader sequence and an apparent E sigma A promoter. A minor promoter also is responsible for transcription of ORF2 and -3. A transcript containing a single ORF is produced in the reverse direction. The reverse ORF overlaps ORF1 and the untranslated comE leader. The comE transcript is present at a very low level during growth and at an elevated level in stationary-phase cells. Conversely, the reverse transcript is present during exponential growth and disappears during stationary phase. The reverse ORF resembles prokaryotic and eukaryotic pyrroline-5'-carboxylate reductases, while ORF2 is similar to several dCMP deaminases. ORF1 and ORF3 are predicted to be integral membrane proteins. The latter is specifically required for DNA uptake but not for binding.

The specific activity of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) increases 30-to 50-fold when dark-grown pea seedlings are shifted into the light. The large subunit (LS) of this multimeric protein is known to be synthesized in the chloroplast, but plastids from dark-grown cells contain relatively low levels of LS. However, despite the low level of LS synthesis in the plastids of dark-grown plants, these organelles contain significant levels of LS mRNA. Hybridization studies showed that the amount of LS mRNA increased about 3-fold, relative to total plant RNA, when dark-grown plants were illuminated. This increase in LS mRNA can be accounted for by a similar increase in chloroplast genome copy number. It was found that the amount of translatable LS mRNA per ltg of plastid RNA is similar when isolated from either dark-grown plants or dark-grown plants subjected to light. These results suggest that although light can increase the level of LS mRNA by increasing the copy number of this gene, the primary regulation of LS synthesis by light in pea chloroplasts is at the level of translation.

Putative membrane invagination sites at which intracytoplasmic photosynthetic membrane growth is initiated in Rhodopseudomonas sphaeroides can be isolated in an upper pigmented fraction by rate-zone sedimentation. The intracellular localization of membranes present in the isolated fraction was investigated with the impermeant surface-labeling reagent pyridoxal 5'-phosphate, which has been shown to diffuse into the periplasmic space and to label proteins of both the peripheral cytoplasmic membrane and the mature intracytoplasmic membrane. A comparison of the extent of labeling at 25 and 0°C was consistent with the possibility that membranes present in the upper pigmented fraction arise from sites near the cell periphery. Pronase digestion of the surface-labeled membranes suggested further that the purified upper fraction consisted largely of open membrane fragments and that the majority of the intracytoplasmic membrane is labeled by this procedure. The pigmented membrane growth initiation sites were separated partially from undifferentiated respiratory cytoplasmic membrane also present in the upper fraction.

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