The nucleotide sequence of the gtJB gene from Streptococcus mutans coding

Bacteria, tomatoes, and trypanosomes all contain genes for a large protein with extensive homology to the regulatory subunit, ClpA, of the ATP-dependent protease of Escherichia coil, COp. AU members of the family have between 756 and 926 amino acids and contain two large regions, of 233 and 192 amino acids, each containing consensus sequences for nucleotide binding. Within these regions there is at least 85% similarity between the most distant members of the family. The high degree of similarity among the ClpA-like proteins suggests that Clp-like proteases are likely to be important participants in energy-dependent proteolysis in prokaryotic and eukaryotic cells.Energy-dependent proteolysis plays a key role in prokaryotic and eukaryotic cells by regulating the availability of certain short-lived regulatory proteins, ensuring the proper stoichiometry for multi-protein complexes, and ridding the cell of abnormal proteins (1)(2)(3). A homologous energy-dependent protease shared by evolutionarily diverse organisms has not been previously described. In this paper we provide evidence that the regulatory subunit of the ATP-dependent Clp protease of Escherichia coli has been conserved to an unusual degree in numerous prokaryotes and eukaryotes. The Clp protease of E. coli is a two-component ATP-dependent protease that contributes to the turnover of abnormal proteins (4-7). The large ClpA subunit (81,000 kDa) has intrinsic ATPase activity. The smaller ClpP subunit (21,000 (7,9).Sequencing of the cipA gene, encoding the regulatory subunit of the Cip protease, allowed us to discover that ClpA is a member of a family of well-conserved proteins with no previously described function. Members ofthe family include a second E. coli gene, called clpB, and genes from plants, trypanosomes, and Gram-positive and Gram-negative bacteria. E. coli contains two members of the family (see below)as does tomato (E.P., unpublished data) (Fig. 1). § § T. brucei has one gene with DNA sequence homology to the family (M.C., unpublished data); other organisms listed in Fig. 1 have not been tested for the presence of a second gene. Most of these sequences have not previously been published; the presence in the data banks of a fragment of the C terminus of the clpB gene (previously called ORF-BG) allowed others of us encountering homologous sequences to contact the author of the first publication (11) and subsequently contact each other. In the absence of a concerted effort to detect Clp-like genes in eukaryotic or other prokaryotic organisms, the serendipitous discovery of seven such genes and a fragment of an eighth suggests that the family is extremely widespread. The degree of conservation strongly suggests that the members of this family all represent the regulatory subunit for Clp-like energy-dependent proteases, although the possibility exists that the ClpA protein has evolved other ATPdependent regulatory functions as well.The proteins predicted from the DNA sequences of members of this family have two regions of particularly hig...

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The gtfB gene coding for a glucosyltransferase (GTF) activity of Streptococcus mutans GS-5 was isolated on a 15.4-kilobase DNA fragment by using a lambda L47.1 gene library. The activity was catalyzed by gene products of 150 and 145 kilodaltons which reacted with antibodies directed against both soluble and insoluble glucan-synthesizing GTFs. The enzyme present in crude Escherichia coli extracts synthesized both soluble and insoluble glucans. The enzyme was partially purified from lysates of the lambda DS-76 clone and synthesized both types of glucans in a primer-independent fashion. In addition, the purified enzyme exhibited a pI of approximately 5.0. Southern blot analysis indicated that the cloned GTF gene represented a contiguous nucleotide sequence on the strain GS-5 chromosome. Furthermore, evidence for the existence of a distinct gene sharing partial homology with gtfB was also obtained. The gtfB gene was subcloned into plasmid pACYC184 into E. coli and exhibited GTF activity when carried on GS-5 inserts as small as 5 kilobases. The approximate location of the GTF promoter and the direction of gene transcription were also determined. The cloned enzyme was not secreted through the cytoplasmic membrane of E. coli, since most of the activity was found in the cytoplasm and, in lesser amounts, associated with the cytoplasmic membrane. The gtfB gene was insertionally inactivated by introducing a gene fragment coding for erythromycin resistance into the GTF coding region. After transformation of strain GS-5 with the altered gene, transformants defective in insoluble glucan synthesis were identified. These results indicate that the gtfB gene codes for a GTF involved in insoluble glucan synthesis in strain GS-5.

The nucleotide sequence of the ftf gene from Streptococcus mutants GS-5 was determined. The deduced amino acid sequence indicates that the unprocessed fructosyltransferase gene product has a molecular weight of 87,600. A typical streptococcal signal sequence is present at the amino terminus of the protein. The processed enzyme is relatively hydrophilic and has a pI of 5.66. An inverted repeat structure was detected upstream from the ftf gene and may function in the regulation of fructosyltransferase expression. Sequencing of the regions flanking the gene revealed the presence of four other putative open reading frames (ORFs). Two of these, ORFs 2 and 3, appear to code for low-molecular-weight proteins containing amino acid sequences sharing homology with several gram-positive bacterial DNA-binding proteins. In addition, ORF 3 is transcribed from the ftf DNA coding strand. Partial sequencing of ORF 4 suggests that its gene product may be an extracellular protein.