A partial nucleotide sequence that included 1,693 base pairs of the M12 (emml2) gene of group A streptococci (strain CS24) and adjacent upstream DNA was determined. Type 12 M protein-specific mRNA of strain CS24 is transcribed from two promoters (P, and P3) (35) it has been shown that the switch from high to low levels of M-protein expression occurs at high frequency and is reversible, which are characteristics of phase variation described for other bacterial species (7,26). In addition to phase variation of M-protein expression (35), streptococci also exhibit both antigenic variation (more than 70 strains with antigenically distinct M proteins have been described) and size variation of this protein (11). The mechanisms that control phase and antigenic variation and the relationship between them is not understood. Although the nature of M protein as an antiphagocytic molecule has been demonstrated, a role for the M-state in the survival of the organism has not been identified. Phase variation has been postulated to be a virulence factor that controls the expression of the type 1 pilus of Escherichia coli (6, 7) and the pilus of Neisseria gonorrhoeae (26 report results of a study of the role of upstream neighboring sequences in the regulation of expression of the emmi2 gene of strain CS24 in E. coli, describe the physical relationship between the deletion in strain CS64 and the 5' end of the emml2 gene, and study the transcriptional activity of the emml2 gene by Northern and primer extension analyses. We found that transcription of the emml2 gene in strain CS64 is diminished by more than 100-fold and that the deletion is more than 400 bases upstream from the transcription start sites of the emml2 gene. MATERIALS AND METHODSBacterial strains, plasmids, bacteriophage, and media. M type 12 group A streptococcal strain CS24 and variant CS64 have been described previously (3,39). Liquid cultures of group A streptococci were grown in Todd-Hewitt broth supplemented with 1% Neopeptone (Difco Laboratories, Detroit, Mich.) for 15 to 18 h at 37°C. E. coli JM83, JM103, and JM110, carrying constructs of plasmids pUC19 and pUC18, were propagated in L broth or on L agar containing ampicillin (30 ,ug/ml) and 5-bromo-4-chloro-3-indolyl-P-Dgalactoside (0.03%) at 37°C. All plasmids used and constructed in this study are listed in Fig. 1

In the unicellular biflagellate green alga Chlamydomonas reinhardtii each basal body is linked to the nucleus by a fibrous nucleus-basal body connector (NBBC) that contains the calcium-binding protein centrin. (Wright et al.: Journal of Cell Biology 101:1903-1912.; Salisbury et al.: Journal of Cell Biology 107:635-642; Huang et al.: Journal of Cell Biology 107:121-131). In order to explore the cellular function of the NBBC we used antiserum directed against centrin to examine a number of mutants known to be defective for basal body assembly and/or localization. Of three variable flagella-number mutants examined, one, vfl-2, is dramatically defective with respect to the NBBC in that 1) the union between basal bodies and nucleus is very labile, 2) there is no detectible centrin in the NBBC region, and 3) total cellular centrin levels are reduced 75-80% relative to wild type. The existence of these defects in a mutant incapable of maintaining normal flagellar number supports the view that the NBBC plays an important role in determining proper basal body localization and/or segregation. In contrast to vfl-2, the mutants vfl-1, vfl-3, uni-1, and bald-2 contain approximately normal levels of centrin and possess stable NBBCs. The observation of NBBCs in the mutant bald-2, which lacks all but very rudimentary basal bodies, indicates that the assembly of the NBBC does not require fully formed basal bodies and that such assembly may not require basal bodies at all. Finally, the possibility that the NBBC is required for induction of gene expression following deflagellation was tested by examining vfl-2 for such induction. Results indicate that the connector does not play a necessary role in the induction process.

The New York Bight extends seaward some 80 to 100 miles (ca. 129 to 161 km) from the Long Island and New Jersey shorelines to the edge of the continental shelf. Over 14 x 106 m3 of sewage sludge, dredge spoils, acid wastes, and cellar dirt are discharged into this area each year. Large populations of Bacillus sp. resistant

The M protein is the antigen on the surface of group A streptococci that allows these bacteria to resist phagocytosis. DNA encoding the M12 protein was cloned into Escherichia coli and used as an isotopically labeled hybridization probe to compare genomic DNA's isolated from M+ and M- isogenic cultures in an effort to elucidate the genetic basis of this variation. DNA's from two spontaneous, independent M- variants contained small (approximately 50 base pairs) deletions which were mapped to identical restriction fragments within or adjacent to the M protein coding sequence. Taken together with the pleiotropic nature of these deletions, this suggests that they define a regulatory switch.