An extracellular protease of Staphylococcus aureus, strain V8, previously shown to cleave specifically the peptide bonds on the carboxyl-terminal side of either aspartate or glutamate residues in phosphate buffer (pH 7.8) hydrolyzes only glutamoyl bonds in either ammonium bicarbonate (pH 7.8) or ammonium acetate (pH 4.0). Of all aspartoyl bonds tested, only the Asp-Gly linkage is cleaved at a detectable rate. The staphylococcal protease hydrolyzes all of the seventeen different glutamoyl bonds studied, although those involving hydrophobic aminoacid residues with bulky side chains are cleaved at a lower rate.-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~T he purification of a proteolytic enzyme from the culture filtrates of Staphylococcus aureus, strain V8, has recently been reported (1). Partial characterization of this protease indicated that it had a molecular weight of 12,000, was inhibited by diisopropyl fluorophosphate, and was active over the pH range of 3.5-9.5, but exhibited maximal activity at pH 4.0 and 7.8 with hemoglobin as substrate. Digestion of oxidized ribonuclease by' the protease, followed by identification of the amino-and carboxyl-terminal residues of the liberated peptides, revealed that the protease cleaved specifically peptide bonds on the carboxyl-terminal side of either aspartic acid or glutamic acid. The specificity of the protease for bonds involving only dicarboxylic amino acids was also demonstrated by the inability of the enzyme to degrade casein in which all of its carboxyl groups had been coupled with glycine ethyl ester in amide linkage, whereas unmodified casein was readily digested.At pH 7.8, in sodium or potassium phosphate buffer, both aspartoyl and glutamoyl peptide bonds were cleaved (1). 2,3; 9,10; 49,50; 86,87; and 111,112. Apparently, none of the five aspartoyl bonds also present in ribonuclease had been hydrolyzed.

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A new gene, designated rcsF, was located adjacent to drpA at the 5.2-min position of the genetic map of Escherichia coli. The deduced amino acid sequence encoded by the rcsF gene indicates a small protein of 133 amino acid residues with a calculated pI of 10.8 that is rich in proline, serine, alanine, and cysteine residues. When overexpressed as a result of its presence on a multicopy plasmid, rcsF confers a mucoid phenotype and restores colony formation to ftsZ84 mutant cells on L agar medium containing no added NaCl. because of the presence of high levels of RcsA is not expressed at 37°C, and it has been suggested that this is due to a temperature-sensitive RcsA-RcsB interaction (unpublished observation cited in reference 9). The activation of the cps genes also depends on rcsC, a gene postulated to code for a membrane protein acting as a sensor that transduces an environmental signal by phosphorylating RcsB. This role for RcsC was deduced from the observation that RcsB is believed to be constitutively phosphorylated in a mutant strain harboring the rcsC137 allele (5). In our laboratory, we have observed that a presumably mutated EnvZ or PhoM protein can activate RcsB by cross talk. However, since this increase in the level of RcsB activity is reduced when the cell harbors a multicopy rcsC plasmid, it was proposed that wild-type RcsC is primarily a phosphatase enzyme (8).In the present study, we identify a new gene, which we designate rcsF, that when overexpressed suppresses the ftsZ84 mutation and stimulates colanic acid synthesis in the wild type but not in an rcsB mutant. It is suggested that the role of RcsF is to promote the phosphorylation of RcsB. MATERIALS AND METHODSMedia, chemicals, strains, and plasmids. The media and chemicals used were the same as those described previously (8). L broth contained 1.0% tryptone (NZ-Amine A; ICN Biochemicals Inc.), 0.5% yeast extract, and, unless otherwise specified, 0.5% NaCl. Before use, each new batch of the yeast extract was tested for its low salt content by estimating whether or not it promoted the growth of an ftsZ84 mutant in L agar without the addition of NaCl.Ampicillin and kanamycin were added at 100 ,ug/ml, and chloramphenicol and tetracycline were added at 20 and 15 ,ug/ml, respectively. 5-Bromo-4-chloro-3-indolyl-o-D-galactopyranoside (X-Gal) was added at 20 ,ug/ml. The tempera-8016

Wild-type genes which, when overexpressed, are capable of restoring the growth deficiency of the division mutantftsZ84 ofEscherichia coli on L medium containing no added NaCl have been isolated. One of these genes is rcsB, a positive regulator of colanic acid biosynthesis. A direct relationship between rcsB expression and FtsZ activity was observed, suggesting that RcsB specifically increases transcription offtsZ, thus accounting for the restoration of colony formation byftsZ84 mutant cells. Analysis of the 5' upstream sequence of rcsB revealed, in addition to the &-4 promoter sequence previously reported, a presumptive &r70 promoter and LexA-binding site plus an upstream sequence that is found to be essential for the expression of rcsB on a plasmid. The absence of the c54 factor does not have a negative effect on the transcription of rcsB. The RcsB protein is an activator of its own synthesis, particularly in the presence of NaCl. Evidence which suggests that RcsB can be phosphorylated by a presumably modified EnvZ or PhoM sensor protein leading to a suppression of the growth deficiency of ftsZ84 mutant cells and to an increase in colanic acid production was obtained. We also demonstrated that the level of colanic acid is reduced when the cells carry a multicopy rcsC plasmid, suggesting that the RcsC sensor has phosphatase activity.Considerable evidence demonstrating a pivotal role for the ftsZ gene in the control of cell division in Escherichia coli has accumulated (for a review, see reference 21). One would therefore expect ftsZ expression or FtsZ activity to be tightly controlled and even to be subject to a multitude of regulatory signals. The genetic structure of ftsZ involves several transcriptional units with promoters initiating transcription from upstream genes, while no transcriptional terminators have been found (1, 34). Thus, it is likely that the extent of transcription from each one of these promoters would be influenced by the presence of regulatory signals whose concentrations are likely to be adjusted depending upon growth and environmental conditions. It has been proposed that the FtsZ activity of ftsZ84 mutant cells is too low for growth to occur on L agar containing no added NaCl (25). Indeed, the presence of a low-copy-number plasmid carrying the ftsZ84 allele can restore colony-forming abilities on this medium. Such a conclusion is in agreement with reports demonstrating that FtsZ activity is limiting for cell division (3) and that no growth occurs when its level is decreased below a certain threshold level (6).In our laboratory, we are engaged in the identification of regulator molecules which increase ftsZ expression or FtsZ activity. Our high levels, was capable of restoring division activity in a ftsZ84 strain at the nonpermissive temperature. This factor was shown to increase the cellular concentration of the FtsZ protein. These findings add further support to the contention that FtsZ is the limiting factor for cell division in theftsZ84 mutant (3). They also suggest that any factor ...