Using an expression cloning technique, we isolated cDNAs for eight M phase phosphoproteins (MPPs 4-11). We then used affinity-purified antibodies to fusion proteins to characterize the intracellular localization and some biochemical properties of these proteins and two others that we identified previously (MPPs 1-2). Each antibody immunoprecipitated one or two protein species of a characteristic size ranging from 17,000 to 220,000 Da. Each MPP, when immunoprecipitated from lysates of M phase cells, was reactive with MPM2, a monoclonal antibody that recognizes a group of related M phase phosphorylation sites, including F-phosphoT-P-L-Q. This reactivity indicated that all the MPPS encoded genuine M phase phosphoproteins. When antibodies to the MPPS were used for immunofluorescence microscopy, each anti-MPP gave a characteristic pattern of localization. In interphase, several of the MPPs were nuclear proteins, whereas others were cytoplasmic or distributed throughout the cell. Three MPPS were strikingly localized to interphase structures: MPP7 to centers of DNA replication, MPP9 to the Golgi complex, and MPP10 to nucleoli. In mitosis, most of the MPPs were distributed throughout the cells. Further studies of the 10 MPPs, most of which are previously undescribed, are expected to provide new understandings of the process of cell division.

The cysteine regulons of Salmonella typhimurium and Escherichia coli are positively regulated by CysB protein and either O-acetyl-L-serine or N-acetyl-L-serine, both of which act as inducers. Gel mobility shift assays and DNase I footprinting experiments showed that CysB protein binds to the S. typhimurium cysK promoter at two sites, one, designated CBS-K1, at positions -78 to -39 relative to the major transcription start site, and the other, designated CBS-K2, at positions -115 to -79. The S. typhimurium cysJIH promoter was found to contain a single binding site, designated CBS-JH, at positions -76 to -35. Acetyl-L-serine stimulated binding to CBS-K1 and CBS-J and inhibited binding to CBS-K2. In the absence of acetyl-L-serine, CysB protein bound to both CBS-K1 and CBS-K2 and gave a complex that migrated more slowly during gel electrophoresis than did that formed in the presence of acetyl-L-serine, in which case CysB protein bound only to CBS-K1. Complexes formed with DNA containing the two binding sites either at the middle or at one end of the fragment migrated differently, suggesting that DNA was bent in the slow complex formed in the absence of acetyl-L-serine and that DNA in the fast complex was less bent or not bent at all. An analysis of upstream deletions of the cysK promoter showed that only CBS-K1 is required for in vivo promoter activity. CBS-J is analogous in position to CBS-K1 and is probably also required for activity of the cysJIH promoter. CBS-K2 has no known function but may help sequester CysB protein at the cysK promoter.Genes of the cysteine regulon in Salmonella typhimurium and Escherichia coli are positively regulated by the CysB protein (13, 15, 16), a tetramer of identical 36-kDa subunits (20,24) that is a member of the LysR family of bacterial activator proteins (9). In addition to requiring CysB protein, derepression of this biosynthetic pathway requires sulfur limitation and the L-cysteine precursor O-acetyl-L-serine, which acts as an inducer (14,15). N-Acetyl-L-serine is also an inducer (25) and is derived nonenzymatically from O-acetyl-L-serine by an intramolecular O-to-N-acetyl shift (6). The requirement for sulfur limitation appears to be due to an anti-inducer effect of sulfide, which interferes with the ability of CysB protein and inducer to activate gene expression both in vivo and in vitro (14,15,26).In vitro studies have shown that transcription initiation from the cysJIH promoter requires CysB and either N-acetyl-L-serine or O-acetyl-L-serine (25) promoter activity. Evidence is also presented that indicates that binding of CysB protein to the cysK promoter induces DNA bending in the absence of inducer but little or no bending in the presence of inducer. MATERIALS AND METHODSBacterial strains, plasmids, and media. E. coli JM105 was the transformation recipient for isolating pUC19 derivatives (36) and the host for M13 phage propagation. NM522 [hsdA5 A(lac-pro)(F' pro' lacIqZAMJ5)] was the host for pT7T3 phagemid derivatives. S. typhimurium strains included the wild-type LT2 st...

Nucleotide sequences of the cysK regions of SalmoneUa typhimurium and Escherichia coli have been determined. A total of 3,812 and 2,595 nucleotides were sequenced from S. typhimurium and E. coli, respectively. Open reading frames of 323 codons were found in both species and were identified as those of cysK by comparison of deduced amino acid sequences with amino-and carboxyl-terminal amino acid analyses of the S. typhimurium cysK gene product O-acetylserine (thiol)-lyase A. The two cysK DNA sequences were 85% identical, and the deduced amino acid sequences were 96% identical. The major transcription initiation sites for cysK were found to be virtually identical in the two organisms, by using primer extension and Si nuclease protection techniques. The -35 region corresponding to the major transcription start site was TTCCCC in S. typhimurium and TTCCGC in E. coli. The deviation of these sequences from the consensus sequence TTGACA may reflect the fact that cysK is subject to positive control and requires the cysB regulatory protein for expression. Sequences downstream of cysK were found to include ptsH and a portion of ptsl, thus establishing the exact relationship of cysK with these two genes. A 290-codon open reading frame, which may represent the cysZ gene, was identified upstream of cysK.Salmonella typhimurium and Escherichia coli synthesize L-cysteine from O-acetyl-L-serine and sulfide in a reaction catalyzed by O-acetylserine (thiol)-lyase (formerly designated O-acetylserine sulfhydrylase [4,24]). Two such enzymes, termed A and B, are found in S. typhimurium (5) and are coded for by cysK and cysM, respectively (18,19). Both cysK and cysM are closely linked to cysA and to the crr ptsI ptsH cluster located at 49 min on the S. typhimurium chromosome (41) and at 52 min on the E. coli chromosome (3). Native O-acetylserine (thiol)-lyase A has a molecular weight of 68,000, contains 2 molecules of pyridoxal phosphate and is composed of two identical 34-kilodalton subunits (4, 6). The large excess of the A isozyme over the B isozyme during aerobic growth indicates that, under these conditions, the former accounts for the majority of Lcysteine synthesis from O-acetyl-L-serine and sulfide (18,23). Unlike the A isozyme, O-acetylserine (thiol)-lyase B also catalyzes the formation of S-sulfocysteine from 0-acetyl-L-serine and thiosulfate (30, 31) and is required for efficient cysteine biosynthesis during anaerobic growth (12). Both enzymes are presumed to exist in E. coli as well, even though only cysK and its product have been demonstrated in this species (6,13).Genes for the enzymes of cysteine biosynthesis are scattered widely on the bacterial chromosome and are regulated by a system of positive control termed the cysteine regulon. Derepression of the cysteine regulon requires a combination of sulfur starvation, the L-cysteine precursor and coinducer 0-acetyl-L-serine, and the protein encoded by the cysB regulatory gene (21-23). The elucidation of the molecular mechanisms involved in this system of positive regulation...

We have prepared a library of Salmoneila typhimurium genonic fragments cloned in pBR322 and packaged in P22HT capsids. Plasmids carrying 24 of 26 specific genes searched for were isolated by transduction at frequencies of 1 to 344 per 106 plasmid transductants. All 11 known genes of the cysteine regulon were isolated from this library, indcluding cysK, which we had previously been unable to clone in a recombinant plasmid with an Escherichia coli host. This library provides a simple and rapid method for isolating most S. typhimurium genes by using S. typhimurium itself as a host and should be particularly useful for cloning genes that might be deleterious to E. coli.Genes from enteric bacteria are often cloned in Escherichia. coli hosts owing to high transformation efficiencies and the availability of strains with a large variety of mutant markers. Selection for specific genes is mnade possible by a high degree of genetic relatedness between species, which usually allows a gene product from one to replace the function of its counterpart in another. Exceptions to this rule might be expected for proteins that interact with other species-specific macromolecules, such as those found in mtiembranes or cell walls, and for components of DNA restriction-modification systems (1). Another possible exception is our inability to subclone the Salmonella typhimurium cysK gene in pBR322 by selecting for Cys+ in a cysK E. coli host, even though it was easily obtained in a X phage vector (10). As a result, we investigated the possibility of cloning cysK and other cys genes as well in pBR322 by using S. typhimurium itself as a host. Poor transformation efficiencies of cys mutants led us to create a plasmid library of S. typhimurium DNA fragments packaged in P22HT capsids. We describe the construction of this library and document its content and the ease with which it can be screened for specific genes by transduction. MATERIALS AND METHODSStrains and culture media. All strains were derivatives of S. typhirnuriumn LT2. LB5000 is metA22 metE551 trpD2 leu hsdLT hsdSA hsdSB and is m+ for all three modification systems; LB5010 is a galE derivative of LB5000. Both strains Were obtained from L. Bullas (3). DW378 is trpC109 cysK1772 cysMl770 (11), and SB2309 is trpB223 A(cysK ptsHI41) (5). Other auxotrophic strains used for screening our phage P22 plasmid library were from P. Hartman or K. E. Sanderson, Salmonella Genetic Stock Centre, University of Calgary. Phage P22HT int3 was obtained from M.Stem. The pBR322 derivative pRSM3 was constructed in this laboratory by R. Monroe and carries the cysB region of S. typhimurium LT2 on a 2.7-kilobase (kb) SafI fragment that appears to be identical to that isolated from the LT7 strain (14).Medium E of Vogel and Bonner (25) (19) was used as the rich medium and was supplemented with 0.1 mM L-cystine for the growth of cysteine auxotrophs. Solid medium contained 1.5% agar and either ampicillin (25 ,ug/ml) or tetracycline (12.5 ,ug/ml) when required. Transformation. The method of Hanahan (9) was modifie...