Cloning and physical mapping of the cysB region of Salmonella typhimurium.

The cysB region of Salmonella typhimurium was cloned in pBR322 and localized to a 1.75-kilobase HincII fragment. Two-dimensional protein electropherograms showed levels of the cysB polypeptide chain that were several fold higher in plasmid-bearing strains than in the wild type. Fully derepressed levels of sulfite reductase and O-acetylserine sulfhydrylase in cysB plasmid-bearing strains were only 25% higher than in the wild type, suggesting that the product of this regulatory gene ordinarily is not a limiting factor in the expression of the cysteine regulon. The mapping of cysB deletions by Southern blots showed a good correlation between the genetic and the physical maps of this gene. The supX gene was initially cloned with cysB and is within 0.7 kilobase of cysB.     

The regulatory cysK mutant of S. typhimurium.

A triazole-resistant mutant, cysK1358, showing novel properties was isolated. Biochemical analysis of this strain suggests a regulatory character of the cysK1358 mutation. The cysK1358 mutation has a pleiotropic effect: the expression of the other gene, cysA, is also altered.     

Analysis of merodiploids of the cysB region in Salmonella typhimurium.

Summary We have studied the regulation of two cysteine biosynthetic enzymes in S. typhimurium merodiploid strains which are heterozygous at the cysB regulatory locus. This gene codes for an element of positive control which is necessary for the expression of the enzymes of the biosynthetic pathway. Under conditions of sulfur deprivation levels of sulfite reductase (coded for by cysI, cysJ and cysG) and of O-acetylserine sulfhydrylase (coded for by cysK) are derepressed in cysB ⁺ haploid strains, but not in cysB ^- haploid strains. Growth on a rich sulfur source such as l-cystine results in low levels of both enzyme activities in cysB ⁺ and cysB ^- haploid strains but not in cysB ^c haploid strains, where enzyme expression is constitutive, i.e. substantially greater than in a cysB ⁺ strain grown on l-cystine, regardless of the nutrients used for growth.We find that cysB ^-/F cysB ⁺ merodiploid strains can be derepressed for sulfite reductase and O-acetylserine sulfhydrylase by growth on a poor sulfur source, and therefore cysB ⁺ is dominant to cysB ^-. Enzyme levels are also derepressed in l-cystine-grown cysB ^c/F cysB ⁺ strains indicating that cysB ^c is dominant to cysB ⁺. The cysB484 allele is known to be cysB ^- in regard to the regulation of sulfite reductase activity, but cysB ^c with respect to O-acetylserine sulfhydrylase. In a cysB484/F cysB ⁺ strain the cysB ^- character of cysB484 is recessive to cysB ⁺, while cysB ^c is dominant to cysB ⁺.Merodiploids of the type cysB ^-/F cysB ⁺, bearing chromosomal point mutations are derepressed by sulfur deprivation to levels which are either less than, equal to, or greater than those of wild type. These results can be explained by assuming a multimeric structure for the cysB protein and the formation in merodiploids of cysB ^-/cysB ⁺ hybrid molecules with altered capacities for gene activation. The dominance of cysB ^c over cysB ⁺ indicates that in contrast to the araC regulatory protein, which acts as both a gene activator and repressor, the cysB protein serves only as an element of positive control.     

Fine-structure genetic map of the cysB locus in Salmonella typhimurium.

A genetic map of the cysB region of the Salmonella typhimurium chromosome was constructed using bacteriophage P22-mediated transduction. Strains bearing delta (supX cysB) mutations were employed to divide this regulatory locus into 12 segments containing a total of 39 single-site mutations. Twenty-five of these single-site mutations were further ordered by reciprocal three-point crosses. The results do not support the concept of multiple cistrons at cysB and suggest that the abortive transductants previously observed in crosses between certain cysB mutants were due to intracistronic complementation. The prototrophic cys-1352 mutation, which causes the constitutive expression of the cysteine biosynthetic enzymes, was found to lie within the cysB region itself. It is bracketed by mutations, which lead to an inability to derepress for these enzymes and result in auxotrophy for cysteine.     

Identification of the Salmonella typhimurium cysB gene product by two-dimensional protein electrophoresis.

Examination of two-dimensional electropherograms of proteins from wild-type Salmonella typhimurium and 16 different cysB strains permitted the identification of a single 34,500-dalton polypeptide chain with a pI of 7.6 that was the product of cysB. Exclusion chromatography indicated that the native cysB protein is a multimer of at least two and probably four or more such subunits.     

Constitutive mutation of cysJIH operon in a cysB deletion strain of Salmonella typhimurium.

Summary In a cysB deletion strain a new mutation, denoted cys-2332 was isolated, which causes the constitutive expression of the cysJIH operon. cys-2332 is closely linked to cysJIH and presumably is located in the initiator region of this operon, rendering its expression independent of the cysB gene product and the internal inducer O-acetyl-L-serine. The presence of salfite reductase (encoded by cysI and cysJ) activity in a cysB ^- cys-2332 double mutant indicates that cysG, which is not linked to cysJIH but is required for the synthesis of the sulfite reductase co-factor siroheme, is not controlled by cysB.     

Involvement of cysB and cysE genes in the sensitivity of Salmonella typhimurium to mecillinam.

cysB and cysE strains were obtained as spontaneous mecillinam-resistant mutants of Salmonella typhimurium. The resistance to mecillinam was caused by the cys mutations which also conferred tolerance to lethal cell shape mutations. Most, but not all, cysB and cysE mutations from other origins displayed the same behavior. Resistance was abolished by O- and N-acetylserine in cysE mutants; by thiosulfate, sulfite, and sulfide in cysB mutants; and by cysteine in both types of mutants. It is concluded that an event involved in mecillinam action requires the inducer and the activator protein of the cysteine regulon.     

DNA sequences of the cysB regions of Salmonella typhimurium and Escherichia coli

Nucleotide sequences of the cysB region of Salmonella typhimurium and Escherichia coli have been determined and compared. A total of 1759 nucleotides were sequenced in S. typhimurium and 1840 in E. coli. Both contain a 972-nucleotide open reading frame identified as the coding region for the cysB regulatory protein on the basis of sequence homology and by comparison of the deduced amino acid sequences with known physicochemical properties of this protein. The DNA sequence identity for the cysB coding region in the two species is 80.5%. The deduced amino acid sequences are 95% identical. The predicted cysB polypeptide molecular weights are 36,013 for S. typhimurium and 36,150 for E. coli. For both proteins a helix-turn-helix region similar to that found in other DNA-binding proteins is predicted from the deduced amino acid sequence. Sequences upstream to cysB contain open reading frames which represent the carboxyl-terminal end of the topA gene product, DNA topoisomerase I. A pattern of highly conserved nucleotide sequences in the 151 nucleotides immediately preceding the cysB initiator codon in both species suggests that this region may contain multiple signals for the regulation of cysB expression.     

Analysis of Drosophila proboscipedia mutant alleles.

Proboscipedia (PB) is a HOX protein required for adult maxillary palp and proboscis formation. To identify domains of PB important for function, 21 pb point mutant alleles were sequenced. Twelve pb alleles had DNA sequence changes that encode an altered PB protein product. The DNA sequence changes of these 12 alleles fell into 2 categories: missense alleles that effect the PB homeodomain (HD), and nonsense or frameshift alleles that result in C-terminal truncations of the PB protein. The phenotypic analysis of the pb homeobox missense alleles suggests that the PB HD is required for maxillary palp and proboscis development and pb - Sex combs reduced (Scr) genetic interaction. The phenotypic analysis of the pb nonsense or frameshift alleles suggests that the C-terminus is an important region required for maxillary palp and proboscis development and pb-Scr genetic interaction. PB and SCR do not interact directly with one another in a co-immunoprecipitation assay and in a yeast two-hybrid analysis, which suggests the pb-Scr genetic interaction is not mediated by a direct interaction between PB and SCR.     

Expression of Escherichia coli fol alleles in Salmonella typhimurium.

Studies of the expression of Escherichia coli fol alleles in Salmonella typhimurium indicated that fol regulatory functions are highly conserved between these bacterial species.     

Radiovaccine of S. typhimurium cells.

Gamma-irradiation of S. typhimurium cells up to a dose of 500 krad significantly reduces their toxicity. However, the antigenicity of these cells is not altered, which suggests that these cells could be used as a vaccine. The protection offered by the irradiated cells is comparable to that of formalin-treated cells. The radio-vaccine, however, offers an additional advantage of significant detoxification of the endotoxin, thereby minimizing side effects. The lipopolysaccharide extracted from the irradiated S. typhimurium cells offered cross-protection against other Salmonella species tested.     

Characterization of human glucocerebrosidase from different mutant alleles.

Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from normal enzyme in its inhibition by both conduritol B epoxide and glucosphingosine demonstrating that the 370 mutant enzyme has an abnormal catalytic site. In addition, the 370 mutant enzyme is less activated by saposin C, but more stimulated by phosphatidylserine than the wild type enzyme. The Arg463----Cys mutant protein was normal with respect to conduritol B epoxide and glucosphingosine inhibition, but was less activated by both saposin C and phosphatidylserine. The Arg120----Gln mutant protein was catalytically inactive. The Leu444----Pro, the pseudopattern, and the Pro415----Arg mutants appear to have reduced amounts of enzyme protein in cells. The studies demonstrated that mutations in the gene for glucocerebrosidase have different effects on the catalytic activity and stability of the enzyme.