Mutational analysis of the histidine operon promoter of Salmonella typhimurium.

We isolated a collection of 67 independent, spontaneous Salmonella typhimurium his operon promoter mutants with decreased his expression. The mutants were isolated by selecting for resistance to the toxic lactose analog o-nitrophenyl-beta-D-thiogalactoside in a his-lac fusion strain. The collection included base pair substitutions. small insertions, a deletion, and one large insertion identified as IS30 (IS121), which is resident on the Mu d1 cts(Apr lac) phage used to construct the his-lac fusion. Of the 37 mutations that were sequenced, 14 were unique. Six of the 14 were isolated more than once, with the IS30 insertion occurring 16 times. The mutations were located throughout the his promoter region, with two in the conserved - 35 hexamer sequence, four in the conserved - 10 hexamer sequence (Pribnow box), seven in the spacer between the - 10 and -35 hexamer sequences, and the IS30 insertions just upstream of the -35 hexamer sequence. Four of the five substitution mutations changed a consensus base pair recognized by E sigma 70 RNA polymerase in the -10 or -35 hexamer. Decreased his expression caused by the 14 different his promoter mutations was measured in vivo. Relative to the wild-type promoter, the mutations resulted in as little as a 4-fold decrease to as much as a 357-fold decrease in his expression, with the largest decreases resulting from changes in the most highly conserved features of E sigma 70 promoters.     

The hisD-hisC gene border of the Salmonella typhimurium histidine operon

Summary We have sequenced the hisD-hisC gene border of the Salmonella typhimurium histidine operon. The translation termination codon of the hisD gene overlaps with the translation initiation codon of the hisC gene in the manner . The Shine-Dalgarno sequence of the hisC gene is contained entirely within hisD and there is no intercistronic space since all of the bases are utilized in coding. Two mutations that alter the hisD-hisC gene border are analyzed. Both mutations simultaneously abolish the termination codon of hisD and modify the initiation codon of hisC. One of the mutations changes the hisC initiation codon from AUG to AUU. The AUU codon is 10 to 20% as efficient as AUG for initiation of translation of the hisC gene. The mutant hisC ribosome binding site is compared to the ribosome binding site of the Escherichia coli infC gene which has been reported to contain an AUU initiation codon. The role of overlapping termination/initiation codons in regulating translation of polycistronic mRNAs in bacterial operons is discussed.     

Genetic characterization of a phi80 transducing bacteriophage carrying the histidine operon of Salmonella typhimurium.

A phi 80 transducing phage, phi 80imm lambdadhis, carrying the Salmonella his-gnd region, was characterized by immunity studies, tonB deletion analysis, and marker rescue analysis. Phi 80imm lambdadhis retains the phage immunity region of the phi 80-lambda hybrid phage from which it was derived. Bacterial genes replace most late phage genes. Deletion analysis shows the prophage gene order to be immlambda-his-gnd and indicates the orientation of the his operon to be hisOGDCBHAFIE-gnd. The structure of phi 80imm lambdadhis is remarkably similar to two independently isolated phi 80 phages that carry the his-gnd region of Escherichia coli and that, like phi80imm lambdahis, were derived by directed gene transposition to the tonB locus. A derivative of phi 80imm lambdadhis that is phi 80 immune is also reported.     

Metabolic regulation of the histidine operon in Escherichia coli and Salmonella typhimurium

Expression of the histidine operon in Escherichia coli cells in contrast to the one in Salmonella typhimurium is changed proportionally to cells growth rate on the different carbon sources. The specific activity of histidinol-dehydrogenase is repressed by addition of 19 amino acids both in Escherichia coli and Salmonella typhimurium independent of the growth medium used. Using of Escherichia coli and Salmonella typhimurium strains containing the heterologous histidine operons made possible to demonstrate the dependence of the histidine operon metabolic regulation to be determined by the operon itself but not by the specificity of the recipient cells. ppGpp was shown to be a positive regulator of the histidine operon expression in Escherichia coli.     

Promoter- and attenuator-related metabolic regulation of the Salmonella typhimurium histidine operon.

Expression of the histidine (his) operon in Salmonella typhimurium was found to be positively correlated with the intracellular level of guanosine tetraphosphate (ppGpp). Limitation for amino acids other than histidine elicited a histidine-independent metabolic regulation of the operon. In bacteria grown at decreased growth rates, his operon expression was metabolically regulated up to a point, after which further decreases in growth rate no longer resulted in further enhancement of operon expression. Studies using strains carrying various regulatory and deletion mutations indicated that metabolic regulation is achieved predominantly by increased RNA chain initiations at the primary (P1) and internal (P2) promoters. Metabolic regulation ordinarly did not involve changes in RNA chain terminations at the attenuator site of the his operon. A model is proposed that involves ppGpp-induced changes in RNA polymerase initiation specificity at particular promoters. A second, special form of metabolic regulation may operate which also is histidine independent, but does involve relief of attenuation.     

Mutation leading to gene fusion in the histidine operon of Salmonella typhimurium

A spontaneous polar mutation located in the region of an intercistronic border in the hiatidine operon of Salmonella was isolated in our laboratory. The mutant, R81, tests as a frameshift in reversion experiments but is prototrophic, capable of growth without histidine supplements despite lowered levels of certain histidine enzymes. The mutation affects the operator distal end of the D gene, causing production of an active histidinol dehydrogenase enzyme with an altered C-terminus. The mutation severely affects expression of the immediately succeeding gene in the translation sequence, hisC, suggesting either that the D–C border and possibly hisC are physically altered or that their normal function in translation is seriously impaired. We have previously described the fortuitous production from R81 of a non-polar derivative with fused D and C genes. This strain produces a bifunctional enzyme with normally separate dehydrogenase and aminotransferase activities present on dimers or multimers of a single fused polypeptide chain. We have now investigated in greater detail the R81 mutation by amino acid sequencing of the C-terminus of altered histidinol dehydrogenase. We find that the R81 mutation causes the addition of a “tail” of four amino acid residues to an otherwise normal dehydrogenase polypeptide chain. The results support our previous suggestion that the R81 mutation profoundly effects the D–C gene border and that this effect is prerequisite to gene fusion.     

Tandem duplications of the histidine operon observed following generalized transduction in Salmonella typhimurium

Unstable merodiploid transductants may be observed among the progeny of certain generalized transductional crosses between complementing mutations in the histidine operon of Salmonella typhimurium. In the presence of a functional recombination system, these transductants are unstable and they segregate His^? clones of both parental genotypes. The properties of these His⁺ transductants suggest that they contain tandem duplications of a region of DNA which includes the histidine operon, such that each copy of the duplication contains one of the two complementing mutations involved in the transduction. Transductional duplications have been observed from 14 pairs of his mutations, but only with complementing pairs of parental mutations. The length of duplicated material may be quite large: two duplications were found to include genetic markers ten minutes removed from the histidine operon on the Salmonella chromosomal map.These transductants appear to arise in a subpopulation of recipient cells which contain pre-existing tandem duplications of the histidine operon. As much as 0.01 to 0.1% of the cell population appears to be tandemly duplicated for a chromosomal region which includes the histidine operon.     

Tandem genetic duplications in Salmonella typhimurium: amplification of the histidine operon.

Bovine pancreatic phospholipase A₂ (M_r = 14,000) has been crystallized and its three-dimensional structure determined by X-ray diffraction analysis to a resolution of 2.4 Å. Three heavy-atom derivatives were used in the phase calculations with inclusion of the anomalous dispersion differences. The resulting electron density map allowed an easy and unambiguous tracing of the peptide chain. Two of the seven disulfide connections appeared to be different from what was suggested by the earlier chemical and structural work. The bovine phospholipase A₂ structure contains about 50% α-helix and 10% β-structure. The bovine enzyme structure was found to deviate substantially from the previously published porcine prophospholipase structure.     

Role of the intercistronic region in post-transcriptional control of gene expression in the histidine transport operon of Salmonella typhimurium: involvement of REP sequences

The high-affinity histidine permease of Salmonella typhimurium is encoded by a four-gene operon containing a large intercistronic region located between the first gene (hisJ) and the three distal genes (hisQ, hisM, hisP). The level of expression of hisJ is 30-fold greater than that of hisP. In order to investigate the role of the intercistronic region in intra-operonic control of gene expression, we have isolated MudII-mediated lacZ gene fusions to hisQ, hisM and hisP. We have used these fusions to isolate and analyse mutants that have altered levels of expression of the hisQ gene, the first gene downstream from the intercistronic region. The results indicate that intra-operonic regulation is due to a combination of factors including efficiency of translational initiation, mRNA degradation, and retroregulation of hisJ expression. They also suggest that the REP (Repetitive Extragenic Palindromic) sequences, which are located in the hisJ-hisQ intercistronic region, may interfere with translation of the hisQ gene and affect upstream messenger RNA stability by protecting it from 3' to 5' nuclease degradation (in agreement with data presented by Newbury et al., 1987).     

Isolation of F' plasmids carrying a portion of the Salmonella typhimurium histidine transport operon.

The transposable drug resistance element Tn10 was employed as a region of homology to direct the insertion of Tn10-containing derivatives of F'ts114 lac into the chromosome of a Salmonella typhimurium strain that carries a Tn10 insertion in the histidine transport operon. Based on the direction of transfer of the resulting Hfr strains, the chromosomal Tn10 insertion was determined to be in orientation "A." New F' plasmids were selectively generated from one of the Hfr strains. The F' factors carry an intact dhuA hisJ portion of the histidine transport operon. A Southern hybridization revealed that one of the F' plasmids was formed by a type II excision event.     

Cloning and expression of the histidine operon of Salmonella typhimurium in cells of Escherichia coli

The histidine operon of Salmonella typhimurium and its fragments were cloned in Escherichia coli cells on a multicopy plasmid. Expression of the cloned genes and histidine production by the variants possessing the hisG mutation which desensibilizes the ATP phosphoribosyl transferase for histidine were studied. Amplification of the complete operon including the hisG gene enables histidine accumulation of 2-3 g/l after 72 hours of fermentation.     

Cloning and restriction map of the first part of the histidine operon of Salmonella typhimurium.

The first part of the histidine operon of Salmonella typhimurium, hisGpeaGD, has been cloned onto the vector plasmid mini-ColE1 (pVH51). The resulting plasmid, pWB91, has a single EcoRI site and is 11,500 base pairs in size. The HindII restriction map was determined by the method of two-dimensional cross-annealing between a partial digest pattern and a complete digest pattern. The restriction fragment containing the genetic control region was identified with the aid of the small (35-base pair) internal deletion 01242 and the observation that heteroduplexed restriction fragments containing this deletion have markedly reduced mobility on polyacrylamide gels. The genetic control region was then mapped in more detail with other restriction enzymes. The genetic orientation of the restriction map was determined with the aid of several deletions of integral HindII fragments generated in vitro.