A study in evolution: the histidine utilization genes of enteric bacteria.

The histidine utilization (hut) system, MIGCPUH, comprises two operons, whose promoters are M and P. The DNA segments of phage λ vectors coding for the hut genes of Salmonella typhimurium and Klebsiella aerogenes were compared by using electron microscopy to study the nature of the hut DNA homology. The hut S. typhimurium/hut K. aerogenes DNA heteroduplexes were spread and mounted for electron microscopy under more and more denaturing conditions. The homology between hut S. typhimurium and hut K. aerogenes is extensive but, as denaturing conditions make base-pairing more difficult, the regions of lower homology are melted, while the regions of higher homology remain base-paired. The denaturation map could be correlated with the genetic map by the use of λhut S. typhimurium phages carrying different portions of the hut genome. The sequences with the highest homology are in the structural genes for the four enzymes, while the regulatory regions, the promoters and the hut repressor gene, are much less homologous.     

Discrimination of promoters by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> RNA polymerase σ<Superscript>A</Superscript> and σ<Superscript>H</Superscript> holoenzymes is associated with periodic patterns in promoter nucleotide sequences

Bacillus subtilis promoters recognized by σ^A and σ^H RNA polymerases were found to have different periodic patterns of nucleotide disposition. Using a special version of Fourier analysis for symbolic sequences, Fourier spectra were obtained for the primary structure of promoters used by the two holoenzymes. Stepwise discriminant analysis with jackknife testing was performed for two promoter data sets (small and large). Based on the spectral patterns of the nucleotide sequences, the data sets could be sorted with 100% accuracy into two classes: promoters recognized by Eσ^A and by Eσ^H. Correlations were obtained between the promoter strength and the characteristics of their Fourier spectra. Moreover, perfect separation was achieved even when the “consensus” ?35 and ?10 sites were replaced by sequences of “blank symbols.” Thus, the periodicity in nucleotide distribution along the DNA chain is itself an attribute sufficient for selective recognition of the cognate promoter by RNA polymerase.     

Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis

HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg²⁺ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn²⁺, Zn²⁺ and Cd²⁺, followed by Mg²⁺, Co²⁺ and Ni²⁺, while Cu²⁺, Yb²⁺ and Hg²⁺ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg²⁺, Mn²⁺ and Ba²⁺), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition.     

Genetic and physical maps of Klebsiella aerogenes genes for histidine utilization (hut)

Summary Deletion derivatives of the hut-containing plasmid pCB101 were tested against point mutants defective in individual genes of the histidine utilization (hut) operons using a complementation/recombination assay. Location of the genes of the right operon, hutU and hutH, was confirmed by direct assay of the gene products, urocanase and histidase; location of the repressor gene was identified by measuring the ability of the plasmid-carried genes to repress the formation of histidase from a chromosomal location. The analysis of eight deletion plasmids unambiguously confirms the map order of the hut genes as hutI-G-C-U-H, and demonstrates that, in Klebsiella aerogenes, the hutU and hutH genes are transcribed from their own promoter. In addition, the genetic map of hut can be aligned with the restriction map of the hut DNA in plasmid pCB101. One of the deletion plasmids studied apparently encodes a defective histidase subunit that is trans-dominant to active histidase. Another deletion, which completely removes the left operon, hutIG, allows high level expression of the hutUH operon and thus overproduction of a toxic intermediate.     

Tuning the Expression Level of a Gene Located on a Bacterial Chromosome

A new method of constructing a set of bacterial cell clones varying in the strength of a promoter upstream of the gene of interest was developed with the use of Escherichia coli MG1655 and lacZ as a reporter. The gist of it lies in constructing a set of DNA fragments with tac-like promoters by means of PCR with the consensus promoter P _tac and primers ensuring randomization of the four central nucleotides in the ?35 region. DNA fragments containing the tac-like promoters and a selective marker (Cm ^R) were used to replace lacI and the regulatory region of the lactose operon in E. coli MG1655. Direct LacZ activity assays with independent integrant clones revealed 14 new promoters (out of 4⁴ = 256 possible variants), whose strength varied by two orders of magnitude: LacZ activity in the corresponding strains gradually varied from 10² Miller units with the weakest promoter to 10⁴ Miller units with consensus P _tac Sequencing of the modified promoters showed that randomization of three positions in the ?35 region is sufficient for generating a representative promoter library, which reduces the number of possible variants from 256 to 64. The method of constructing a set of clones varying in expression of the gene or operon of interest is promising for modern metabolic engineering.