The role of gene fusions in the evolution of metabolic pathways: the histidine biosynthesis case

Background

Histidine biosynthesis is one of the best characterized anabolic pathways. There is a large body of genetic and biochemical information available, including operon structure, gene expression, and increasingly larger sequence databases. For over forty years this pathway has been the subject of extensive studies, mainly in Escherichia coli and Salmonella enterica, in both of which details of histidine biosynthesis appear to be identical. In these two enterobacteria the pathway is unbranched, includes a number of unusual reactions, and consists of nine intermediates; his genes are arranged in a compact operon (hisGDC [NB]HAF [IE]), with three of them (hisNB, hisD and hisIE) coding for bifunctional enzymes. We performed a detailed analysis of his gene fusions in available genomes to understand the role of gene fusions in shaping this pathway.

Results

The analysis of HisA structures revealed that several gene elongation events are at the root of this protein family: internal duplication have been identified by structural superposition of the modules composing the TIM-barrel protein.Several his gene fusions happened in distinct taxonomic lineages; hisNB originated within γ-proteobacteria and after its appearance it was transferred to Campylobacter species (ε-proteobacteria) and to some Bacteria belonging to the CFB group. The transfer involved the entire his operon. The hisIE gene fusion was found in several taxonomic lineages and our results suggest that it probably happened several times in distinct lineages.Gene fusions involving hisIE and hisD genes (HIS4) and hisH and hisF genes (HIS7) took place in the Eukarya domain; the latter has been transferred to some δ-proteobacteria.

Conclusion

Gene duplication is the most widely known mechanism responsible for the origin and evolution of metabolic pathways; however, several other mechanisms might concur in the process of pathway assembly and gene fusion appeared to be one of the most important and common.

     

his-Linked Hydrogen Sulfide Locus in Salmonella typhimurium

Investigation of strains of Salmonella typhimurium having extended his deletion mutations indicates that a genetic site (or sites) affecting H₂S production is located in the region of the chromosome adjacent to the operator end of the his operon. This site is co-transducible with the his genes. Experimental data indicate that the site is also present on an F'his factor derived from S. typhimurium, FS401. Evidence is presented for the existence of another site affecting H₂S production which is not linked to his.     

Deletion mutants of nitrogen fixation in Klebsiella pneumoniae: Mapping of a cluster of nif genes essential for nitrogenase activity

Deletions of the nitrogen fixation (nif) region of the Klebsiella genome were isolated by selecting for resistance to virulent phages whose resistance loci are adjacent to nif. The extent of the various deletions was monitored by assaying several different enzymes or gene products coded for by this segment of DNA. Three classes of deletion mutants were detected: (1) gluconate-6-phosphate dehydrogenase minus (gnd^?), histidine minus but histidinol dehydrogenase plus (his^?, his D⁺), nitrogenase plus (nif⁺), shikimate utilization plus (shu⁺); (2) gnd^?, his D^?, nif^?, shu⁺; (3) gnd^?, his D^?, nif^?, shu^?. From these studies we conclude that the cluster of nif genes essential for nitrogenase activity is located on the genetic linkage map of Klebsiella between his and shu; the gene order in this region in thus phage-resistance locus (rfb?), gnd, his operon, nif, shu. Genetic analysis substantiates the finding that the nif cluster is located proximally to the operator end of the his operon.     

Genetic fusions that help define a transcription termination region in Escherichia coli

The trpA gene product was analyzed from a class of strains of Escherichia coli K12 in which the lac operon has been fused by deletion to the trp operon. These are strains that have retained the ability to synthesize tryptophan. Two of these strains are shown to make a wild-type trpA product; these strains retain intact all structural genes of the ttrp operon. It is proposed that the lac operon in these strains is fused to a region of the trp operon between trpA, the last gene in the operon, and the region where trp messenger RNA synthesis terminates. The region where trp messenger RNA synthesis terminates thus is distinct from the trp structural genes.     

Rearrangement of the Bacterial Chromosome Using Tn10 as a Region of Homology

The transposable tetracycline resistance element, Tn10, can serve as a region of homology to promote rec-dependent deletion, duplication and directed transposition of bacterial genes. Tn10 insertions in regions of the chromosome near the histidine operon (his) have been isolated and characterized in Salmonella typhimurium. When strains are constructed containing two Tn10 insertions flanking the his operon in the same orientation (Tn10-his-Tn10), recombination can occur between Tn10 sequences resulting in the deletion of the intervening his region. The sites of the Tn10 insertions determine the endpoints of the deletion. In crosses designed to construct strains carrying Tn10-his-Tn10, another class of unstable recombinants arises in which the his region exists in tandem duplication, with a Tn10 insertion joining the duplicated copies (his-Tn10-his). The sites of the parental Tn10 insertions mark the endpoints of the duplication. When a strain carrying Tn10-his-Tn10 is used as a donor of his⁺ in conjugation or P22-mediated transduction, recombinants can arise in which the his region has been transposed to the site of any Tn10 insertion, far from the normal location of his in the recipient chromosome. In this manner, the his operon has been moved to the site of a pyrB::Tn10 insertion and has been placed on F'' plasmids. At these new locations, the his⁺ character shows the rec-dependent deletion of his⁺ expected for a Tn10-his-Tn10 duplication. These methods should be generally useful for the manipulation of bacterial genes.     

Genetic fusions defining trp and lac operon regulatory elements

Two procedures for easily isolating deletions that fuse the trp and lac operons are described. Using these procedures, a large number of fusion deletions have been isolated. The lac ends of these deletions extend varying distances into the lacI gene and the lac promoter-operator region. Therefore, contrary to a previous report, there does not appear to be a messenger-termination signal at the C-terminal end of the lacI gene.The trp ends of fusion deletions do not have to extend into the trp structural genes to effect fusion, suggesting that mRNA synthesis initiated at the trp promoter proceeds some distance beyond the trp structural genes before a messenger termination signal is reached. Deletions that extend a short distance into the C-terminus of trpA, the last gene in the trp operon, do not completely abolish activity of the trpA product.The procedures described for isolating fusions of the trp and lac operons can be generalized to other systems.     

A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu

A general procedure is described for transposing the lac genes to selected locations on the Escherichia coli chromosome. These transpositions were designed so that the lac² genes could be fused to nearby promoters. In particular, the lac genes were fused to the promoters for the araBAD, araC and leu genes. In these fusions the lac genes are regulated by the controls of the genes to which they are fused. These fusions are therefore useful in discovering new types of regulation of gene expression, as was found in the case of the araC gene. λ transducing phage carrying the fusion as well as nearby genes can easily be isolated. Some of these fusions may result in the formation of hybrid proteins.     

The Evolution of Histidine Biosynthesis in Archaea: Insights into the <Emphasis Type="Italic">his</Emphasis> Genes Structure and Organization in LUCA

The available sequences of genes encoding the enzymes associated with histidine biosynthesis suggest that this is an ancient metabolic pathway that was assembled prior to the diversification of Bacteria, Archaea, and Eucarya. Paralogous duplication, gene elongation, and fusion events of several different his genes have played a major role in shaping this biosynthetic route. We have analyzed the structure and organization of histidine biosynthetic genes from 55 complete archaeal genomes and combined it with phylogenetic inference in order to investigate the mechanisms responsible for the assembly of the his pathway and the origin of his operons. We show that a wide variety of different organizations of his genes exists in Archaea and that some his genes or entire his (sub-)operons have been likely transferred horizontally between Archaea and Bacteria. However, we show that, in most Archaea, his genes are monofunctional (except for hisD) and scattered throughout the genome, suggesting that his operons might have been assembled multiple times during evolution and that in some cases they are the result of recent evolutionary events. An evolutionary model for the structure and organization of his genes in LUCA is proposed.     

Accumulation of single-stranded regions in DNA and the block to replication in a human cell line alkylated with methyl methane sulfonate

Once a permeability barrier is overcome, proflavin is highly mutagenic for Salmonella typhimurium and Escherichia coli. Mutagenesis of the his operon (in Salmonella) and lac operon (in E. coli) depends on derepression of the respective operons. No proflavin mutagenesis was detected in rec^? strains. Over 100 proflavin-induced his mutants have been classified: 50% are base substitution types, 30% are stable (10% demonstrably multisite), and only 20% are probably frameshift mutations. None of the proflavin-induced frameshift mutations is of the type previously shown to be suppressed by frameshift suppressor mutations.     

Reversion and immobilization of phage Mud1 cts (Apr lac) insertion mutations in Salmonella typhimurium

Summary Phage Mud1 cts (Ap^r lac), or Mud1, insertion mutations may be accompanied by adjacent deletion formation which can complicate use of lac fusions generated with this phage for gene regulatory studies. As for phage Mu insertion mutations, phage Mud1 insertions fail to revert at significant frequency (whether or not accompanied by an adjacent deletion). We describe isolation of revertible (X mutant) derivatives of phage Mud1 in Salmonella typhimurium. The X mutant derivatives allow use of reversion as a simple test to determine whether a Mud1 insertion has occurred precisely without an adjacent deletion that may have fused the lac genes to a promoter outside of the gene of interest. In addition, a simple method for stabilizing Mud1 generated lac fusions against subsequent transposition is described.     

A high-resolution electron microscopy study of nucleosomes from simian virus 40 chromatin

The DNA sequence changes of 31 mutations altering the attenuation control mechanism of the histidine operon are presented. These mutations are discussed in terms of a model for operon regulation that involves a his leader peptide gene whose translation regulates formation of alternative stem-loop structures in the his leader messenger RNA. Three suppressible mutations generate nonsense codons (ochre and UGA) in the his leader peptide gene, demonstrating that translation of this gene is essential for operon expression. Eight mutations presumably reduce the efficiency of translation initiation of the his leader peptide gene, causing reduced levels of operon expression. Five of these mutations directly alter the leader peptide gene initiator codon (AUG). Three mutations alter sequences just in front of the initiator codon and presumably alter the ribosome recognition site. Fourteen mutations reduce the stability of the his leader mRNA stem-loop structures that are alternatives to the attenuator stem. The properties of these mutations provide support for the role of these stem-loop structures in preventing formation of the attenuator stem. Finally, we show that mutations that alter the attenuator stem suppress hisO mutations. This lends support to the proposal that these hisO mutations cause reduced levels of operon expression due to excessive attenuator stem formation. The properties of these 31 mutations provide substantial support for the model of his operon regulation described in this paper.     

Genetic analysis of the histidine operon control region of Salmonella typhimurium

Mutants of the histidine operon control region (hisO) include two classes: (1) those completely unable to express the operon (His auxotrophs), and (2) prototrophs that are unable to achieve fully induced levels of operon expression (still His⁺ but sensitive to the drug amino-triazole). Using new, as well as previously existing hisO mutants, we constructed a fine-structure deletion map of hisO. Mutations that presumably alter the his promoter map at one end of hisO; mutations that alter the his attenuator map at the other end of hisO. Between the promoter and the attenuator lie a number of mutations that affect either the translation of the his leader peptide gene, or the formation and stability of his leader messenger RNA structures. All of the point mutations mapping in this central region revert to His⁺ at a very high frequency (10^?5 to 10^?6); this frequency is increased by both base substitution and frameshift-inducing mutagens. Many of the His^? mutants are suppressed by informational suppressors; all three types of nonsense mutations have been identified, demonstrating that translation of a region of hisO between the promoter and attenuator is essential for his operon expression. All of the hisO mutations tested are cis-dominant.     

Pleiotropic effect of his gene mutations on nitrogen fixation in Klebsiella pneumoniae

Several his mutations were found to influence nitrogen fixation in Klebsiella pneumoniae: hisB, hisC, and hisD mutants had 50% of wild-type levels of nitrogenase activity when supplied with 30 μg or less histidine/ml although this concentration did not limit protein synthesis and the mutants retained a Nif⁺ plate phenotype. A hisA mutation had a similar but more dramatic effect. At low concentrations of histidine the hisA mutant strain had only 5% of the nitrogenase activity found at high histidine concentration or in a his⁺ strain, and was also Nif^- on low histidine agar plates. Addition of adenine restored nitrogenase activity in the hisA but not the hisB, hisC, or hisD mutants. Low levels of intracellular ATP, a consequence of hisG enzyme activity, correlated with loss of nitrogen-fixing ability in the hisA mutant which failed to sustain nif gene expression under these conditions. Synthesis of other major cell proteins was relatively unaffected indicating that nif gene expression is selectively regulated by the energy status of the organism.     

Fusions of the lac operon to the transfer RNA gene tyrT of Escherichia coli.

The tyrT gene codes for one of the tyrosirie tRNA species. Using the Casadabatn (1976a) technique, strains of Escherichia coli were isolated in which the lac structural genes are fused to the promoter of the tyrT gene. This procedure involved obtaining a number of insertions of phage Mu DNA in the tyrT gene, lysogenizing the Mu insertion strains with a λplac-Mu hybrid phage, and selecting Lac⁺ derivatives of such lysogens. In a number of Lac⁺ strains thus obtained, the synthesis of β-galactosidase, the product of the lacZ gene, is regulated in a similar fashion to the synthesis of stable RNA. The fusion strains were shown directly to be tyrT-lac fusions by demonstrating that a Mu insertion in the tyrT gene when genetically recombined into the presumed fusion, inactivates the expression of the lac genes. This result shows that tyrT gene sequences are fused to and control the expression of the lac genes in these strains. This is the first report in which genes which code for proteins have been fused to a stable RNA gene in vivo.     

Regulation of the regulatory gene for the arabinose pathway,araC

The promoter of the araC gene was fused to the structural genes of the lac operon using the techniques described in the preceding paper. The resulting fusion strains were used to study the regulation of the araC gene by assaying the fused lac gene products. It was found that the expression of the fused lac genes was repressed by the product of the araC gene and was regulated by the cyclic AMP catabolite control system. This implies that the araC gene itself is repressed by its own product and is catabolite regulated. These findings introduce a new level of complexity in the regulation of the arabinose pathway of Escherichia coli.