The Origin and Evolution of Operons: The Piecewise Building of the Proteobacterial Histidine Operon

The structure and organization of 470 histidine biosynthetic genes from 47 different proteobacteria were combined with phylogenetic inference to investigate the mechanisms responsible for assembly of the his pathway and the origin of his operons. Data obtained in this work showed that a wide variety of different organization strategies of his gene arrays exist and that some his genes or entire his operons are likely to have been horizontally transferred between bacteria of the same or different proteobacterial branches. We propose a piecewise model for the origin and evolution of proteobacterial his operons, according to which the initially scattered his genes of the ancestor of proteobacteria coded for monofunctional enzymes (except possibly for hisD) and underwent a stepwise compacting process that reached its culmination in some -proteobacteria. The initial step of operon buildup was the formation of the his core, a cluster consisting of four genes (hisBHAF) whose products interconnect histidine biosynthesis to both de novo synthesis of purine metabolism and that occurred in the common ancestor of the // branches, possibly after its separation from the one. The following step was the formation of three mini-operons (hisGDC, hisBHAF, hisIE) transcribed from independent promoters, that very likely occurred in the ancestor of the /-branch, after its separation from the one. Then the three mini-operons joined together to give a compact operon. In most -proteobacteria the two fusions involving the gene pairs hisN–B and hisI–E occurred. Finally the -proteobacterial his operon was horizontally transferred to other proteobacteria, such as Campylobacter jejuni. The biological significance of clustering of his genes is also discussed.[Reviewing Editor: Dr. Martin Kreitman]     

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.     

A secondary promoter for elongation factor Tu synthesis in the str ribosomal protein operon of Escherichia coli

Summary The str operon of Escherichia coli contains genes for ribosomal proteins S12 and S7 and for elongation factors EF-G and EF-Tu (Jaskunas et al. 1975). We have subcloned various segments of DNA from this operon onto multicopy plasmids. We found that cells carrying a recombinant plasmid which lacks the major promoter for the str operon but contains the 5 portion of the EF-Tu gene synthesize a novel protein which we have identified as a truncated EF-Tu molecule. Moreover, cells carrying plasmids with an intact EF-Tu gene synthesize the elongation factor at a 3-to 5-fold higher rate than haploid cells. Thus the EF-Tu gene can be expressed in the absence of the major promoter for the str operon. This expression is not due to read-through from plasmid promoters, but it is dependent on the presence of the distal portion of the EF-G gene on the plasmids. These results indicate that there is a secondary promoter for EF-Tu expression, apparently located within the structural gene for elongation factor EF-G.     

Binding of lac repressor to the secondary lac operator in Escherichia coli.

Summary In the lac operon, the existence of a secondary repressor binding site, inside Z gene, had been inferred from in vitro binding studies (Reznikoff et al., 1974; Gilbert et al., 1975).A serie of deletions have been constructed from a lac transducing bacteriophage. Some of those deleted bacteriophages have still the property of derepressing a chromosomal lac operon, even though they do not contain any more the lac operator. This phenomenon is an indication that the secondary repressor binding site is also active in vivo.     

Flagellin genes of Methanococcus vannielii : amplification by the Polymerase Chain Reaction, demonstration of signal peptides and identification of major components of the flagellar filament

The highly conserved nature of the 5′-termini of all archaeal flagellin genes was exploited by polymerase chain reaction (PCR) techniques to amplify the sequence of a portion of a flagellin gene family from the archaeon Methanococcus vannielii. Subsequent inverse PCR experiments generated fragments that permitted the sequencing of a total of three flagellin genes, which, by comparison with flagellin genes that have been sequenced, from other archaea appear to be equivalent to flaB1, flaB2, and flaB3 of M. voltae. Analysis of purified M. vannielii flagellar filaments by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed two major flagellins (M_r= 30 800 and 28 600), whose N-terminal sequences identified them as the products of the flaB1 and flaB2 genes, respectively. The gene product of flaB3 could not be detected in flagellar filaments by SDS-PAGE. The protein sequence data, coupled with the DNA sequences, demonstrated that both FlaB1 and FlaB2 flagellins are translated with a 12-amino acid signal peptide which is absent from the mature protein incorporated into the flagellar filament. These data suggest that archaeal flagellin export differs significantly from that of bacterial flagellins. Received: 27 November 1997 / Accepted: 19 March 1998     

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.

     

A critical evaluation of the predicted and X-ray structures of alpha-lactalbumin

The rapidly increasing availability of protein amino-acid sequences, many of which have been determined from the corresponding gene sequences, has intensified interest in the prediction of related protein structures when the three-dimensional structure of another member of the family is known. The study of bovine -Lactalbumin provides a classic example in which the three-dimensional structure was predicted, first by Browneet al. (1969) and later by Warmeet al. (1974), from the three-dimensional structure of hen-egg-white lysozyme (Blakeet al., 1965), taking into account the striking relationship between the amino acid sequences of the two proteins. A comprehensive comparison of these models with the structure of baboon -Lactalbumin derived from X-ray crystallography (Acharyaet al., 1989) is presented. The models mostly compare well with the experimentally determined structure except in the flexible C-terminal region of the molecule (rms deviation on C of residues 1–95, 1.1 Å).     

Revised locations of the hisI and pru (proline utilization) genes on the Pseudomonas aeruginosa chromosome map

Summary The location of genes in the vicinity of the major FP2 origin on the chromosome of Pseudomonas aeruginosa PAO has been revised. The markers hisI (a transduction group of histidine biosynthetic genes) and pru (a gene cluster encoding proline utilization functions) were located in the 90 to 95/0 min chromosome region by a series of plate matings mediated by R68.45. Three-factor-crosses using this plasmid established the following marker order: pur-67 pru hisI/cys-59 proB ilvB/C. Genetic evidence is presented to confirm the previous observations that FP2 can mobilize the chromosome from at least two origins near proB and in both directions. Thus, when markers in this chromosome region are analyzed by FP2 crosses only, the mapping data may be difficult to interpret. This complication can be overcome by the use of R68.45 and Tfr (transposon-facilitated recombination) or Hfr donors.     

Mutagenesis and functional analysis of the Escherichia coli tRNALeu1 promoter

The leuV promoter which produces tRNA^Leu₁ in Escherichia coli has been extensively mutagenized in order to determine the effects of altered sequences on promoter efficiency (strength) and on growth-rate-dependent regulation (GDR). Each mutant promoter was ligated with a β-galactosidase reporter gene into the chromosome of a host cell by phage lambda lysogenization. Reporter gene activities were measured for cells growing in selected media at various growth rates. Sequences which flank the ?10 consensus region, when altered, caused remarkable up-promoter effects, increasing efficiency in some cases almost 10-fold. One up mutation which had five successive T residues in the‘discriminator’region completely abolished GDR, whereas several mutations with single base changes in the discriminator had little or no effect on GDR. Another mutation which changed one base in the ?35 region to bring it to consensus increased promoter strength 18-fold and sharply reduced GDR. Chimaeric promoters in which segments of leuV were replaced by segments of the his operon showed that only when the discriminator of leuV is replaced by the his discriminator was GDR-disturbed. AM upstream sequences which were replaced by his sequences had little effect on GDR. Overall, there appeared to be little correlation between promoter efficiency and GDR.     

DNA sequence analysis of spontaneous histidine mutations in a polA1 strain of Escherichia coli K12 suggests a specific role of the GTGG sequence

Summary Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5-GCTGGCTGGCTGGCTG-3. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong imitator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5-GTGG-3 sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.     

Origin of replication, oriC, of the Escherichia coli chromosome: mapping of genes relative to R.EcoRI cleavage sites in the oriC region.

Summary A precise genetic-physical map of the tnailv region at 82 min on the genetic map of E. coli is obtained through deletion mapping and analysis by restriction endonuclease EcoRI of plasmids, derived from an F carrying the genes between aroE and ilv.A locus, designated het, which in its diploid state results in slow growth and heterogeneity of cell size due to distorted cell division, maps between bglB and asn, 30–45 kb counterclockwise of ilv.The pattern of R.EcolRI cleavage sites in the het region is identical with the pattern obtained by Marsh and worcel (1977) who analyzed DNA labeled preferentially in the region of the DNA replication origin (oriC). We suggest that oriC is identical with the het site and that it can be allocated to a position 32 kb counterclockwise of the ilv operon.Abbreviations CCC covalently closed circular - kb kilobases - MD 10⁶ Daltons - mw molecular weight - R.EcoRI restriction endonuclease EcoRI New Genetic Symbols het heterogeneity of cell size distribution (this study) - maf maintenance of F (Wada et al., 1977) - oriC chromosomal origin of replication (Hiraga, 1976) - oriV origin of vegetative replication of F (Guyer et al., 1976) - oriT origin of transfer replication of F (Guyer et al., 1976) - poh permissive on Hfr (Hiraga, 1976)     

Isolation and characterization of ?80dgal transducing phages that carry gal operator-promoter insertion mutations

Summary 80dgal transducing bacteriophages have been isolated by the F-fusion technique of Press et al. (1971) and gal-operator-promoter insertion mutations have been introduced by homogenote formation.Five different 80dgal isolates have been studied in more detail. One of the 80 phages transduces the gal operon and gene aroG as well as at least part of the trp-operon; the gal operon of another 80dgal transducing phage is inverted with respect to the 80dgal sequences. Heteroduplex DNA mapping indicates that one of the 80dgal isolates in addition to the gal operon and a portion of the adjacent chromosomal region carries an IS2-element which is derived from the F'gal episome.The isolated 80dgal phages may be utilized for preparing pure gal mRNA and insertion-RNA as well as pure gal operon DNA.     

Electron microscopy of polar insertions in the lac operon of Escherichia coli

Summary Several strongly polar mutations in the omega region of the z gene of the lac operon result from insertions consisting of only two specific sequences of DNA, one about 870 and the other 1170 nucleotide pairs long (based on single-strand measurements). No sequence homology was detected between the shorter (IS1) and longer (IS3) insertions. The IS1 insertion was shown to possess a specific attachment site, but it can be inserted with either orientation at several sites in the z gene. Four insertion sites in the omega region of gene z were identified and the position of the lac5 substitution and the SR2 deletion in the plac DNA were determined by heteroduplex mapping.     

Intron-containing tRNA genes ofSulfolobus solfataricus

Summary Nucleotide sequences of four tRNA genes from the archaebacteriumSulfolobus solfataricus have been determined. Based upon DNA sequence analysis, three of the four genes contain presumptive intervening sequences (introns) in their anticodon loops. The three introns can form similar, but not identical, secondary structures. The cleavage site at the 3 end of all three introns occurs in a three-base bulge loop. All four genes lack an encoded 3 CCA terminus and are flanked by A+T-rich DNA sequences. Two of the genes are located on antiparallel DNA strands, with their 3 termini separated by 414 bp of sequence. Including two previously published sequences, a total of five introns have now been detected among sixS. solfataricus tRNA genes. Occurrence of introns at corresponding locations in both archaebacterial and eukaryotic tRNA genes suggests that the intron/exon form of gene structure predates the evolutionary divergence of the archaebacteria and the eukaryotes.     

An unlinked 5 S ribosomal RNA gene in the archaebacterium, Thermococcus celer

Summary We have determined the nucleotide sequence of an unlinked 5 S rRNA gene region from a thermophilic archaebacterium, Thermococcus celer. This 5 S rRNA gene is flanked by a single tRNA^Asp sequence and appears to be transcribed as part of a very short operon consisting of only two gene sequences. Comparative studies indicate features in the 5 and 3 flanking sequences, which bear similarity with promoter and termination signals in eubacteria, but also reflect unusual features found in at least some archaebacteria. The evolution of this unlinked operon and the unusual features are discussed.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila

We have cloned a 1.6-kb region of chromosomal DNA from Thermoplasma acidophilum into Escherichia coli using as a probe part of the Methanococcus vannielii fus-gene. The sequence of the clone was highly homologous to part of the corresponding Methanococcus vannielii gene. By chromosome walking, a 4.7-kb EcoRI fragment containing the complete gene was isolated. Nucleotide sequencing revealed an open reading frame of 2196 nucleotides. The deduced amino acid sequence contains the known peptide sequence around the ADP-ribosylation site of T. acidophilum elongation factor 2, which unequivocally confirms that the fus-gene has been cloned. The amino acid sequence was compared to that of hamster and E. coli, as well as to known archaebacterial EF-2 sequences.