SUMMARY The seven conserved enzymatic domains required for tryptophan (Trp) biosynthesis are encoded in seven genetic regions that are organized differently (whole-pathway operons, multiple partial-pathway operons, and dispersed genes) in prokaryotes. A comparative bioinformatics evaluation of the conservation and organization of the genes of Trp biosynthesis in prokaryotic operons should serve as an excellent model for assessing the feasibility of predicting the evolutionary histories of genes and operons associated with other biochemical pathways. These comparisons should provide a better understanding of possible explanations for differences in operon organization in different organisms at a genomics level. These analyses may also permit identification of some of the prevailing forces that dictated specific gene rearrangements during the course of evolution. Operons concerned with Trp biosynthesis in prokaryotes have been in a dynamic state of flux. Analysis of closely related organisms among the Bacteria at various phylogenetic nodes reveals many examples of operon scission, gene dispersal, gene fusion, gene scrambling, and gene loss from which the direction of evolutionary events can be deduced. Two milestone evolutionary events have been mapped to the 16S rRNA tree of Bacteria, one splitting the operon in two, and the other rejoining it by gene fusion. The Archaea, though less resolved due to a lesser genome representation, appear to exhibit more gene scrambling than the Bacteria. The trp operon appears to have been an ancient innovation; it was already present in the common ancestor of Bacteria and Archaea. Although the operon has been subjected, even in recent times, to dynamic changes in gene rearrangement, the ancestral gene order can be deduced with confidence. The evolutionary history of the genes of the pathway is discernible in rough outline as a vertical line of descent, with events of lateral gene transfer or paralogy enriching the analysis as interesting features that can be distinguished. As additional genomes are thoroughly analyzed, an increasingly refined resolution of the sequential evolutionary steps is clearly possible. These comparisons suggest that present-day trp operons that possess finely tuned regulatory features are under strong positive selection and are able to resist the disruptive evolutionary events that may be experienced by simpler, poorly regulated operons.

SUMMARY Aspartokinase (Ask) exists within a variable network that supports the synthesis of 9 amino acids and a number of other important metabolites. Lysine, isoleucine, aromatic amino acids, and dipicolinate may arise from the ASK network or from alternative pathways. Ask proteins were subjected to cohesion group analysis, a methodology that sorts a given protein assemblage into groups in which evolutionary continuity is assured. Two subhomology divisions, ASKα and ASKβ, have been recognized. The ASKα subhomology division is the most ancient, being widely distributed throughout the Archaea and Eukarya and in some Bacteria. Within an indel region of about 75 amino acids near the N terminus, ASKβ sequences differ from ASKα sequences by the possession of a proposed ancient deletion. ASKβ sequences are present in most Bacteria and usually exhibit an in-frame internal translational start site that can generate a small Ask subunit that is identical to the C-terminal portion of the larger subunit of a heterodimeric unit. Particularly novel are ask genes embedded in gene contexts that imply specialization for ectoine (osmotic agent) or aromatic amino acids. The cohesion group approach is well suited for the easy recognition of relatively recent lateral gene transfer (LGT) events, and many examples of these are described. Given the current density of genome representation for Proteobacteria, it is possible to reconstruct more ancient landmark LGT events. Thus, a plausible scenario in which the three well-studied and iconic Ask homologs of Escherichia coli are not within the vertical genealogy of Gammaproteobacteria, but rather originated via LGT from a Bacteroidetes donor, is supported.

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Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic Escherichia coli paralogs are AroA I proteins, but many members of the Bacteria possess the AroA II class of enzyme, sometimes in combination with AroA I proteins. AroA II DAHP synthases until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the Xanthomonas campestris AroA II protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroA II was cloned in E. coli by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (>99%) for the unstable protein. The recombinant AroA II protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector. L-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroA II of higher plants, X. campestris AroA II was not hysteretically activated by dithiols. Compared to plant AroA II proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroA II originated within the Bacteria domain, and it seems probable that higher-plant plastids acquired AroA II from a gram-negative bacterium via endosymbiosis. The X. campestris AroA II protein is suggested to exemplify a case of analog displacement whereby an ancestral aroA I species was discarded, with the aroA II replacement providing an alternative pattern of allosteric control. Three subgroups of AroA II proteins can be recognized: a large, central group containing the plant enzymes and that from X. campestris, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream.Microorganisms and plants engage 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) as the initial catalyst for the commitment of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) to the biosynthesis of aromatic amino acids, vitamin K, folic acid, and ubiquinone (4). The pathway contributes heavily to the biochemical individuality of organisms, with an enormous variety of peripheral branch connections known. Various pigments, siderophores, signaling compounds, defense metabolites, structural compounds, antibiotics, and other secondary metabolites are derived from this pathway (4).DAHP synthases fall into two distinct homology families. The AroA I family is divided into subfamilies AroA I␣ and AroA I␤ (32). The AroA I␣ subfamily is widely represented in gram-negative bacteria in which differentially regulated paralogs are commonly exp...

We previously proposed that in Chlamydiaceae rapid vegetative growth and a quiescent state of survival (persistence) depend upon alternative protein translational profiles dictated by host tryptophan (Trp) availability. These alternative profiles correspond, respectively, with a set of chlamydial proteins having higher-than-predicted contents of Trp (“Up-Trp” selection), or with another set exhibiting lower-than-predicted contents of Trp (“Down-Trp” selection). A comparative evaluation of Chlamydiaceae proteomes for Trp content has now been extended to a number of other taxon families within the Chlamydiales Order. At the Order level, elevated Trp content occurs for transporters of nucleotides, S-adenosylmethionine (SAM), dicarboxylate substrates, and Trp itself. For Trp and nucleotide transporters, this is even more pronounced in other chlamydiae families (Parachlamydiaceae, Waddliaceae, and Simkaniaceae) due to extensive paralog expansion. This suggests that intracellular Trp availability served as an ancient survival cue for enhancement or restraint of chlamydial metabolism in the common Chlamydiales ancestor. The Chlamydiaceae Family further strengthened Up-Trp selection for proteins that function in cell division, lipopolysaccharide biosynthesis, and methyltransferase reactions. Some proteins that exhibit Up-Trp selection are uniquely present in the Chlamydiaceae, e.g., cytotoxin and the paralog families of polymorphic membrane proteins (Pmp's). A striking instance of Down-Trp selection in the Chlamydiaceae is the chorismate biosynthesis pathway and the connecting menaquinone pathway. The newly recognized 1,4-dihydroxy-6-napthoate pathway of menaquinone biosynthesis operates in Chlamydiaceae, whereas the classic 2-napthoate pathway is used in the other Chlamydiales families. Because of the extreme Down-Trp selection, it would appear that menaquinone biosynthesis is particularly important to the integrity of the persistent state maintained under conditions of severe Trp limitation, and may thus be critical for perpetuation of chronic disease states.