Structural, evolutionary and genetic analysis of the histidine biosynthetic “core” in the genus Burkholderia Maria Cristiana Papaleo a , Edda Russo a , Marco Fondi a , Giovanni Emiliani b , Antonio Frandi a , Matteo Brilli c , Roberta Pastorelli d , Renato Fani a, ⁎ a Department of Evolutionary Biology, Via Romana 17-19, University of Florence, 50125 Florence, Italy b Department of Environmental and Forestry Sciences, via S. Bonaventura 13, 50145 University of Florence, Italy c UMR CNRS 5558 – LBBE “Biométrie et Biologie évolutive”, UCB Lyon 1 - Bât. Grégor Mendel, 43 bd du 11 novembre 1918, 69622 Villeurbanne cedex d Research Centre of Agrobiology and Pedology, Piazza M. D'Azeglio 30, Agricultural Research Council (CRA) Florence, Italy abstract article info Article history: Received 27 April 2009 Received in revised form 25 July 2009 Accepted 5 August 2009 Available online 13 August 2009 Received by R. Britton Keywords: Operon evolution Alien genes Histidine genes In this work a detailed analysis of the structure, the expression and the organization of his genes belonging to the core of histidine biosynthesis (hisBHAF) in 40 newly determined and 13 available sequences of Bur- kholderia strains was carried out. Data obtained revealed a strong conservation of the structure and organization of these genes through the entire genus. The phylogenetic analysis showed the monophyletic origin of this gene cluster and indicated that it did not undergo horizontal gene transfer events. The analysis of the intergenic regions, based on the substitution rate, entropy plot and bendability suggested the existence of a putative transcription promoter upstream of hisB, that was supported by the genetic analysis that showed that this cluster was able to complement Escherichia coli hisA, hisB, and hisF mutations. Moreover, a preliminary transcriptional analysis and the analysis of microarray data revealed that the expression of the his core was constitutive. These findings are in agreement with the fact that the entire Burkholderia his operon is heterogeneous, in that it contains “alien” genes apparently not involved in histidine biosynthesis. Besides, they also support the idea that the proteobacterial his operon was piece- wisely assembled, i.e. through accretion of smaller units containing only some of the genes (eventually together with their own promoters) involved in this biosynthetic route. The correlation existing between the structure, organization and regulation of his “core” genes and the function(s) they perform in cellular metabolism is discussed. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Histidine biosynthesis is one of the most studied anabolic pathways. It has been studied for over 40 years in Escherichia coli and its close relative Salmonella enterica (formerly Salmonella typhimurium), leading to the accumulation of a very large body of biochemical, genetic, molecular and physiological data (Alifano et al., 1996). Histidine biosynthesis consists of nine intermediates and of eight distinct proteins that in the two enterobacterial species are encoded by eight genes organized in a very compact operon and arranged in the order hisGDC(NB)HAF(IE)(Alifano et al., 1996; Fani et al., 1997, 2006). Four of the his genes (hisBHAF) are particularly interesting from an evolutionary viewpoint and form the so-called “core” of the pathway (Fig. 1), which plays an important role in cellular metabolism. Indeed, it is a metabolic cross-point inter- connecting histidine biosynthesis to both nitrogen metabolism and de novo synthesis of purines. The available information also showed that after the assembly of the entire pathway, the structure and/or organization of his genes underwent major rearrangements in the three domains, which generated a wide variety of structural and/or clustering strategies of his genes (Fani et al., 1998, 2005). Thus, the analysis of the structure and organization of his genes might help in shedding some light on the origin and evolution of operons (Fani et al., 2005; Price et al., 2006). Recently, we proposed that the proteobacterial his operon might be a recent invention of evolution and was piece-wisely constructed (Fani et al., 2005, 2006). According to the model proposed, the his genes, scattered on the genome of proteobacterial ancestor, underwent a progressive clustering that culminated in some γ-proteobacteria where the Gene 448 (2009) 16–28 Abbreviations: hisG, ATP phosphoribosyl transferase (EC 2.4.2.17); hisD, histidinol dehydrogenase (EC 1.1.1.23); hisC, histidinol-phosphate aminotransferase (EC 2.6.1.9); hisN, histidinol-phosphate phosphatase (EC 3.1.3.15); hisB, imidazoleglycerol-phos- phate dehydratase (EC 4.2.1.19); hisH, G-type glutamine amidotransferase; hisA,[N- (5′-phosphoribosyl) formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerise (EC 5.3.1.16); hisF, imidazole glycerol phosphate synthase subunit HisF (EC 4.1.3.-); hisI, phosphoribosyl-AMP cyclohydrolase (EC 3.5.4.19); hisE, phosphoribosyl- ATP pyrophosphatase (EC 3.6.1.31); murA, UDP-N-acetylglucosamine 1-carboxyvinyl- transferase; marC, integral membrane protein; Bcc, Burkholderia cepacia complex. ⁎ Corresponding author. Tel.: +39 55 2288244; fax: +39 55 2288250. E-mail address: renato.fani@unifi.it (R. Fani). 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.08.002 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene