Structural, evolutionary and genetic analysis of the histidine biosynthetic “core” in the genus Burkholderia

Abstract

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 Burkholderia 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.

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, 1996, Fani, 1997, Fani et al., 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 interconnecting 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, Fani et al., 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, Fani et al., 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 operons are very compact and include fused and/or overlapping genes. The first step of the operon construction was the formation of the his “core” (Alifano, 1996, Fani et al., 1995), an event likely occurring in the ancestor of α/β/γ proteobacteria (or even predating its appearance). Then, the ancestral hisBHAF operon joined two other independently formed sub-operons (hisGDC and hisIE) giving an almost complete operon. A corollary of this model is the following: if the evolution of long his compact operons actually happened this way, one can imagine intermediate stages in which the his genes were organized in less compact operons, eventually containing one or more functionally unrelated (“alien”) genes (heterogeneous operons), which during evolution were excised and/or transposed to other chromosomal locations. In principle, since the heterogeneous operons contain genes not involved in histidine biosynthesis, the pathway should not be tuned as finely as in E. coli and S. enterica and might be constitutively expressed. Lastly, if the piece-wise model (Fani et al., 2005) is correct, in these operons the internal promoters identified in E. coli and S. enterica and located upstream of hisB and/or hisI might still be present and functional. Even though this idea might be intuitive, in our knowledge it has not been demonstrated (at least) for histidine biosynthesis. The only data available are those concerning the α-proteobacterium Azospirillum brasilense, possessing a his heterogeneous cluster hisBHorf1AFEhit where a transcription promoter located just upstream of hisB allows the constitutive expression of the downstream genes (Fani, 1989, Fani, 1993).

Additionally, it is not clear yet whether the gene structure, organization and/or regulation are linked to the taxonomical position of a given strain/species/genus and/or to the metabolism of the organism(s). In some cases (Fondi et al., 2007) the appearance of a given gene structure (i.e. gene fusion) and/or organization can be mapped in a taxonomical branch, whereas in others (Fani et al., 2005) it appears much less related to the phylogeny. Thus, the analysis of genes belonging to a heterogeneous his operon at multiple taxonomic scales, ranging from strain to genus level, which has not been carried out until now, can shed some light on these issues. In this context the his “core” represents an interesting case study. Thus, the aim of this work was to study the his “core” from different “non-model” strains harboring a heterogeneous his operon by an approach combining both bioinformatic and experimental methods. Such an analysis may provide useful hints on: i) the degree of conservation of structure and organization of genes belonging to the his core at the genus, species and strain level; ii) the degree of conservation/divergence of intergenic sequences; iii) the presence of transcription regulatory signals within them and the degree of their conservation at either taxonomic (between strains belonging to the same or to different species/genus) or ecological level (between strains occupying different environmental niches); iv) the regulation of his “core” transcription in microorganisms different from enterobacteria and whether expression is constitutive or either regulated by histidine concentrations; v) the presence and the nature of alien genes; and vi) the validity of the piece-wise model of the origin and evolution of proteobacterial his operons. To this purpose, we focused our attention on β-proteobacteria, a taxon including bacteria harboring homogeneous as well as heterogeneous his operons and representing key organisms in the construction of compact proteobacterial his operons.Within β-proteobacteria, the Burkholderia cepacia complex (Bcc) is relevant in ecology and human health. Indeed, these bacteria are important opportunistic pathogens causing lung infections in immuno-compromised patients, especially those with cystic fibrosis (CF). In contrast to these pathogenic properties, Bcc organisms are also found in natural habitats and have a considerable ecological and commercial importance. In fact, some Bcc strains are able to catabolize many toxic compounds and to produce a number of substances that antagonize soil borne plant pathogens. Therefore, the Bcc represents an interesting and heterogeneous taxonomical entity, comprising strains from different environments and with different metabolic abilities. Last, the different ecological niches occupied by Bcc strains and/or species may influence the regulation of his genes and provide some insights into the conservation/divergence of transcriptional regulatory signals. A very preliminary analysis of the organization of histidine biosynthetic genes carried out in a very limited number of Burkholderia strains (Fani et al., 2005) revealed that the his genes are organized in a heterogeneous operon comprising 16 genes, seven apparently not belonging to the known histidine biosynthesis chain of reactions (Fig. 1).

Thus, in this work the structure of the histidine biosynthetic core (hisBHAF) was studied within the genus Burkholderia at multiple taxonomic levels by analyzing it in a panel of 40 Bcc strains representative of nine species from both clinical and environmental sources as well as in the 13 sequences available in databases.