Buchnera aphidicola, associated with most of the aphids (Hemiptera: aphididae), is a fascinating bacterium, both because of its apparent minimalist physiology and because of its intermediate status between an autonomous cell and an intracellular organelle. Shaped by some 150–200 million years of intracellular evolution, its genome and regulatory system have evolved to fit the evolutionary constraints imposed by the symbiotic partnership [1, 2]. This work is a comparative genomic analysis of cis- and trans-regulators encoded in the Buchnera genomes of five different aphid strains, Acyrthosiphon pisum (BAp), Schizaphis graminum (BSg), Baizongia pistacea (BBp), Cinara cedri (BCc) and Cinara tujafilina (BCt), combined with analyses of the intrinsic physical topological properties of their DNA molecules. The main objective was to decipher the regulatory mechanisms underlying gene regulation in these bacteria.
The Buchnera genomes from the five aphid species share certain properties: (1) a small size, from 416 kb for BCc to 641 kb for BAp[1, 3–6]; (2) a low GC-content of about 25%; (3) a standard bacterial gene density of about 85% of coding DNA; (4) the conservation of most genes encoding enzymes from the biosynthesis of essential amino acids that Buchnera furnish to their hosts [7, 8]. The differences between these five aphid species are related to the physiology of the symbiotic interactions that created specific evolutionary constraints, contributing to the differentiation of the Buchnera gene repertoires . BCc offers an example of evolution with an extremely reduced genome, probably linked to the presence in their aphid host of the co-primary endosymbiont “Candidatus Serratia symbiotica” with which they show a strong dependency and partially share genes of several amino acid biosynthetic pathways [10, 11].
Gene regulation in Buchnera has been a matter of controversy in recent years. Global transcriptomic analyses revealed a weak transcriptional response to various stresses applied on the host, such as heat shock  and single amino acid excess  in BSg, as well as aromatic and essential amino acid depletions in BAp. However, stronger effects were observed when the transcriptional responses were compared between Buchnera populations from embryonic and maternal aphid compartments , somehow reflecting two different physiological growing states of Buchnera. Finally, following the kinetics of the response in BAp, a specific induction (repression) of the genes of the leucine biosynthetic pathway was observed after one day of treatment following a depletion (excess) of the leucine concentration in the aphid diet, although the transcriptional response was not significant after seven days of treatment .
In bacteria, two main interrelated processes govern gene transcription . There is the “classical” mechanism, involving sigma and specific transcription factors binding DNA sequences located in the proximity of the transcrip-tion initiation site of a gene and able to induce or repress transcription initiation by the RNA polymerase . Then there is a more recently discovered mechanism based on the regulation of DNA topology controlled by Nucleoid Associated Proteins (NAP) and topoisomerases [19–23] and partially characterized by several physical parameters, such as DNA stability, curvature and supercoiling [24, 25]. Both processes involve trans-regulatory factors (i.e., proteins binding to DNA) interacting, with varying degrees of specificity, with the cis-regulatory elements (i.e., DNA sequences). The regulatory mechanisms controlling transcription initiation are the most thoroughly described in free-living bacteria  and genome organisation suggests that they have also been conserved in Buchnera. Other events responsible for transcriptional regulation, like termination, mRNA maturation and stability control, as well as translation regulation, are also important targets for gene expression regulation, possibly involving small RNAs [27, 28]. These mechanisms have not been taken into account in this work. Finally, post-translational modification by reversible Nε-lysine acetylation of transcription factors has been recently reported in bacteria and might directly affect gene expression . However, it seems unlikely that this mechanism exists in Buchnera as the corresponding acetyltransferase (Pat or YfiQ) and the NAD-dependant deacetylase (CobB), described in Salmonella enterica and Escherichia coli, are lacking in Buchnera.
The bacterial chromosome is known to be associated with proteins which allow for a massive compaction and, at the same time, are able to dynamically regulate the DNA molecule, rendering rapidly accessible those DNA regions which need to be transcribed [30, 31]. NAPs have been extensively described in E. coli[32, 33]. They participate in the chromosome structuring and also in all the processes involving DNA transactions (replication, recombination and transcription). NAPs are basic, small molecular weight proteins and their relative abundance is dynamic and dependent on the cell physiology. For example, different bacterial growing phases are characterised by specific expression patterns of the different NAPs [34, 35]. Although more than 12 NAPs have been described in E. coli, almost all the literature centres on only four of them: H-NS (Histone-like Nucleoid Structuring protein), HU (Heat Unstable nucleoid protein), IHF (Integration Host Factor) and FIS (Factor for Inversion Stimulation). Apart from NAPs, the maintenance of the chromosome supercoiling in bacteria is controlled by topoisomerases that either relax the negative supercoils (type I topoisomerase) or serve to introduce them (ATP consuming type II topoisomerase), hence linking the energetic metabolism of the cell with the DNA topology .
Negative supercoiling is essential for chromosome compaction and for the survival of the bacteria [21, 37]. Local supercoiling variations in the DNA modulate the polymerase affinity for promoters. Hence, DNA supercoiling is often considered as a true transcriptional regulator that is sensitive to the environmental conditions [19, 23, 35, 38] and that uses the ATP/ADP ratio governing gyrase activity as a sensor of the energetic level of the cell [36, 39]. Curvature, flexibility and stability, contrary to supercoiling, are properties which are highly correlated with the primary sequence of the DNA molecule, although it has been suggested that nucleoid proteins might also influence these parameters . Curvature and flexibility (estimated in this work by the base-pair propeller twisting) are essential for the initiation of transcription since the promoter affinity for polymerase, and for the associated transcription factors, is sensitive to the topology of the DNA molecule . The double-strand stability of the DNA molecule is also very important, particularly in the promoter region, for transcription initiation. In this study, we have estimated the base stacking energy, which is a direct measure of the base pair affinity within the DNA molecule , and the Stress-Induced DNA Duplex Destabilization (SIDD)  to assess the stability of the double strand DNA molecule in Buchnera.
The aim of this work is to give an initial description of the structure and of the evolution of the gene regulatory network in Buchnera using a genomic comparative analysis. Regulatory networks are known to evolve quickly and both the DNA-binding domains of transcription factors and their target sequence sets are highly dynamic (i.e., orthologous regulators are often regulating non-orthologous targets), making comparative studies difficult [43, 44]. The Buchnera model is interesting in this respect, first because the bacteria evolved for millions of years sequestrated within aphids, preventing any contact with other bacterial populations and, hence, any horizontal gene transfer and, secondly, because Buchnera were almost uniformly constrained by the intracellular conditions (which relaxes the selection of some genes that become superfluous) and by the physiological requirements imposed by their symbiotic association with aphids (mostly concerning the biosynthesis of essential metabolites, such as amino acids). Thus, after analysing the selective constraints exerted on the regulatory genes, we performed a systematic characterization of the cis- and trans-regulatory elements predicted in the Buchnera genomes from the five sequenced strains. These analyses were then coupled with the characterisation of some intrinsic topological properties of the Buchnera DNA chromosome, which allowed us to formulate certain hypotheses regarding their possible involvement in gene transcription regulation in these bacteria.