The acidithiobacilli are sulfur-oxidizing acidophilic bacteria that thrive in both natural and anthropogenic low pH environments. They contribute to processes that lead to the generation of acid rock drainage in several different geoclimatic contexts, and their properties have long been harnessed for the biotechnological processing of minerals. Presently, the genus is composed of seven validated species, described between 1922 and 2015: Acidithiobacillus thiooxidans, A. ferrooxidans, A. albertensis, A. caldus, A. ferrivorans, A. ferridurans, and A. ferriphilus. However, a large number of Acidithiobacillus strains and sequence clones have been obtained from a variety of ecological niches over the years, and many isolates are thought to vary in phenotypic properties and cognate genetic traits. Moreover, many isolates remain unclassified and several conflicting specific assignments muddle the picture from an evolutionary standpoint. Here we revise the phylogenetic relationships within this species complex and determine the phylogenetic species boundaries using three different typing approaches with varying degrees of resolution: 16S rRNA gene-based ribotyping, oligotyping, and multi-locus sequencing analysis (MLSA). To this end, the 580 16S rRNA gene sequences affiliated to the Acidithiobacillus spp. were collected from public and private databases and subjected to a comprehensive phylogenetic analysis. Oligotyping was used to profile high-entropy nucleotide positions and resolve meaningful differences between closely related strains at the 16S rRNA gene level. Due to its greater discriminatory power, MLSA was used as a proxy for genome-wide divergence in a smaller but representative set of strains. Results obtained indicate that there is still considerable unexplored diversity within this genus. At least six new lineages or phylotypes, supported by the different methods used herein, are evident within the Acidithiobacillus species complex. Although the diagnostic characteristics of these subgroups of strains are as yet unresolved, correlations to specific metadata hint to the mechanisms behind econiche-driven divergence of some of the species/phylotypes identified. The emerging phylogenetic structure for the genus outlined in this study can be used to guide isolate selection for future population genomics and evolutionary studies in this important acidophile model.

While a functional quorum sensing system has been identified in the acidophilic chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 23270^T and shown to modulate cell adhesion to solid substrates, nothing is known about the genes it regulates. To address the question of how quorum sensing controls biofilm formation in A. ferrooxidans^T, the transcriptome of this organism in conditions in which quorum sensing response is stimulated by a synthetic superagonist AHL (N-acyl homoserine lactones) analog has been studied. First, the effect on biofilm formation of a synthetic AHL tetrazolic analog, tetrazole 9c, known for its agonistic QS activity, was assessed by fluorescence and electron microscopy. A fast adherence of A. ferrooxidans^T cells on sulfur coupons was observed. Then, tetrazole 9c was used in DNA microarray experiments that allowed the identification of genes regulated by quorum sensing signaling, and more particularly, those involved in early biofilm formation. Interestingly, afeI gene, encoding the AHL synthase, but not the A. ferrooxidans quorum sensing transcriptional regulator AfeR encoding gene, was shown to be regulated by quorum sensing. Data indicated that quorum sensing network represents at least 4.5% (141 genes) of the ATCC 23270^T genome of which 42.5% (60 genes) are related to biofilm formation. Finally, AfeR was shown to bind specifically to the regulatory region of the afeI gene at the level of the palindromic sequence predicted to be the AfeR binding site. Our results give new insights on the response of A. ferrooxidans to quorum sensing and on biofilm biogenesis.