Genomic Species Are Ecological Species as Revealed by Comparative Genomics in Agrobacterium tumefaciens

The definition of bacterial species is based on genomic similarities, giving rise to the operational concept of genomic species, but the reasons of the occurrence of differentiated genomic species remain largely unknown. We used the Agrobacterium tumefaciens species complex and particularly the genomic species presently called genomovar G8, which includes the sequenced strain C58, to test the hypothesis of genomic species having specific ecological adaptations possibly involved in the speciation process. We analyzed the gene repertoire specific to G8 to identify potential adaptive genes. By hybridizing 25 strains of A. tumefaciens on DNA microarrays spanning the C58 genome, we highlighted the presence and absence of genes homologous to C58 in the taxon. We found 196 genes specific to genomovar G8 that were mostly clustered into seven genomic islands on the C58 genome—one on the circular chromosome and six on the linear chromosome—suggesting higher plasticity and a major adaptive role of the latter. Clusters encoded putative functional units, four of which had been verified experimentally. The combination of G8-specific functions defines a hypothetical species primary niche for G8 related to commensal interaction with a host plant. This supports that the G8 ancestor was able to exploit a new ecological niche, maybe initiating ecological isolation and thus speciation. Searching genomic data for synapomorphic traits is a powerful way to describe bacterial species. This procedure allowed us to find such phenotypic traits specific to genomovar G8 and thus propose a Latin binomial, Agrobacterium fabrum, for this bona fide genomic species

Coordinated Regulation of Species-Specific Hydroxycinnamic Acid Degradation and Siderophore Biosynthesis Pathways in Agrobacterium fabrum

International audienceThe rhizosphere-inhabiting species Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to degrade hydroxycinnamic acids (HCAs), especially ferulic acid and p-coumaric acid, via the novel A. fabrum HCA degradation pathway. Gene expression profiles of A. fabrum strain C58 were investigated in the presence of HCAs, using a C58 whole-genome oligoarray. Both ferulic acid and p-coumaric acid caused variations in the expression of more than 10% of the C58 genes. Genes of the A. fabrum HCA degradation pathway, together with the genes involved in iron acquisition, were among the most highly induced in the presence of HCAs. Two operons coding for the biosynthesis of a particular siderophore, as well as genes of the A. fabrum HCA degradation pathway, have been described as being specific to the species. We demonstrate here their coordinated expression, emphasizing the interdependence between the iron concentration in the growth medium and the rate at which ferulic acid is degraded by cells. The coordinated expression of these functions may be advantageous in HCA-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. The present results confirm that there is cooperation between the A. fabrum-specific genes, defining a particular ecological niche. IMPORTANCE: We previously identified seven genomic regions in Agrobacterium fabrum that were specifically present in all of the members of this species only. Here we demonstrated that two of these regions, encoding the hydroxycinnamic acid degradation pathway and the iron acquisition pathway, were regulated in a coordinated manner. The coexpression of these functions may be advantageous in hydroxycinnamic acid-rich but iron-starved environments in which microorganisms have to compete for both iron and carbon sources, such as in plant roots. These data support the view that bacterial genomic species emerged from a bacterial population by acquiring specific functions that allowed them to outcompete their closest relatives. In conclusion, bacterial species could be defined not only as genomic species but also as ecological species

Antigen specific activation of cytotoxic CD8+ T cells by Staphylococcus aureus infected dendritic cells

International audienceStaphylococcus aureus ( S. aureus ) is a pathogen associated with a wide variety of diseases, from minor to life-threatening infections. Antibiotic-resistant strains have emerged, leading to increasing concern about the control of S. aureus infections. The development of vaccines may be one way to overcome these resistant strains. However, S. aureus ability to internalize into cells – and thus to form a reservoir escaping humoral immunity – is a challenge for vaccine development. A role of T cells in the elimination of persistent S. aureus has been established in mice but it remains to be established if CD8 + T cells could display a cytotoxic activity against S. aureus infected cells. We examined in vitro the ability of CD8 + T cells to recognize and kill dendritic cells infected with S. aureus. We first evidenced that both primary mouse dendritic cells and DC2.4 cell line can be infected with S. aureus . We then generated a strain of S. aureus expressing a model CD8 epitope and transgenic F5 CD8 + T cells recognizing this model epitope were used as reporter T cells. In response to S. aureus -infected dendritic cells, F5 CD8 + T cells produced IFN-γ in an antigen-specific manner and displayed an increased ability to kill infected cells. Altogether, these results demonstrate that cells infected by S. aureus display bacteria-derived epitopes at their surface that are recognized by CD8 + T cells. This paves the way for the development of CD8 + T cell-based therapies against S. aureus

Impact of currently marketed tampons and menstrual cups on Staphylococcus aureus growth and TSST-1 production in vitro

International audienceFifteen currently marketed intravaginal protection products (11 types of tampon and four menstrual cups) were tested by the modified tampon sac method to determine their effect on Staphylococcus aureus growth and toxic shock toxin 1 (TSST-1) production. Most tampons reduced S. aureus growth and TSST-1 production, with differences based on brand and composition, and S. aureus growth was higher in de-structured than in unaltered tampons. We observed higher S. aureus growth and toxin production in menstrual cups than in tampons, potentially due to the additional air introduced to the bag by cups, with differences based on cup composition and size.Importance Menstrual toxic shock syndrome is a rare but severe disease. It occurs in healthy women vaginally colonized by Staphylococcus aureus producing toxic shock syndrome toxin 1 using intravaginal protection such as tampons or menstrual cups. Intravaginal protection induces TSS production by collecting catamenial products which act as a growth medium for S. aureus Previous studies have evaluated the impact of tampon composition on S. aureus producing toxic shock syndrome toxin 1, but they are not recent and did not include menstrual cups. This study demonstrates that highly reproducible results for S. aureus growth and TSST-1 production can be obtained using a simple protocol that reproduces the physiological conditions of tampon and cup usage as closely as possible, providing recommendations for tampon or cup use to both manufacturers and consumers. Notably, our results do not show that menstrual cups are safer than tampons and suggest that they require similar precautions

RsaC sRNA modulates the oxidative stress response of Staphylococcus aureus during manganese starvation

International audienceThe human opportunistic pathogen Staphylococcus aureus produces numerous small regulatory RNAs (sRNAs) for which functions are still poorly understood. Here, we focused on an atypical and large sRNA called RsaC. Its length varies between different isolates due to the presence of repeated sequences at the 5′ end while its 3′ part is structurally independent and highly conserved. Using MS2-affinity purification coupled with RNA sequencing (MAPS) and quantitative differential proteomics, sodA mRNA was identified as a primary target of RsaC sRNA. SodA is a Mn-dependent superoxide dismutase involved in oxidative stress response. Remarkably, rsaC gene is co-transcribed with the major manganese ABC transporter MntABC and, consequently, RsaC is mainly produced in response to Mn starvation. This 3′UTR-derived sRNA is released from mntABC-RsaC precursor after cleavage by RNase III. The mature and stable form of RsaC inhibits the synthesis of the Mn-containing enzyme SodA synthesis and favors the oxidative stress response mediated by SodM, an alternative SOD enzyme using either Mn or Fe as co-factor. In addition, other putative targets of RsaC are involved in oxidative stress (ROS and NOS) and metal homeostasis (Fe and Zn). Consequently, RsaC may balance two interconnected defensive responses, i.e. oxidative stress and metal-dependent nutritional immunity

What is a supercoiling-sensitive gene? Insights from topoisomerase I inhibition in the Gram-negative bacterium Dickeya dadantii

International audienceDNA supercoiling is an essential mechanism of bacterial chromosome compaction, whose level is mainly regulated by topoisomerase I and DNA gyrase. Inhibiting either of these enzymes with antibiotics leads to global supercoiling modifications and subsequent changes in global gene expression. In previous studies, genes responding to DNA relaxation induced by DNA gyrase inhibition were categorised as ‘supercoiling-sensitive’. Here, we studied the opposite variation of DNA supercoiling in the phytopathogen Dickeya dadantii using the non-marketed antibiotic seconeolitsine. We showed that the drug is active against topoisomerase I from this species, and analysed the first transcriptomic response of a Gram-negative bacterium to topoisomerase I inhibition. We find that the responding genes essentially differ from those observed after DNA relaxation, and further depend on the growth phase. We characterised these genes at the functional level, and also detected distinct patterns in terms of expression level, spatial and orientational organisation along the chromosome. Altogether, these results highlight that the supercoiling-sensitivity is a complex feature, which depends on the action of specific topoisomerases, on the physiological conditions, and on their genomic context. Based on previous in vitro expression data of several promoters, we propose a qualitative model of SC-dependent regulation that accounts for many of the contrasting transcriptomic features observed after DNA gyrase or topoisomerase I inhibition

Analysis of hydroxycinnamic acids degradation in Agrobacterium fabrum reveals a CoA-dependent, beta-oxidative deacetylation pathway

International audienceThe soil and rhizosphere inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the A. tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized by genetic and analytical means intermediates of degradation as feruloyl-CoA, 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl-CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl-CoA, vanillic acid and protocatechuic acid. Genes atu1416, atu1417 and atu1420 have been experimentally shown necessary for the degradation of ferulic acid. Moreover, genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a HMPKP-CoA β-keto-thiolase, respectively. We thus demonstrated that the A. fabrum-hydroxycinnamic degradation pathway is an original coenzyme-A-dependent β-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables metabolism of toxic compounds from plants and their use for growth, likely providing this species an ecological advantage in hydroxycinnamic-rich environments such as plant roots or decaying plant materials

Toward an ecological concept for bacterial species in Agrobacterium

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Toward an ecological concept for bacterial species in Agrobacterium

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