In C. crescentus, iron metabolism is mainly controlled by the transcription factor Fur (ferric uptake regulator). Iron-bound Fur represses genes related to iron uptake and can directly activate the expression of genes for iron-containing proteins. In this work, we used total RNA sequencing (RNA-seq) of wild type C. crescentus growing in minimal medium under iron limitation and a fur mutant strain to expand the known Fur regulon, and to identify novel iron-regulated genes. The RNA-seq of cultures treated with the iron chelator 2-2-dypiridyl (DP) allowed identifying 256 upregulated genes and 236 downregulated genes, being 176 and 204 newly identified, respectively. Sixteen transcription factors and seven sRNAs were upregulated in iron limitation, suggesting that the response to low iron triggers a complex regulatory network. Notably, lexA along with most of its target genes were upregulated, suggesting that DP treatment caused DNA damage, and the SOS DNA repair response was activated in a RecA-dependent manner, as confirmed by RT-qPCR. Fluorescence microscopy assays using an oxidation-sensitive dye showed that wild type cells in iron limitation and the fur mutant were under endogenous oxidative stress, and a direct measurement of cellular H2O2 showed that cells in iron-limited media present a higher amount of endogenous H2O2. A mutagenesis assay using the rpoB gene as a reporter showed that iron limitation led to an increase in the mutagenesis rate. These results showed that iron deficiency causes C. crescentus cells to suffer oxidative stress and to activate the SOS response, indicating an increase in DNA damage.

Inflammation is a driven force in modulating microbial communities, but little is known about the interplay between colonizing microorganisms and the immune response in periodontitis. Since local and systemic inflammation may play a whole role in disease, we aimed to evaluate the oral and fecal microbiome of patients with periodontitis and to correlate the oral microbiome data with levels of inflammatory mediator in saliva.Methods: Nine patients with periodontitis (P) in Stage 3/Grade B and nine age-matched non-affected controls (H) were evaluated. Microbial communities of oral biofilms (the supra and subgingival from affected and non-affected sites) and feces were determined by sequencing analysis of the 16SrRNA V3–V4 region. Salivary levels of 40 chemokines and cytokines were correlated with oral microbiome data.Results: Supragingival microbial communities of P differed from H (Pielou's evenness index, and Beta diversity, and weighted UniFrac), since relative abundance (RA) of Defluviitaleaceae, Desulfobulbaceae, Mycoplasmataceae, Peptostreococcales-Tissierellales, and Campylobacteraceae was higher in P, whereas Muribaculaceae and Streptococcaceae were more abundant in H. Subgingival non-affected sites of P did not differ from H, except for a lower abundance of Gemellaceae. The microbiome of affected periodontitis sites (PD ≥ 4 mm) clustered apart from the subgingival sites of H. Oral pathobionts was more abundant in sub and supragingival biofilms of P than H. Fecal samples of P were enriched with Acidaminococcus, Clostridium, Lactobacillus, Bifidobacterium, Megasphaera, and Romboutsia when compared to H. The salivary levels of interleukin 6 (IL-6) and inflammatory chemokines were positively correlated with the RA of several recognized and putative pathobionts, whereas the RA of beneficial species, such as Rothia aeria and Haemophilus parainfluenzae was negatively correlated with the levels of Chemokine C-C motif Ligand 2 (CCL2), which is considered protective. Dysbiosis in patients with periodontitis was not restricted to periodontal pockets but was also seen in the supragingival and subgingival non-affected sites and feces. Subgingival dysbiosis revealed microbial signatures characteristic of different immune profiles, suggesting a role for candidate pathogens and beneficial organisms in the inflammatory process of periodontitis.

BackgroundAs bacterial cells enter stationary phase, they adjust their growth rate to comply with nutrient restriction and acquire increased resistance to several stresses. These events are regulated by controlling gene expression at this phase, changing the mode of exponential growth into that of growth arrest, and increasing the expression of proteins involved in stress resistance. The two-component system SpdR/SpdS is required for the activation of transcription of the Caulobacter crescentus cspD gene at the onset of stationary phase.ResultsIn this work, we showed that both SpdR and SpdS are also induced upon entry into stationary phase, and this induction is partly mediated by ppGpp and it is not auto-regulated. Global transcriptional analysis at early stationary phase of a spdR null mutant strain compared to the wild type strain was carried out by DNA microarray. Twenty-three genes showed at least twofold decreased expression in the spdR deletion mutant strain relative to its parental strain, including cspD, while five genes showed increased expression in the mutant. The expression of a set of nine genes was evaluated by quantitative real time PCR, validating the microarray data, and indicating an important role for SpdR at stationary phase. Several of the differentially expressed genes can be involved in modulating gene expression, including four transcriptional regulators, and the RNA regulatory protein Hfq. The ribosomal proteins NusE and NusG, which also have additional regulatory functions in transcription and translation, were also downregulated in the spdR mutant, as well as the ParE1 toxin. The purified SpdR protein was shown to bind to the regulatory region of CC0517 by Electrophoretic Mobility Shift Assay, and the SpdR-regulated gene CC0731 was shown to be expressed at a lower level in the null cspD mutant, suggesting that at least part of the effect of SpdR on the expression of this gene is indirect.ConclusionsThe results indicate that SpdR regulates several genes encoding proteins of regulatory function, which in turn may be required for the expression of other genes important for the transition to stationary phase.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0682-y) contains supplementary material, which is available to authorized users.

Small RNAs are important for post-transcriptional regulation of gene expression, affecting stability and activity of their target mRNAs. The bacterial Sm-like protein Hfq is required to promote pairing between both RNAs when their sequence complementarity is limited. To provide a first global view on the post-transcriptional landscape of the α-proteobacterium Caulobacter crescentus, we have identified the Hfq-binding RNAs employing High-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP). A total of 261 RNAs, including 3 unannotated RNAs, were successfully identified and classified according to putative function. Moreover, possible interactions between the identified sRNAs with mRNA targets were postulated through computational target predictions.

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