The estimation of a postmortem interval (PMI) is particularly important for forensic investigations. The aim of this study was to assess the succession of bacterial communities associated with the decomposition of mouse cadavers and determine the most important biomarker taxa for estimating PMIs. High-throughput sequencing was used to investigate the bacterial communities of gravesoil samples with different PMIs, and a random forest model was used to identify biomarker taxa. Redundancy analysis was used to determine the significance of environmental factors that were related to bacterial communities. Our data showed that the relative abundance of Proteobacteria, Bacteroidetes and Firmicutes showed an increasing trend during decomposition, but that of Acidobacteria, Actinobacteria and Chloroflexi decreased. At the genus level, Pseudomonas was the most abundant bacterial group, showing a trend similar to that of Proteobacteria. Soil temperature, total nitrogen, NH₄⁺-N and NO₃⁻-N levels were significantly related to the relative abundance of bacterial communities. Random forest models could predict PMIs with a mean absolute error of 1.27 days within 36 days of decomposition and identified 18 important biomarker taxa, such as Sphingobacterium, Solirubrobacter and Pseudomonas. Our results highlighted that microbiome data combined with machine learning algorithms could provide accurate models for predicting PMIs in forensic science and provide a better understanding of decomposition processes.

Azorhizobium caulinodans is a symbiotic nitrogen-fixing bacterium that forms both root and stem nodules on Sesbania rostrata. During nodule formation, bacteria have to withstand organic peroxides that are produced by plant. Previous studies have elaborated on resistance to these oxygen radicals in several bacteria; however, to the best of our knowledge, none have investigated this process in A. caulinodans. In this study, we identified and characterised the organic hydroperoxide resistance gene ohr (AZC_2977) and its regulator ohrR (AZC_3555) in A. caulinodans ORS571. Hypersensitivity to organic hydroperoxide was observed in an ohr mutant. While using a lacZ-based reporter system, we revealed that OhrR repressed the expression of ohr. Moreover, electrophoretic mobility shift assays demonstrated that OhrR regulated ohr by direct binding to its promoter region. We showed that this binding was prevented by OhrR oxidation under aerobic conditions, which promoted OhrR dimerization and the activation of ohr. Furthermore, we showed that one of the two conserved cysteine residues in OhrR, Cys₁₁, was critical for the sensitivity to organic hydroperoxides. Plant assays revealed that the inactivation of Ohr decreased the number of stem nodules and nitrogenase activity. Our data demonstrated that Ohr and OhrR are required for protecting A. caulinodans from organic hydroperoxide stress and play an important role in the interaction of the bacterium with plants. The results that were obtained in our study suggested that a thiol-based switch in A. caulinodans might sense host organic peroxide signals and enhance symbiosis.

Glutaredoxin (GRX) plays an essential role in the control of the cellular redox state and related pathways in many organisms. There is limited information on GRXs from the model nitrogen (N₂)-fixing bacterium Azorhizobium caulinodans. In the present work, we identified and performed functional analyses of monothiol and dithiol GRXs in A. caulinodans in the free-living state and during symbiosis with Sesbania rostrata. Our data show that monothiol GRXs may be very important for bacterial growth under normal conditions and in response to oxidative stress due to imbalance of the redox state in grx mutants of A. caulinodans. Functional redundancies were also observed within monothiol and dithiol GRXs in terms of different physiological functions. The changes in catalase activity and iron content in grx mutants were assumed to favor the maintenance of bacterial resistance against oxidants, nodulation, and N₂ fixation efficiency in this bacterium. Furthermore, the monothiol GRX12 and dithiol GRX34 play a collective role in symbiotic associations between A. caulinodans and Sesbania rostrata. Our study provided systematic evidence that further investigations are required to understand the importance of glutaredoxins in A. caulinodans and other rhizobia.

Azorhizobium caulinodans ORS571 contains an 87.6 kb integrative and conjugative element (ICE^Ac) that conjugatively transfers symbiosis genes to other rhizobia. Many hypothetical redundant gene fragments (rgfs) are abundant in ICE^Ac, but their potential function in horizontal gene transfer (HGT) is unknown. Molecular biological methods were employed to delete hypothetical rgfs, expecting to acquire a minimal ICE^Ac and consider non-functional rgfs as editable regions for inserting genes related to new symbiotic functions. We determined the significance of rgf4 in HGT and identified the physiological function of genes designated rihF1a (AZC_3879), rihF1b (AZC_RS26200), and rihR (AZC_3881). In-frame deletion and complementation assays revealed that rihF1a and rihF1b work as a unit (rihF1) that positively affects HGT frequency. The EMSA assay and lacZ-based reporter system showed that the XRE-family protein RihR is not a regulator of rihF1 but promotes the expression of the integrase (intC) that has been reported to be upregulated by the LysR-family protein, AhaR, through sensing host’s flavonoid. Overall, a conservative module containing rihF1 and rihR was characterized, eliminating the size of ICE^Ac by 18.5%. We propose the feasibility of constructing a minimal ICE^Ac element to facilitate the exchange of new genetic components essential for symbiosis or other metabolic functions between soil bacteria.

Many rhizobia use quorum sensing (QS) systems to detect their population density and modify their symbiotic behavior with the legume host. There are three LuxRI-type QS systems in Rhizobium etli CFN42, and CinR plays a key role in symbiotic performance. However, the details of how CinR regulates the symbiotic process remain unknown. In this study, we employed the RNA-Seq method to screen differentially expressed genes between the wild-type strain and the ΔcinR mutant of R. etli CFN42. We found that most of the genes related to reactive oxygen species (ROS) were expressed at lower levels in the ΔcinR mutant than in CFN42. We also found that the ΔcinR mutant was more sensitive to H₂O₂ than to CFN42. We then showed that CinR positively regulated katG expression and possessed an affinity to bind the katG promoter in the absence of the AHL ligand. The addition of AHLs promoted CinR binding to the katG promoter and enhanced katG expression. Accumulation of H₂O₂ and O₂^•− was observed in root nodules formed by the ΔcinR mutant. Crucially, katG overexpression rescued the H₂O₂-sensitive phenotype in vitro and partially restored defective symbiotic performance in nodules formed by the ΔcinR mutant on the common bean. These results suggest that CinR globally regulates ROS scavenging gene expression in order to balance oxidative stress within root nodules, promoting nitrogenase activity of R. etli CFN42.