Search

Microbial communities inhabiting caves in quartz-rich rocks are still underexplored, despite their possible role in the silica cycle. The world's longest orthoquartzite cave, Imawarì Yeuta, represents a perfect arena for the investigation of the interactions between microorganisms and silica in non-thermal environments due to the presence of extraordinary amounts of amorphous silica speleothems of different kinds. In this work, the microbial diversity of Imawarì Yeuta was dissected by analyzing nineteen samples collected from different locations representative of different silica amorphization phases and types of samples. Specifically, we investigated the major ecological patterns in cave biodiversity, specific taxa enrichment, and the main ecological clusters through co-occurrence network analysis. Water content greatly contributed to the microbial communities' composition and structures in the cave leading to the sample clustering into three groups DRY, WET, and WATER. Each of these groups was enriched in members of Actinobacteriota , Acidobacteriota , and Gammaproteobacteria , respectively. Alpha diversity analysis showed the highest value of diversity and richness for the WET samples, while the DRY group had the lowest. This was accompanied by the presence of correlation patterns including either orders belonging to various phyla from WET samples or orders belonging to the Actinobacteriota and Firmicutes phyla from DRY group samples. The phylogenetic analysis of the dominant species in WET and DRY samples showed that Acidobacteriota and Actinobacteriota strains were affiliated with uncultured bacteria retrieved from various oligotrophic and silica/quartz-rich environments, not only associated with subterranean sites. Our results suggest that the water content greatly contributes to shaping the microbial diversity within a subterranean quartzite environment. Further, the phylogenetic affiliation between Imawarì Yeuta dominant microbes and reference strains retrieved from both surface and subsurface silica- and/or CO 2 /CO-rich environments, underlines the selective pressure applied by quartz as rock substrate. Oligotrophy probably in association with the geochemistry of silica/quartz low pH buffering activity and alternative energy sources led to the colonization of specific silica-associated microorganisms. This study provides clues for a better comprehension of the poorly known microbial life in subsurface and surface quartz-dominated environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ghezzi, Foschi, Firrincieli, Hong, Vergara, De Waele, Sauro and Cappelletti.)

Arsenic (As) ranks among the priority metal(loid)s that are of public health concern. In the environment, arsenic is present in different forms, organic or inorganic, featured by various toxicity levels. Bacteria have developed different strategies to deal with this toxicity involving different resistance genetic determinants. Bacterial strains of Rhodococcus genus, and more in general Actinobacteria phylum, have the ability to cope with high concentrations of toxic metalloids, although little is known on the molecular and genetic bases of these metabolic features. Here we show that Rhodococcus aetherivorans BCP1, an extremophilic actinobacterial strain able to tolerate high concentrations of organic solvents and toxic metalloids, can grow in the presence of high concentrations of As(V) (up to 240 mM) under aerobic growth conditions using glucose as sole carbon and energy source. Notably, BCP1 cells improved their growth performance as well as their capacity of reducing As(V) into As(III) when the concentration of As(V) is within 30-100 mM As(V). Genomic analysis of BCP1 compared to other actinobacterial strains revealed the presence of three gene clusters responsible for organic and inorganic arsenic resistance. In particular, two adjacent and divergently oriented ars gene clusters include three arsenate reductase genes ( arsC 1/2/3) involved in resistance mechanisms against As(V). A sequence similarity network (SSN) and phylogenetic analysis of these arsenate reductase genes indicated that two of them (ArsC2/3) are functionally related to thioredoxin (Trx)/thioredoxin reductase (TrxR)-dependent class and one of them (ArsC1) to the mycothiol (MSH)/mycoredoxin (Mrx)-dependent class. A targeted transcriptomic analysis performed by RT-qPCR indicated that the arsenate reductase genes as well as other genes included in the ars gene cluster (possible regulator gene, arsR , and arsenite extrusion genes, arsA, acr3 , and arsD ) are transcriptionally induced when BCP1 cells were exposed to As(V) supplied at two different sub-lethal concentrations. This work provides for the first time insights into the arsenic resistance mechanisms of a Rhodococcus strain, revealing some of the unique metabolic requirements for the environmental persistence of this bacterial genus and its possible use in bioremediation procedures of toxic metal contaminated sites.

Naphthenic acids (NAs) are an important group of toxic organic compounds naturally occurring in hydrocarbon deposits. This work shows that Rhodococcus aetherivorans BCP1 cells not only utilize a mixture of eight different NAs (8XNAs) for growth but they are also capable of marked degradation of two model NAs, cyclohexanecarboxylic acid (CHCA) and cyclopentanecarboxylic acid (CPCA) when supplied at concentrations from 50 to 500 mgL -1 . The growth curves of BCP1 on 8XNAs, CHCA, and CPCA showed an initial lag phase not present in growth on glucose, which presumably was related to the toxic effects of NAs on the cell membrane permeability. BCP1 cell adaptation responses that allowed survival on NAs included changes in cell morphology, production of intracellular bodies and changes in fatty acid composition. Transmission electron microscopy (TEM) analysis of BCP1 cells grown on CHCA or CPCA showed a slight reduction in the cell size, the production of EPS-like material and intracellular electron-transparent and electron-dense inclusion bodies. The electron-transparent inclusions increased in the amount and size in NA-grown BCP1 cells under nitrogen limiting conditions and contained storage lipids as suggested by cell staining with the lipophilic Nile Blue A dye. Lipidomic analyses revealed significant changes with increases of methyl-branched (MBFA) and polyunsaturated fatty acids (PUFA) examining the fatty acid composition of NAs-growing BCP1 cells. PUFA biosynthesis is not usual in bacteria and, together with MBFA, can influence structural and functional processes with resulting effects on cell vitality. Finally, through the use of RT (Reverse Transcription)-qPCR, a gene cluster ( chcpca ) was found to be transcriptionally induced during the growth on CHCA and CPCA. Based on the expression and bioinformatics results, the predicted products of the chcpca gene cluster are proposed to be involved in aerobic NA degradation in R. aetherivorans BCP1. This study provides first insights into the genetic and metabolic mechanisms allowing a Rhodococcus strain to aerobically degrade NAs.

Pseudomonas pseudoalcaligenes KF707 is a soil bacterium which is known for its capacity to aerobically degrade harmful organic compounds such as polychlorinated biphenyls (PCBs) using biphenyl as co-metabolite. Here we provide the first genetic and functional analysis of the KF707 respiratory terminal oxidases in cells grown with two different carbon sources: glucose and biphenyl. We identified five terminal oxidases in KF707: two c(c)aa 3 type oxidases (Caa 3 and Ccaa 3 ), two cbb 3 type oxidases (Cbb 3 1 and Cbb 3 2), and one bd type cyanide-insensitive quinol oxidase (CIO). While the activity and expression of both Cbb 3 1 and Cbb 3 2 oxidases was prevalent in glucose grown cells as compared to the other oxidases, the activity and expression of the Caa 3 oxidase increased considerably only when biphenyl was used as carbon source in contrast to the Cbb 3 2 oxidase which was repressed. Further, the respiratory activity and expression of CIO was up-regulated in a Cbb 3 1 deletion strain as compared to W.T. whereas the CIO up-regulation was not present in Cbb 3 2 and C(c)aa 3 deletion mutants. These results, together, reveal that both function and expression of cbb 3 and caa 3 type oxidases in KF707 are modulated by biphenyl which is the co-metabolite needed for the activation of the PCBs-degradation pathway.

Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD-coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation-were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria-Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step.