Introduction. Oral bacteria, such as Rothia dentocariosa (Rd), can modulate external aqueous phase orthophosphate (Pi) concentrations via Pi internalization and accumulate high amounts of Pi in the form of polyphosphate (polyP).Hypothesis/Gap Statement. This study investigates whether the carbohydrate metabolism of Rd that generates acid and solubilizes Pi, derived from phosphorus-32 (32P)-labelled tooth enamel, can be internalized and incorporated into intracellular polyP. The study investigated the hypothesis that the internalization of tooth-derived Pi and polyP synthesis could be attenuated.Aim. A radionuclide 32P-labelled remineralized model examined the influence of gallein, an inhibitor of polyP kinases, on the ingestion of tooth-derived Pi and on the bacterium's cultivability.Methodology. Radionuclide 32P-labelled remineralized bovine enamel (n=6) was exposed to Rd (ATCC 17931) incubated in brain heart infusion (BHI) broth containing glucose. Attenuated total reflectance Fourier transform infrared (FTIR) spectroscopy and cross-polarization optical coherence tomography assessed the surface and subsurface characteristics of the remineralized and demineralized enamel surface. Liquid scintillation traced the 32P release from the tooth to the intracellular polyP in galleinpos/neg samples. Cell-media, supernatant, cell resuspension, and extracted polyP, from Rd exposed to the radioisotope-labelled remineralized enamel, were analysed for 32P activity at 10 and 22 h time points.Results. FTIR determined that 32P was incorporated in dental enamel lesions and was released after exposure to an acidic buffer (pH=4.5) or Rd. After 24 h of exposure, galleinpos reduced Rd's planktonic growth and increased pH by 7.45% and 7.19%, respectively, compared to galleinneg. Gallein inhibited the cultivability of planktonic Rd on BHI agar plates with a 13- to 60-fold reduction of c.f.u. ml-1 (25 µM-13.03×, 50 µM-19.58× and 100 µM-60.57×) compared to galleinneg samples. Isotope labelling demonstrated that Rd used Pi derived directly from the tooth for the benefit of the bacterium's fitness through the formation of polyP.Conclusion. Rd used the Pi derived directly from the tooth for the benefit of the bacterium's fitness. Gallein attenuated polyP accumulation, reduced bacterial cultivability and may be a potential therapy option in the prevention and treatment of primary and secondary caries.

Microorganisms are important catalysts for the oxidation of reduced inorganic sulfur compounds. One environmentally important source of reduced sulfur is metal sulfide minerals that occur in economic mineral deposits and mine waste. Previous research found that Sulfuriferula spp. were abundant and active in long-term weathering experiments with simulated waste rock and tailings from the Duluth Complex, Northern Minnesota. We, therefore, isolated several strains of Sulfuriferula spp. from these long-term experiments and characterized their metabolic and genomic properties to provide insight into microbe-mineral interactions and the microbial biogeochemistry in these and other moderately acidic to circumneutral environments. The Sulfuriferula strains are all obligate chemolithoautotrophs capable of oxidizing inorganic sulfur compounds and ferrous iron. The strains grew over different pH ranges, but all grew between pH 4.5 and 7, matching the weathering conditions of the Duluth Complex rocks. All strains grew on the iron-sulfide mineral pyrrhotite (Fe1 - xS, 0 < x < 0.125) as the sole energy source, as well as hydrogen sulfide and thiosulfate, which are products of sulfide mineral breakdown. Despite their metabolic similarities, each strain encodes a distinct pathway for the oxidation of reduced inorganic sulfur compounds as well as differences in nitrogen metabolism that reveal diverse genomic capabilities among the group. Our results show that Sulfuriferula spp. are primary producers that likely play a role in sulfide mineral breakdown in moderately acidic to circumneutral mine waste, and the metabolic diversity within the genus may explain their success in sulfide mineral-rich and other sulfidic environments. IMPORTANCE:Metal sulfide minerals, such as pyrite and pyrrhotite, are one of the main sources of reduced sulfur in the global sulfur cycle. The chemolithotrophic microorganisms that break down these minerals in natural and engineered settings are catalysts for biogeochemical sulfur cycling and have important applications in biotechnological processes such as biomining and bioremediation. Sulfuriferula is a recently described genus of sulfur-oxidizing bacteria that are abundant primary producers in diverse terrestrial environments, including waste rock and tailings from metal mining operations. In this study, we explored the genomic and metabolic properties of new isolates from this genus, and the implications for their ecophysiology and biotechnological potential in ore and waste from economic mineral deposits.

Microorganisms are important catalysts for the oxidation of reduced inorganic sulfur compounds. One environmentally important source of reduced sulfur is metal sulfide minerals that occur in economic mineral deposits and mine waste. Previous research found that Sulfuriferula spp. were abundant and active in long-term weathering experiments with simulated waste rock and tailings from the Duluth Complex, Northern Minnesota. We therefore isolated several strains of Sulfuriferula spp. from these long-term experiments and characterized their metabolic and genomic properties to provide insight into microbe-mineral interactions and the microbial biogeochemistry in these and other moderately acidic to circumneutral environments. The Sulfuriferula strains are all obligate chemolithoautotrophs capable of oxidizing inorganic sulfur compounds and ferrous iron. The strains grew over different pH ranges, but all grew between pH 4.5-7, matching the weathering conditions of the Duluth Complex rocks. All strains grew on the iron-sulfide mineral pyrrhotite (Fe1-xS, 0 < x < 0.125) as the sole energy source, as well as hydrogen sulfide and thiosulfate, which are products of sulfide mineral breakdown. Despite their metabolic similarities, each strain encodes a distinct pathway for the oxidation of reduced inorganic sulfur compounds as well as differences in nitrogen metabolism that reveal diverse genomic capabilities among the group. Our results show that Sulfuriferula spp. are primary producers that likely play a role in sulfide mineral breakdown in moderately acidic to circumneutral mine waste, and the metabolic diversity within the genus likely explains their success in sulfide mineral-rich and other sulfidic environments.

Inorganic polyphosphates (polyPs) are energy-storing biopolymers synthesized by all three domains of life. PolyP accumulation has been well studied with respect to its role in stress response, but its role in dental disease has received less attention. Dental decay can be promoted by changes in pH as well as the chemical activity of ions such as phosphate in oral fluids at the enamel interface. Previous work has shown that the drawdown of phosphate from biofilm fluids can alter the saturation state of oral fluids to thermodynamically favour mineral dissolution. The members of the Lactobacillaceae are known to accumulate polyP and play a role in early-stage and late-stage dental caries. In this study, we examined the effects of potential metabolic inhibitors on polyP accumulation in Lacticaseibacillus rhamnosus. We observed that two inhibitors of the enzyme responsible for polyP synthesis, gallein and fluoride, inhibited polyP accumulation in a balanced medium. However, fluoride and gallein treatments amended with either glucose or lactate were found to enhance polyP accumulation. These results illustrate the potential complexity of polyP metabolism in the oral environment.

The Duluth Complex (DC) contains sulfide-rich magmatic intrusions that represent one of the largest known economic deposits of copper, nickel, and platinum group elements. Previous work showed that microbial communities associated with experimentally-weathered DC waste rock and tailings were dominated by uncultivated taxa and organisms not typically associated with mine waste. However, those experiments were designed for kinetic testing and do not necessarily represent the conditions expected for long-term environmental weathering. We used 16S rRNA gene methods to characterize the microbial communities present on the surfaces of naturally-weathered and historically disturbed outcrops of DC material. Rock surfaces were dominated by diverse uncultured Ktedonobacteria, Acetobacteria, and Actinobacteria, with abundant algae and other phototrophs. These communities were distinct from microbial assemblages from experimentally-weathered DC rocks, suggesting different energy and nutrient resources in environmental samples. Sulfide mineral incubations performed with and without algae showed that photosynthetic microorganisms could have an inhibitory effect on autotrophic populations, resulting in slightly lower sulfate release and differences in dominant microorganisms. The microbial assemblages from these weathered outcrops show how communities develop during weathering of sulfide-rich DC rocks and represent baseline data that could evaluate the effectiveness of future reclamation of waste produced by large-scale mining operations.

The uptake and storage of extracellular orthophosphate (P-i) by polyphosphate (polyP) accumulating bacteria may contribute to mineral dissolution in the oral cavity. To test the effect of potential inhibitors of polyP kinases on Rothia dentocariosa, gallein (0, 25, 50, and 100 mu M) and fluoride (0, 50, and 100 ppm) were added to R. dentocariosa cultures grown in brain-heart infusion broth. At a late log growth phase (8 h), extracellular P-i was measured using an ascorbic acid assay, and polyP was isolated from bacterial cells treated with RNA/DNAases using a neutral phenol/chloroform extraction. Extracts were hydrolyzed and quantified as above. Gallein and fluoride had minor effects on bacterial growth with NaF having a direct effect on media pH. Gallein (>= 25 mu M) and fluoride (>= 50 ppm) attenuated the bacterial drawdown of extracellular P-i by 56.7% (P < 0.05) and 37.3% (P < 0.01). There was a corresponding polyP synthesis decrease of 73.2% (P < 0.0001) from gallein and 83.1% (P < 0.0001) from fluoride. Attenuated total reflectance-Fourier-transform infrared spectroscopy validated the presence of polyP and its reduced concentration in R. dentocariosa bacterial cells following gallein and fluoride treatment. Rothia dentocariosa can directly change extracellular Pi and accumulate intracellular polyP, but the mechanism is attenuated by gallein and NaF.

Until recently, the cellular envelopes of bacteria were regarded as static and rigid relative to those of eukaryotes. While investigating peptidoglycan synthesis in populations of giant sulfur bacteria, Candidatus Thiomargarita spp., we observed internal vesicle-like features (VLFs). VLFs, as imaged following the active incorporation of D-amino acids, appear to begin as invaginations and delaminations of the cellular envelope. Staining with wheat germ agglutinin confirmed the presence of peptidoglycan in VLFs, while polymyxin B revealed that the outer membrane is present in some VLFs. Transmission electron microscopy revealed a complex network of interconnected VLFs. Genomes of Ca . Thiomargarita nelsonii lack a canonical divisome, while possessing homologs to genes such as actin, membrane scaffolding proteins, and dynamins that are associated with phagocytosis in eukaryotes. The physiological role of VLFs remains unclear, but the presence of sulfur globules in some suggests compartmentalization of metabolism and energy production. This is the first report of peptidoglycan and outer membrane bound intracellular vesicles within prokaryotic cells. These findings transform the canonical view of the inflexible bacterial cell envelope and further narrow the divide between prokaryotes and eukaryotes. ### Competing Interest Statement The authors have declared no competing interest.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Modern phosphogenesis occurs on continental margins influenced by upwelling and high primary productivity. The formation of phosphatic sediments is coupled to global climate fluctuations, biological cycling of phosphorus and local redox conditions. Although the processes involved in phosphogenesis are well described, high-resolution data on the redox and stable isotope systematics in Recent in-situ phosphorites are scarce. In this contribution, we investigate the trace element and sulfur, nitrogen and organic carbon stable isotope composition of Recent in-situ phosphatic sediments off the coast of Namibia. Also, we examine the reliability of different widely used geochemical proxies in phosphatic sediments. Our results suggest a shift from sulfidic to suboxic conditions, coinciding with the maximum in solid calcium phosphate mineral concentration. This shift is accompanied by unidirectional changes in Mo and Re enrichments and TOC abundance. Relatively low pyrite delta S-34 values (ca -20%) of phosphatic sediments indicate open system fractionation during phosphogenesis. The initiation of phosphogenesis is also accompanied by negative shifts in sedimentary delta C-13(org) and delta N-15 values. Phosphate associated sulfate (PAS) d34S values are lower than modern seawater sulfate values, suggesting the involvement of chemolithotrophic sulfur oxidation. Our results show a shift in redox conditions from sulfidic to (sub)oxic, coupled with active sulfur cycling are prerequisites for phosphogenesis. Phosphatic sediments show substantial enrichments in U and V highlighting the complexity of using these elements, as well as V/(V + Ni) and V/Cr, as redox proxies particularly in phosphorites and phosphatic sediments. (C) 2021 Elsevier Ltd. All rights reserved.

Micro-organisms have long been implicated in the construction of stromatolites. Yet, establishing a microbial role in modern stromatolite growth via molecular analysis is not always straightforward because DNA in stromatolites can have multiple origins. For example, the genomic material could represent the microbes responsible for the construction of the stromatolite (i.e., "builders"), microbes that inhabited the structure after it was built (i.e., "tenants"), or microbes/organic matter that were passively incorporated after construction from the water column or later diagenetic fluids (i.e., "squatters"). Disentangling the role of micro-organisms in stromatolite construction, already difficult in modern systems, becomes more difficult as organic signatures degrade, and their context is obscured. To evaluate our ability to accurately decipher the role of micro-organisms in stromatolite formation in geologically recent settings, 16/18S SSU rRNA gene sequences were analyzed from three systems where the context of growth was well understood: (a) an actively growing stromatolite from a silicic hot spring in Yellowstone National Park, Wyoming, where the construction of the structure is controlled by cyanobacteria; (b) a mixed carbonate and silica precipitate from Little Hot Creek, a hot spring in the Long Valley Caldera of California that has both abiogenic and biogenic components to accretion; and (c) a near-modern lacustrine carbonate stromatolite from Walker Lake, Nevada that is likely abiogenic. In all cases, the largest percentage of recovered DNA sequences, especially when focused on the deeper portions of the structures, belonged to either the tenant or squatter communities, not the actual builders. Once removed from their environmental context, correct interpretation of biology's role in stromatolite morphogenesis was difficult. Because high-throughput genomic analysis may easily lead to incorrect assumptions even in these modern and near-modern structures, caution must be exercised when interpreting micro-organismal involvement in the construction of accretionary structures throughout the rock record.

Due to their lithotrophic metabolisms, morphological complexity and conspicuous appearance, members of the Beggiatoaceae have been extensively studied for more than 100 years. These bacteria are known to be primarily sulfur-oxidizing autotrophs that commonly occur in dense mats at redox interfaces. Their large size and the presence of a mucous sheath allows these cells to serve as sites of attachment for communities of other microorganisms. But little is known about their individual niche preferences and attached microbiomes, particularly in marine environments, due to a paucity of cultivars and their prevalence in habitats that are difficult to access and study. Therefore, in this study, we compare Beggiatoaceae strain composition, community composition, and geochemical profiles collected from sulfidic sediments at four marine stations off the coast of Namibia. To elucidate community members that were directly attached and enriched in both filamentous Beggiatoaceae, namely Ca. Marithioploca spp. and Ca. Maribeggiatoa spp., as well as non-filamentous Beggiatoaceae, Ca. Thiomargarita spp., the Beggiatoaceae were pooled by morphotype for community analysis. The Beggiatoaceae samples collected from a highly sulfidic site were enriched in strains of sulfur-oxidizing Campylobacterota, that may promote a more hospitable setting for the Beggiatoaceae, which are known to have a lower tolerance for high sulfide to oxygen ratios. We found just a few host-specific associations with the motile filamentous morphotypes. Conversely, we detected 123 host specific enrichments with non-motile chain forming Beggiatoaceae. Potential metabolisms of the enriched strains include fermentation of host sheath material, syntrophic exchange of H2 and acetate, inorganic sulfur metabolism, and nitrite oxidation. Surprisingly, we did not detect any enrichments of anaerobic ammonium oxidizing bacteria as previously suggested and postulate that less well-studied anaerobic ammonium oxidation pathways may be occurring instead.

Fast, cheap, and simple separation of lipids and hydrocarbons can currently be achieved using thin-layer chromatography. Here, we describe an alternative planar chromatographic method using polyvinylidene difluoride membranes as the stationary phase. The procedure has the same advantages of thin-layer chromatography over other expensive and time-consuming techniques, such as high-performance liquid chromatography or gas chromatography. Polyvinylidene difluoride membranes, however, also provide an immediate support for analyte development via immunodetection, are easy to manipulate, and potentially increase the performance of other detection methods. We show that polyvinylidene difluoride membranes are compatible with a variety of solvents that can migrate by capillarity and redistribute analytes between the membrane and the solvent according to their relative affinities, providing a chromatographic separation. We directly test the developed membranes by immunoblotting using anti-squalene antibodies that cross-react with acyclic isoprenoids. Separations of crude oils and plant extracts under different solvent conditions show the potential to resolve hydrocarbon group types and also to provide characteristic fingerprints of plant pigments and squalene degradation products. Polyvinylidene difluoride membranes prove useful as a stationary phase for planar chromatography and for the subsequent immunodetection of the separated compounds, providing a new and simple chromatographic technique to analyze lipids and hydrocarbons.

Carbonate rocks at marine methane seeps are commonly colonized by sulfur-oxidizing bacteria that co-occur with etch pits that suggest active dissolution. We show that sulfur-oxidizing bacteria are abundant on the surface of an exemplar seep carbonate collected from Del Mar East Methane Seep Field, USA. We then used bioreactors containing aragonite mineral coupons that simulate certain seep conditions to investigate plausible in situ rates of carbonate dissolution associated with sulfur-oxidizing bacteria. Bioreactors inoculated with a sulfur-oxidizing bacterial strain, Celeribacter baekdonensis LH4 , growing on aragonite coupons induced dissolution rates in sulfidic, heterotrophic, and abiotic conditions of 1773.97 (±324.35), 152.81 (±123.27), and 272.99 (±249.96) μmol CaCO 3 • cm −2 • yr −1 , respectively. Steep gradients in pH were also measured within carbonate-attached biofilms using pH-sensitive fluorophores. Together, these results show that the production of acidic microenvironments in biofilms of sulfur-oxidizing bacteria are capable of dissolving carbonate rocks, even under well-buffered marine conditions. Our results support the hypothesis that authigenic carbonate rock dissolution driven by lithotrophic sulfur-oxidation constitutes a previously unknown carbon flux from the rock reservoir to the ocean and atmosphere.

Background Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. Results To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the IS S1 transposon to generate a library of over 1000 mutants in Lactococcus lactis , the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. Conclusions Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

Modern carbonate tufa towers in the alkaline (~pH 9.5) Big Soda Lake (BSL), Nevada, exhibit rapid precipitation rates (exceeding 3 cm/year) and host diverse microbial communities. Geochemical indicators reveal that carbonate precipitation is, in part, promoted by the mixing of calcium‐rich groundwater and carbonate‐rich lake water, such that a microbial role for carbonate precipitation is unknown. Here, we characterize the BSL microbial communities and evaluate their potential effects on carbonate precipitation that may influence fast carbonate precipitation rates of the active tufa mounds of BSL. Small subunit rRNA gene surveys indicate a diverse microbial community living endolithically, in interior voids, and on tufa surfaces. Metagenomic DNA sequencing shows that genes associated with metabolisms that are capable of increasing carbonate saturation (e.g., photosynthesis, ureolysis, and bicarbonate transport) are abundant. Enzyme activity assays revealed that urease and carbonic anhydrase, two microbial enzymes that promote carbonate precipitation, are active in situ in BSL tufa biofilms, and urease also increased calcium carbonate precipitation rates in laboratory incubation analyses. We propose that, although BSL tufas form partially as a result of water mixing, tufa‐inhabiting microbiota promote rapid carbonate authigenesis via ureolysis, and potentially via bicarbonate dehydration and CO2 outgassing by carbonic anhydrase. Microbially induced calcium carbonate precipitation in BSL tufas may generate signatures preserved in the carbonate microfabric, such as stromatolitic layers, which could serve as models for developing potential biosignatures on Earth and elsewhere.

Summary Microbial precipitation of calcium carbonate is a widespread environmental phenomenon that has diverse engineering applications, from building and soil restoration to carbon sequestration. Urease‐mediated ureolysis and CO2 (de)hydration by carbonic anhydrase (CA) are known for their potential to precipitate carbonate minerals, yet many environmental microbial community studies rely on marker gene or metagenomic approaches that are unable to determine in situ activity. Here, we developed fast and cost‐effective tests for the field detection of urease and CA activity using pH‐sensitive strips inside microcentrifuge tubes that change colour in response to the reaction products of urease (NH3) and CA (CO2). The urease assay proved sensitive and useful in the field to detect in situ activity in biofilms from a saline lake, a series of calcareous fens, and ferrous springs, finding relatively high urease activity in lake samples. Incubations of lake microbes with urea resulted in significantly higher CaCO3 precipitation compared to incubations with a urease inhibitor, showing that the rapid assay indicated an on‐site active metabolism potentially mediating carbonate precipitation. The CA assay, however, showed less sensitivity compared to the urease test. While its sensitivity limits its utility, the assay may still be useful as a preliminary indicator given the paucity of other means for detecting CA activity in the field. Field urease, and potentially CA, activity assays complement molecular approaches and facilitate the search for carbonate‐precipitating microbes and their in situ activity, which could be applied toward agriculture, engineering and carbon sequestration technologies.