Outer membrane-derived vesicles (OMVs) have been studied in different phyla of gram-negative bacteria, most extensively in the Pseudomonadota, where they have been shown to participate in diverse biological and environmental processes. To date, the production of OMVs has not been reported in the Chlorobiaceae within the phylum Chlorobiota. Chlorobaculum tepidum is the model organism for the Chlorobiaceae that synthesizes and consumes insoluble extracellular sulfur (S(0)) globules by an unknown mechanism. Here, we report evidence implicating outer membrane vesicles in biogenic S(0) globule synthesis. We demonstrate that Cba. tepidum secretes OMVs in the extracellular milieu and that OMV concentration and size vary with growth conditions, particularly sulfide concentration. A core of 31 proteins involved in diverse biological processes such as cell wall biogenesis, inorganic ion transport, and metabolism was found to be shared between OMVs, extracellular S(0) globules, and Cba. tepidum-intact cells. Multiple analytical methods indicated that OMVs contain S(0) and that OMVs and biogenic S(0) globules share protein and polysaccharide signatures, including lipooligosaccharides. Together, these lines of evidence indicate that Cba. tepidum's OMVs are one component of sulfur transport between cells and extracellular sulfur globules.IMPORTANCEAll living cells must exchange material with their environment while maintaining cellular integrity. This is a particular challenge for materials that are not water-soluble; however, many bacteria utilize insoluble materials for energy conservation and as nutrients for growth. Here, we show that Cba. tepidum makes outer membrane vesicles, and these vesicles are likely involved in the exchange of material with extracellular elemental sulfur globules formed and consumed by Cba. tepidum as part of its energy metabolism based on oxidizing reduced sulfur compounds like hydrogen sulfide. These data expand our basic understanding of Cba. tepidum's metabolism. As elemental sulfur is an industrial by-product with a limited number of uses, the information here may help enable the use of additional sulfur compounds by Cba. tepidum to drive the synthesis of biomass and/or specialty biochemicals from waste elemental sulfur by this autotrophic bacterium.

Outer membrane-derived vesicles (OMVs) have been studied in different phyla of Gram-negative bacteria, most extensively in the Pseudomonadota, where they have been shown to participate in diverse biological and environmental processes. To date, the production of OMVs has not been reported in the Chlorobiaceae within the phylum Chlorobiota. Chlorobaculum. tepidum is the model organism for the Chlorobiaceae that synthesizes and consumes insoluble extracellular sulfur (S(0)) globules by an unknown mechanism. Here, we report evidence implicating outer membrane vesicles in biogenic S(0) globule synthesis. First we demonstrate that Cba. tepidum secretes OMVs in the extracellular milieu, and that OMV yield and size vary with growth conditions, particularly sulfide concentration. A core of 28 proteins involved in diverse biological processes such as cell wall biogenesis, inorganic ion transport and metabolism were found to be shared between OMVs, extracellular S(0) globules and Cba. tepidum intact cells. Multiple analytical methods indicated that OMVs contain S(0) and that OMVs and biogenic S(0) globules share protein and polysaccharide signatures, including lipooligosaccharides. Together these lines of evidence indicate that Cba. tepidum's OMVs are one component of sulfur transport between cells and extracellular sulfur globules. ### Competing Interest Statement Y.Y. is a named inventor on a patent application (PCT/US2023/020215) for the E3technology, which has been licensed exclusively to CDS Analytical LLC (Oxford, PA).

Strain AA17T was isolated from an apparently healthy fragment of Montipora capitata coral from the reef surrounding Moku o Loʻe in Kāne‘ohe Bay, O‘ahu, Hawai‘i, USA, and was taxonomically evaluated using a polyphasic approach. Comparison of a partial 16S rRNA gene sequence found that strain AA17T shared the greatest similarity with Aestuariibacter halophilus JC2043T (96.6

Elemental sulfur (S0) is produced and degraded by phylogenetically diverse groups of microorganisms. For Chlorobaculum tepidum, an anoxygenic phototroph, sulfide is oxidized to produce extracellular S0 globules, which can be further oxidized to sulfate. While some sulfur-oxidizing bacteria (e.g., Allochromatium vinosum) are also capable of growth on commercial S0 as an electron donor, C. tepidum is not. Even colloidal sulfur sols, which appear indistinguishable from biogenic globules, do not support the growth of C. tepidum. Here, we investigate the properties that make biogenic S0 globules distinct from abiotic forms of S0. We found that S0 globules produced by C. tepidum and abiotic S0 sols are quite similar in terms of mineralogy and material properties, but the two are distinguished primarily by the properties of their surfaces. C. tepidum's globules are enveloped by a layer of organics (protein and polysaccharides), which results in a surface that is fundamentally different from that of abiotic S0 sols. The organic coating on the globules appears to slow the aging and crystallization of amorphous sulfur, perhaps providing an extended window of time for microbes in the environment to access the more labile forms of sulfur as needed. Overall, our results suggest that the surface of biogenic S0 globules may be key to cell-sulfur interactions and the reactivity of biogenic S0 in the environment.

Thermus thermophilus is a deep-branching thermophilic aerobe. It is a member of the Deinococcus - Thermus phylum that, together with the Aquificae , constitute the earliest branching aerobic bacterial lineages; therefore, this organism serves as a model for early diverged bacteria (R. K. Hartmann, J. Wolters, B. Kröger, S. Schultze, et al., Syst Appl Microbiol 11:243–249, 1989, https://doi.org/10.1016/S0723-2020(89)80020-7 ) whose natural heated habitat may contain mercury of geological origins (G. G. Geesey, T. Barkay, and S. King, Sci Total Environ 569-570:321–331, 2016, https://doi.org/10.1016/j.scitotenv.2016.06.080 ). T. thermophilus likely arose shortly after the oxidation of the biosphere 2.4 billion years ago. Studying T. thermophilus physiology provides clues about the origin and evolution of mechanisms for mercury and oxidative stress responses, the latter being critical for the survival and function of all extant aerobes.

The green sulfur bacteria (Chlorobiaceae) are anaerobes that use electrons from reduced sulfur compounds (sulfide, S-0, and thiosulfate) as electron donors for photoautotrophic growth. Chlorobaculum tepidum, the model system for the Chlorobiaceae, both produces and consumes extracellular S-0 globules depending on the availability of sulfide in the environment. These physiological changes imply significant changes in gene regulation, which has been observed when sulfide is added to Cba. tepidum growing on thiosulfate. However, the underlying mechanisms driving these gene expression changes, i.e., the specific regulators and promoter elements involved, have not yet been defined. Here, differential RNA sequencing (dRNA-seq) was used to globally identify transcript start sites (TSS) that were present during growth on sulfide, biogenic S-0, and thiosulfate as sole electron donors. TSS positions were used in combination with RNA-seq data from cultures growing on these same electron donors to identify both basal promoter elements and motifs associated with electron donor-dependent transcriptional regulation. These motifs were conserved across homologous Chlorobiaceae promoters. Two lines of evidence suggest that sulfide-mediated repression is the dominant regulatory mode in Cba. tepidum. First, motifs associated with genes regulated by sulfide overlap key basal promoter elements. Second, deletion of the Cba. tepidum 1277 (CT1277) gene, encoding a putative regulatory protein, leads to constitutive overexpression of the sulfide: quinone oxidoreductase CT1087 in the absence of sulfide. The results suggest that sulfide is the master regulator of sulfur metabolism in Cba. tepidum and the Chlorobiaceae. Finally, the identification of basal promoter elements with differing strengths will further the development of synthetic biology in Cba. tepidum and perhaps other Chlorobiaceae. IMPORTANCE Elemental sulfur is a key intermediate in biogeochemical sulfur cycling. The photoautotrophic green sulfur bacterium Chlorobaculum tepidum either produces or consumes elemental sulfur depending on the availability of sulfide in the environment. Our results reveal transcriptional dynamics of Chlorobaculum tepidum on elemental sulfur and increase our understanding of the mechanisms of transcriptional regulation governing growth on different reduced sulfur compounds. This report identifies genes and sequence motifs that likely play significant roles in the production and consumption of elemental sulfur. Beyond this focused impact, this report paves the way for the development of synthetic biology in Chlorobaculum tepidum and other Chlorobiaceae by providing a comprehensive identification of promoter elements for control of gene expression, a key element of strain engineering.

Low-molecular-weight thiols are key metabolites that participate in many basic cellular processes: central metabolism, detoxification, and oxidative stress resistance. Here we describe a new thiol, N-methyl-bacillithiol, found in an anaerobic phototrophic bacterium and identify a gene that is responsible for its synthesis from bacillithiol, the main thiol metabolite in many Gram-positive bacteria. We show that the presence or absence of this gene in a sequenced genome accurately predicts thiol content in distantly related bacteria. On the basis of these results, we analyzed genome data and predict that bacillithiol and its derivatives are the most widely distributed thiol metabolites in biology.

[This corrects the article on p. 682 in vol. 8, PMID: 28484436.].

The Epsilonproteobacteria is the fifth validly described class of the phylum Proteobacteria, known primarily for clinical relevance and for chemolithotrophy in various terrestrial and marine environments, including deep-sea hydrothermal vents. As 16S rRNA gene repositories have expanded and protein marker analysis become more common, the phylogenetic placement of this class has become less certain. A number of recent analyses of the bacterial tree of life using both 16S rRNA and concatenated marker gene analyses have failed to recover the Epsilonproteobacteria as monophyletic with all other classes of Proteobacteria. In order to address this issue, we investigated the phylogenetic placement of this class in the bacterial domain using 16S and 23S rRNA genes, as well as 120 single-copy marker proteins. Single- and concatenated-marker trees were created using a data set of 4,170 bacterial representatives, including 98 Epsilonproteobacteria. Phylogenies were inferred under a variety of tree building methods, with sequential jackknifing of outgroup phyla to ensure robustness of phylogenetic affiliations under differing combinations of bacterial genomes. Based on the assessment of nearly 300 phylogenetic tree topologies, we conclude that the continued inclusion of Epsilonproteobacteria within the Proteobacteria is not warranted, and that this group should be reassigned to a novel phylum for which we propose the name Epsilonbacteraeota (phyl. nov.). We further recommend the reclassification of the order Desulfurellales (Deltaproteobacteria) to a novel class within this phylum and a number of subordinate changes to ensure consistency with the genome-based phylogeny. Phylogenomic analysis of 658 genomes belonging to the newly proposed Epsilonbacteraeota suggests that the ancestor of this phylum was an autotrophic, motile, thermophilic chemolithotroph that likely assimilated nitrogen from ammonium taken up from the environment or generated from environmental nitrate and nitrite by employing a variety of functional redox modules. The emergence of chemoorganoheterotrophic lifestyles in several Epsilonbacteraeota families is the result of multiple independent losses of various ancestral chemolithoautotrophic pathways. Our proposed reclassification of this group resolves an important anomaly in bacterial systematics and ensures that the taxonomy of Proteobacteria remains robust, specifically as genome-based taxonomies become more common.

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate 1b (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, 15m (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid 3 (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment.

10 * These authors contributed equally to this work. 11 # Corresponding Author: 12 Address: Rm. 282 DBI, 15 Innovation Way, Newark, DE 19711 U.S.A. 13 Phone: 1-302-831-3404 14 E-mail: tehanson@udel.edu 15 †-Current address: Noble Research Institute, 2510 Sam Noble Parkway 16 Ardmore, OK 73401, USA; E-mail: vraman@noble.org 17 ‡-Current address: Wesley College, 120 North State Street, Dover, DE 19901 USA: E-mail: 18 kevin.shuman2@wesley.edu 19 §-Current address: United States Naval Research Laboratory, 4555 Overlook Ave. SW, 20 Washington, D.C. 20375; E-mail: Brian.Eddie@nrl.navy.mil 21

The genome sequence of the obligate chemolithoautotroph Hydrogenovibrio crunogenus paradoxically predicts a complete oxidative citric acid cycle (CAC). This prediction was tested by multiple approaches including whole cell carbon assimilation to verify obligate autotrophy, phylogenetic analysis of CAC enzyme sequences and enzyme assays. Hydrogenovibrio crunogenus did not assimilate any of the organic compounds provided (acetate, succinate, glucose, yeast extract, tryptone). Enzyme activities confirmed that its CAC is mostly uncoupled from the NADH pool. 2-Oxoglutarate:ferredoxin oxidoreductase activity is absent, though pyruvate:ferredoxin oxidoreductase is present, indicating that sequence-based predictions of substrate for this oxidoreductase were incorrect, and that H. crunogenus may have an incomplete CAC. Though the H. crunogenus CAC genes encode uncommon enzymes, the taxonomic distribution of their top matches suggests that they were not horizontally acquired. Comparison of H. crunogenus CAC genes to those present in other 'Proteobacteria' reveals that H. crunogenus and other obligate autotrophs lack the functional redundancy for the steps of the CAC typical for facultative autotrophs and heterotrophs, providing another possible mechanism for obligate autotrophy.

ABSTRACT Mercury (Hg), one of the most toxic and widely distributed heavy metals, has a high affinity for thiol groups. Thiol groups reduce and sequester Hg. Therefore, low-molecular-weight (LMW) and protein thiols may be important cell components used in Hg resistance. To date, the role of low-molecular-weight thiols in Hg detoxification remains understudied. The mercury resistance (mer) operon of Thermus thermophilus suggests an evolutionary link between Hg(II) resistance and low-molecular-weight thiol metabolism. The mer operon encodes an enzyme involved in methionine biosynthesis, Oah. Challenge with Hg(II) resulted in increased expression of genes involved in the biosynthesis of multiple low-molecular-weight thiols (cysteine, homocysteine, and bacillithiol), as well as the thioredoxin system. Phenotypic analysis of gene replacement mutants indicated that Oah contributes to Hg resistance under sulfur-limiting conditions, and strains lacking bacillithiol and/or thioredoxins are more sensitive to Hg(II) than the wild type. Growth in the presence of either a thiol-oxidizing agent or a thiol-alkylating agent increased sensitivity to Hg(II). Furthermore, exposure to 3 μM Hg(II) consumed all intracellular reduced bacillithiol and cysteine. Database searches indicate that oah2 is present in all Thermus sp. mer operons. The presence of a thiol-related gene was also detected in some alphaproteobacterial mer operons, in which a glutathione reductase gene was present, supporting the role of thiols in Hg(II) detoxification. These results have led to a working model in which LMW thiols act as Hg(II)-buffering agents while Hg is reduced by MerA. IMPORTANCE The survival of microorganisms in the presence of toxic metals is central to life's sustainability. The affinity of thiol groups for toxic heavy metals drives microbe-metal interactions and modulates metal toxicity. Mercury detoxification (mer) genes likely originated early in microbial evolution in geothermal environments. Little is known about how mer systems interact with cellular thiol systems. Thermus spp. possess a simple mer operon in which a low-molecular-weight thiol biosynthesis gene is present, along with merR and merA. In this study, we present experimental evidence for the role of thiol systems in mercury resistance. Our data suggest that, in T. thermophilus, thiolated compounds may function side by side with mer genes to detoxify mercury. Thus, thiol systems function in consort with mer-mediated resistance to mercury, suggesting exciting new questions for future research.

Low-molecular weight (LMW) thiols are metabolites that mediate redox homeostasis and the detoxification of chemical stressors in cells. LMW thiols are also thought to play a central role in sulfur oxidation pathways in phototrophic bacteria, including the Chlorobiaceae . Fluorescent thiol labeling of metabolite extracts coupled with HPLC showed that Chlorobaculum tepidum contained a novel LMW thiol with a mass of 412 ± 1 Da corresponding to a molecular formula of C 14 H 24 N 2 O 10 S. These data suggested the new thiol is closely related to bacillithiol (BSH), the major LMW thiol from low G+C% Gram-positive bacteria. By comparing the as-isolated bimane adduct with chemically synthesized candidate structures, the Cba. tepidum thiol structure was identified as N-methyl-bacillithiol (N-Me-BSH), methylated on the cysteine nitrogen, a rarely observed modification in metabolism. Orthologs of bacillithiol biosynthetic genes in the Cba. tepidum genome were required for the biosynthesis of N-Me-BSH. Furthermore, the CT1040 gene product was genetically identified as the BSH N-methyltransferase. N-Me-BSH was found in all Chlorobi examined as well as Polaribacter sp. strain MED152, a member of the Bacteroidetes. A comparative genomic analysis indicated that BSH/N-Me-BSH is synthesized not only by members of the Chlorobi , Bacteroidetes , Deinococcus - Thermus , and Firmicutes , but also by Acidobacteria , Chlamydiae , Gemmatimonadetes , and Proteobacteria. Significance Statement Here, N-Me-BSH is shown to be a redox-responsive LMW thiol cofactor in Cba. tepidum and the gene, nmbA , encoding the BSH N-methyltransferase responsible for its synthesis is identified. The co-occurrence of orthologs to BSH biosynthesis genes and bacillithiol N-methyltransferase was confirmed to correctly predict LMW thiol biosynthesis in phylogenetically distant genomes. The analysis indicates that BSH/N-Me-BSH are likely the most widely distributed class of LMW thiols in biology. This finding sheds light on the evolution of LMW thiol metabolism, which is central to redox homeostasis, regulation and stress resistance in all cellular life. It also sheds light on a rare chemical modification. N-Me-BSH is the fourth instance of cysteinyl nitrogen methylation in metabolism. Identification of the BSH N-methyltransferase reported here will enable detailed in vivo and in vitro dissection of the functional consequences of this modification. As a standalone N-methyltransferase, NmbA may be useful as a component of constructed biosynthetic pathways for novel product (bio)synthesis.

Environmental MicrobiologyVolume 18, Issue 4 p. 1094-1095 Highlight Back to the future: function-first metagenomics returns to the fore Thomas E. Hanson, Corresponding Author Thomas E. Hanson School of Marine Science and Policy, Department of Biological Sciences, and Delaware Biotechnology Institute, University of Delaware, Newark, DE, 19716 USAFor correspondence. E-mail: tehanson@UDel.Edu; Tel. 3028313404; Fax 3028313447.Search for more papers by this author Thomas E. Hanson, Corresponding Author Thomas E. Hanson School of Marine Science and Policy, Department of Biological Sciences, and Delaware Biotechnology Institute, University of Delaware, Newark, DE, 19716 USAFor correspondence. E-mail: tehanson@UDel.Edu; Tel. 3028313404; Fax 3028313447.Search for more papers by this author First published: 18 March 2016 https://doi.org/10.1111/1462-2920.13260Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume18, Issue4April 2016Pages 1094-1095 RelatedInformation

ABSTRACT Microbial sulfur metabolism, particularly the formation and consumption of insoluble elemental sulfur (S 0 ), is an important biogeochemical engine that has been harnessed for applications ranging from bioleaching and biomining to remediation of waste streams. Chlorobaculum tepidum , a low-light-adapted photoautolithotrophic sulfur-oxidizing bacterium, oxidizes multiple sulfur species and displays a preference for more reduced electron donors: sulfide > S 0 > thiosulfate. To understand this preference in the context of light energy availability, an “energy landscape” of phototrophic sulfur oxidation was constructed by varying electron donor identity, light flux, and culture duration. Biomass and cellular parameters of C. tepidum cultures grown across this landscape were analyzed. From these data, a correction factor for colorimetric protein assays was developed, enabling more accurate biomass measurements for C. tepidum , as well as other organisms. C. tepidum 's bulk amino acid composition correlated with energy landscape parameters, including a tendency toward less energetically expensive amino acids under reduced light flux. This correlation, paired with an observation of increased cell size and storage carbon production under electron-rich growth conditions, suggests that C. tepidum has evolved to cope with changing energy availability by tuning its proteome for energetic efficiency and storing compounds for leaner times. IMPORTANCE How microbes cope with and adapt to varying energy availability is an important factor in understanding microbial ecology and in designing efficient biotechnological processes. We explored the response of a model phototrophic organism, Chlorobaculum tepidum , across a factorial experimental design that enabled simultaneous variation and analysis of multiple growth conditions, what we term the “energy landscape.” C. tepidum biomass composition shifted toward less energetically expensive amino acids at low light levels. This observation provides experimental evidence for evolved efficiencies in microbial proteomes and emphasizes the role that energy flux may play in the adaptive responses of organisms. From a practical standpoint, our data suggest that bulk biomass amino acid composition could provide a simple proxy to monitor and identify energy stress in microbial systems.

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Soil incorporation of Si-rich rice residues may aid smallholder rice farmers in improving crop yields and may affect As uptake. Here, the biogeochemical impacts of rice residue incorporation into flooded soil without plants were evaluated.

An ammonia-oxidizing bacterium, strain D1FHS, was enriched into pure culture from a sediment sample retrieved in Jiaozhou Bay, a hyper-eutrophic semi-closed water body hosting the metropolitan area of Qingdao, China. Based on initial 16S rRNA gene sequence analysis, strain D1FHS was classified in the genus Nitrosococcus, family Chromatiaceae, order Chromatiales, class Gammaproteobacteria; the 16S rRNA gene sequence with highest level of identity to that of D1FHS was obtained from Nitrosococcus halophilus Nc4(T). The average nucleotide identity between the genomes of strain D1FHS and N. halophilus strain Nc4 is 89.5%. Known species in the genus Nitrosococcus are obligate aerobic chemolithotrophic ammonia-oxidizing bacteria adapted to and restricted to marine environments. The optimum growth (maximum nitrite production) conditions for D1FHS in a minimal salts medium are: 50 mM ammonium and 700 mM NaCl at pH of 7.5 to 8.0 and at 37°C in dark. Because pertinent conditions for other studied Nitrosococcus spp. are 100-200 mM ammonium and <700 mM NaCl at pH of 7.5 to 8.0 and at 28-32°C, D1FHS is physiologically distinct from other Nitrosococcus spp. in terms of substrate, salt, and thermal tolerance.