Edward Jerome Crane Iii

The Salton Sea Trough has a range of geothermal features, including mud volcanoes, pots, and seeps that express fluids at temperatures ranging from ambient to 65 to 100 C. The features produce 99% CO2/0.5% CH4 gas of a thermogenic origin and contain hydrothermally-produced petroleum. The mud/clay from the Davis-Schrimpf mud volcanoes has an elemental sulfur (S0) concentration of 317 microM, suggesting that sulfur metabolism should be important in the system. Surprisingly, there has been very little microbiological characterization of these features. Described here are microbial communities, determined by Illumina sequencing of the 515F – 806R 16S rRNA gene fragment, from mud volcanoes/seeps from the Davis-Schrimph seep field and nearby areas with surface feature temperatures from ambient, 65 C, and 100 C. In addition, we have characterized the communities that developed over 1 month at 65 C in enrichment cultures from the 65 C mud volcanoes incubated with a range of electron acceptors (ferrihydrite, S0, SO42-, and S2O32-) and electron donors (tryptone/yeast/casamino acids, crude oil, and CH3CO2-). The 65 C mud and enrichment culture communities were generally predominated by autotrophic H2-oxidizers and organotrophic H2-producers, acetogens, acetotrophs and heterotrophic S0, SO42-, and S2O32- reducers, including many relatives of microbes previously observed in the deep subsurface and from petroleum fields or production waters. While methanogens were present, they were generally at low levels, and few obvious methylotrophs or anaerobic methane oxidizers were detected. Overall, these results provide evidence for a subsurface lithoautotrophic microbial ecosystem (or SliME) in the Salton Sea trough subsurface.

Although mustards (family, Brassicaceae) are common across southern California, research has not focused on the effects of type-conversion of native California sage scrub (CSS) to areas dominated by invasive mustards. To better understand how mustard invasions, primarily the short-pod mustard, Hirschfeldia incana, impact soil microbial assemblages, we examined microbial abundance and assemblages from intact CSS and adjacent mustard-dominated soils at three sites. We also explored if germination rates for various plant species differed between CSS and mustard soils. We found that mustard invasions reduce soil microbial abundances by more than 50% and alter soil fungal assemblages. Fungal richness, diversity, and evenness did not differ between habitats, highlighting that these habitats harbor unique microbial assemblages. While mustard allelopathy is predicted to be the primary driver of these changes, mustard invasions also increased soil pH. Although functional consequences of these shifts are unknown, low mustard germination in CSS soils supports biological resistance to mustard invasion in CSS. Overall, our results demonstrate that mustard invasions, H. incana in particular, exert a strong selecting force on soil microbial assemblages, which can influence effective CSS restoration and preservation of ecosystem services.

California sage scrub (CSS) is an endangered, shrub-dominated, southern California ecosystem type threatened by increasing fire frequencies and conversion to non-native grasslands. As CSS harbors a rich endemic flora and fauna and is critical for maintaining key ecosystem services; significant attention has been focused on understanding how many key factors (N-deposition, drought, and competition with non-natives) influence post-fire CSS recovery. Unfortunately, few studies have examined the role of herbivores. For five years, we investigated the influence of herbivores on post-fire CSS succession by annually surveying plant assemblages in control (herbivores allowed access) and experimental (herbivore excluded) plots at the Bernard Field Station following a fire in May 2017. We found that herbivores reduce the cover of both invasive grasses and native shrubs, and negatively impact Artemisia californica survivorship. Consequently, post-fire plant restoration efforts should not discount the effects of herbivores. While native forb cover was elevated in plots that permitted herbivores, plots near the fire edge experienced increased herbivory pressure and lower cover of most plants. Despite modifications in plant assemblages, we did not observe differences in soil microbe abundances, fungi assemblage structure, or nutrient concentrations. We suggest that restoration practitioners consider caging ‘shrub regeneration’ areas to facilitate post-fire CSS recovery.

Pollinator studies in the endangered California sage scrub ecosystem have focused on spring insect assemblages, when most plant species bloom. Consequently, the insect assemblages using common fall-blooming sage scrub shrubs Lepidospartum squamatum, Ericameria pinifolia, and Baccharis pilularis remain undescribed. Our study aimed to: (1) document flower-visiting insect assemblages on fall-blooming shrubs, (2) assess the efficacy of three sampling techniques in inventorying insect assemblages, and (3) explore, using DNA metabarcoding, which plants are utilized and the extent to which surrounding suburban habitats’ plants are also used. While elevated sampling is required to inventory flower-visiting insects, we describe a diverse assemblage consisting of 123 species. Insect assemblages differed between L. squamatum and B. pilularis, as well as, E. pinifolia and B. pilularis, but not between L. squamatum and E. pinifolia. Direct sampling approaches (netting and photo documentation) collected 115 species not collected by passive malaise traps, highlighting that active observations are required to describe flower-visiting insect assemblages. Sequencing the ITS2 region of pollen from abundant visitors revealed that a majority of pollen is from the sage scrub ecosystem, highlighting its value. Our results indicate that the presence of fall-blooming shrubs may be critical for maintaining diverse sage scrub insect and pollinator assemblages.

Understanding how wildfires and modification in plant assemblages interact to influence soil bacteria assemblages is a crucial step in understanding how these disturbances may influence ecosystem structure and function. Here, we resampled soil from three study sites previously surveyed in spring 2016 and 2017 and compared soil bacterial assemblages prior to and six months after (spring 2019) the 2018 Woolsey Fire in the Santa Monica Mountain National Recreation Area using Illumina sequencing of the 16S rRNA gene. All sites harbored both native California sage scrub and a non-native (grassland or forbland) habitat, allowing us to examine how fire influenced bacterial assemblages in common southern California habitats. Most results contrasted with oura-priorihypotheses: (1) richness and diversity increased following the fire, (2) heat/drought resistant and sensitive bacteria did not show consistent and differing patterns by increasing and decreasing, respectively, in relative abundance after the fire, and (3) bacterial assemblage structure was only minimally impacted by fire, with no differences being found between 2017 (pre-fire) and 2019 (post-fire) in three of the six habitats sampled. As sage scrub and non-native grasslands consistently harbored unique bacterial assemblages both before and following the fire, modifications in plant compositions will likely have legacy effects on these soils that persist even after a fire. Combined, our results demonstrate that bacterial assemblages in southern California habitats are minimally affected by fire. Because direct impacts of fire are limited, but indirect impacts, e.g., modifications in plant compositions, are significant, plant restoration efforts following a fire should strive to revegetate sage scrub areas to prevent legacy changes in bacterial composition.

NADH-dependent persulfide reductase (Npsr) has been proposed to facilitate dissimilatory sulfur respiration by reducing persulfide or sulfane sulfur-containing substrates to H₂S. The presence of this gene in the sulfate and thiosulfate-reducing Archaeoglobus fulgidus DSM 4304 and other hyperthermophilic Archaeoglobales appears anomalous, as A. fulgidus is unable to respire S⁰ and grow in the presence of elemental sulfur. To assess the role of Npsr in the sulfur metabolism of A. fulgidus DSM 4304, the Npsr from A. fulgidus was characterized. AfNpsr is specific for persulfide and polysulfide as substrates in the oxidative half-reaction, exhibiting on the order of 10⁴ M^-1 s^-1, which is similar to the kinetic parameters observed for hyperthermophilic CoA persulfide reductases. In contrast to the bacterial Npsr, AfNpsr exhibits low disulfide reductase activity with DTNB; however, similar to the bacterial enzymes, it does not show detectable activity with CoA-disulfide, oxidized glutathione, or cystine. The 3.1 Å X-ray structure of AfNpsr reveals access to the tightly bound catalytic CoA, and the active site Cys 42 is restricted by a flexible loop (residues 60-66) that is not seen in the bacterial homologs from Shewanella loihica PV-4 and Bacillus anthracis. Unlike the bacterial enzymes, AfNpsr exhibits NADH oxidase activity and also shows no detectable activity with NADPH. Models suggest steric and electrostatic repulsions of the NADPH 2-phosphate account for the strong preference for NADH. The presence of Npsr in the nonsulfur-reducing A. fulgidus suggests that the enzyme may offer some protection against S⁰ or serve in another metabolic role that has yet to be identified.

Within the family of pyridine nucleotide disulfide oxidoreductase (PNDOR), enzymes are a group of single‐cysteine containing FAD‐dependent reductases that utilize a tightly bound coenzyme A to assist in the NAD(P)H‐dependent reduction of di‐, per‐, and polysulfide substrates in bacteria and archaea. For many of these homodimeric enzymes, it has proved difficult to determine the substrate specificity and metabolic function based on sequence and genome analysis alone. Coenzyme A‐disulfide reductase (CoADR) isolated from Pyrococcus horikoshii (phCoADR) reduces Co‐A per‐ and polysulfides, but, unlike other highly homologous members of this group, is a poor CoA disulfide reductase. The phCoADR structure has a narrower access channel for CoA substrates, which suggested that this restriction might be responsible for the enzyme's poor activity toward the bulky CoA disulfide substrate. To test this hypothesis, the substrate channel was widened by making four mutations along the channel wall (Y65A, Y66A, P67G, and H367G). The structure of the quadruple mutant shows a widened substrate channel, which is supported by a fourfold increase in k_cat for the NAD(P)H‐dependent reduction of CoA disulfide and enhanced activity toward the substrate at lower temperatures. Anaerobic titrations of the enzyme with NADH revealed a half‐site reactivity not observed with the wild‐type enzyme in which one subunit of the enzyme could be fully reduced to an EH₄ state, while the other remained in an EH₂ or EH₂·NADH state. These results suggest that for these closely related enzymes, substrate channel morphology is an important determinant of substrate specificity, and homology modeling will be the preferred technique for predicting function among PNDORs.

A novel anaerobic, hyperthermophilic archaeon was isolated from a mud volcano in the Salton Sea geothermal system in southern California, USA. The isolate, named strain 521T, grew optimally at 90 °C, at pH 5.5–7.3 and with 0–2.0 % (w/v) NaCl, with a generation time of 10 h under optimal conditions. Cells were rod-shaped and non-motile, ranging from 2 to 7 µm in length. Strain 521T grew only in the presence of thiosulfate and/or Fe(III) (ferrihydrite) as terminal electron acceptors under strictly anaerobic conditions, and preferred protein-rich compounds as energy sources, although the isolate was capable of chemolithoautotrophic growth. 16S rRNA gene sequence analysis places this isolate within the crenarchaeal genus Pyrobaculum . To our knowledge, this is the first Pyrobaculum strain to be isolated from an anaerobic mud volcano and to reduce only either thiosulfate or ferric iron. An in silico genome-to-genome distance calculator reported <25 % DNA–DNA hybridization between strain 521T and eight other Pyrobaculum species. Due to its genotypic and phenotypic differences, we conclude that strain 521T represents a novel species, for which the name Pyrobaculum igneiluti sp. nov. is proposed. The type strain is 521T (=DSM 103086T=ATCC TSD-56T).