The Diverse World of Protists-an Ideal Community with which to Introduce Microscopy in the Microbiology Teaching Laboratory.

Protists and other eukaryotes are present in diverse terrestrial and aquatic environments. They can easily be collected from local ponds and streams. Although they are typically larger in size than prokaryotes, making them easy to study with even basic microscopes, microbiology laboratory courses often do not discuss them in detail and may only dedicate a short time to observing preserved samples. This laboratory exercise allows students to develop microscope skills, experience real world application of collecting and processing field samples, and delve deeper into the diverse world of protists and other microbial eukaryotes. Students may also engage by sharing their findings on the website iNaturalist to contribute to the scientific knowledge collected around the world. This laboratory module was initially designed for in-person learning but has been successfully adapted to remote learning and can also be applied to a complete online learning environment.

Poster Development and Presentation to Improve Scientific Inquiry and Broaden Effective Scientific Communication Skills.

We have redesigned a tried-and-true laboratory exercise into an inquiry-based team activity exploring microbial growth control, and implemented this activity as the basis for preparing a scientific poster in a large, multi-section laboratory course. Spanning most of the semester, this project culminates in a poster presentation of data generated from a student-designed experiment. Students use and apply the scientific method and improve written and verbal communication skills. The guided inquiry format of this exercise provides the opportunity for student collaboration through cooperative learning. For each learning objective, a percentage score was tabulated (learning objective score = points awarded/total possible points). A score of 80% was our benchmark for achieving each objective. At least 76% of the student groups participating in this project over two semesters achieved each learning goal. Student perceptions of the project were evaluated using a survey. Nearly 90% of participating students felt they had learned a great deal in the areas of formulating a hypothesis, experimental design, and collecting and analyzing data; 72% of students felt this project had improved their scientific writing skills. In a separate survey, 84% of students who responded felt that peer review was valuable in improving their final poster submission. We designed this inquiry-based poster project to improve student scientific communication skills. This exercise is appropriate for any microbiology laboratory course whose learning outcomes include the development of scientific inquiry and literacy.

Selenate reductase activity in Escherichia coli requires Isc iron-sulfur cluster biosynthesis genes.

The selenate reductase in Escherichia coli is a multi-subunit enzyme predicted to bind Fe-S clusters. In this study, we examined the iron-sulfur cluster biosynthesis genes that are required for selenate reductase activity. Mutants devoid of either the iscU or hscB gene in the Isc iron-sulfur cluster biosynthesis pathway lost the ability to reduce selenate. Genetic complementation by the wild-type sequences restored selenate reductase activity. The results indicate the Isc biosynthetic system plays a key role in selenate reductase Fe-S cofactor assembly and is essential for enzyme activity.

Seleniivibrio woodruffii gen. nov., sp. nov., a selenate- and arsenate-respiring bacterium in the Deferribacteraceae.

A Gram-type-negative, obligately anaerobic, selenate-respiring bacterium, strain S4(T), was isolated from activated sludge of a wastewater treatment plant in New Jersey after enrichment with 10 mM selenate as the sole electron acceptor. In addition to its selenate-respiring capability, strain S4(T) also respired arsenate with acetate as carbon source and electron donor. Fermentative growth was not observed. The optimum growth temperature was 37 °C and optimum pH was pH 7. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain S4(T) is a novel member of the family Deferribacteraceae, with the type strain of Denitrovibrio acetiphilus as its closest cultivated relative, with 91.5 % sequence similarity. The cellular fatty acid profile was composed predominantly of straight-chain fatty acids C14 : 0, C15 : 0, C16 : 0, C17 : 0 and C18 : 0, which distinguishes this organism from its closest relatives. The DNA G+C content was 47.7 mol%. Together, these findings support the conclusion that strain S4(T) represents a novel genus and species, for which the name Seleniivibrio woodruffii gen. nov., sp. nov. is proposed. The type strain of Seleniivibrio woodruffii is S4(T) ( = DSM 24984(T) = ATCC BAA-2290(T)).

Physiological response of Desulfurispirillum indicum S5 to arsenate and nitrate as terminal electron acceptors.

The ability of anaerobic prokaryotes to employ different terminal electron acceptors for respiration enables these organisms to flourish in subsurface ecosystems. Desulfurispirillum indicum strain S5 is an obligate anaerobic bacterium that is able to grow by respiring a range of different electron acceptors, including arsenate and nitrate. Here, we examined the growth, electron acceptor utilization, and gene expression of D. indicum growing under arsenate and nitrate-reducing conditions. Consistent with thermodynamic predictions, the experimental results showed that the reduction of nitrate to ammonium yielded higher cell densities than the reduction of arsenate to arsenite. However, D. indicum grew considerably faster by respiration on arsenate compared with nitrate, with doubling times of 4.3 ± 0.2 h and 19.2 ± 2.0 h, respectively. Desulfurispirillum indicum growing on both electron acceptors exhibited the preferential utilization of arsenate before nitrate. The expression of the arsenate reductase gene arrA was up-regulated approximately 100-fold during arsenate reduction, as determined by qRT-PCR. Conversely, the nitrate reductase genes narG and napA were not differentially regulated under the conditions tested. The results of this study suggest that physiology, rather than thermodynamics, controls the growth rates and hierarchy of electron acceptor utilization in D. indicum.

Energy metabolism and multiple respiratory pathways revealed by genome sequencing of Desulfurispirillum indicum strain S5.

Desulfurispirillum indicum strain S5, a novel obligate anaerobe belonging to the phylum Chrysiogenetes, is a dissimilatory selenate-, selenite-, arsenate-, nitrate- and nitrite-reducing bacterium. The circular genome of this metabolically versatile bacterium is 2.9 Mbp, with a G+C content of 56.1% and 2619 predicted protein-coding genes. Genome analysis uncovered the components of the electron transport chain, providing important insights into the ability of D. indicum to adapt to different conditions, by coupling the oxidation of various electron donors to the reduction of a wide range of electron acceptors. Sequences encoding the subunits of dehydrogenases and enzymes with roles in the oxidation of several electron donors, including acetate, pyruvate and lactate were identified. Furthermore, five terminal oxidoreductase complexes were encoded in the D. indicum genome. Phylogenetic analyses of their catalytic subunits, operon structure and co-transcription of subunit-coding genes indicate a likely role of three of them as respiratory arsenate reductase (Arr), periplasmic nitrate reductase (Nap) and the membrane-bound nitrate reductase (Nar). This study is the first description and annotation of the genome of a dissimilatory selenate- and arsenate-respiring organism, and D. indicum represents the first sequenced member of its phylum. Our analysis demonstrates the complexity of the microorganism's respiratory system, provides the basis for the functional analysis of metalloid oxyanions respiration and expands our knowledge of the deep branching phylum of Chrysiogenetes.

Desulfurispirillum indicum sp. nov., a selenate- and selenite-respiring bacterium isolated from an estuarine canal.

Strain S5(T), a novel bacterium that was isolated for its capability to respire selenate to elemental selenium, is described. In addition to selenate respiration, it was also capable of dissimilatory selenite, arsenate and nitrate reduction with short-chain organic acids such as pyruvate, lactate and acetate as the carbon sources and electron donors. The isolate was unable to grow fermentatively. Strain S5(T) was isolated from sediment of an estuarine canal in Chennai, India. Phylogenetic analysis of the 16S rRNA gene of this novel isolate revealed that it belonged to the family Chrysiogenaceae with sequence similarities of 92 and 98  %, respectively, with the type strains of Chrysiogenes arsenatis and Desulfurispirillum alkaliphilum, its closest known relatives. Strain S5(T) and D. alkaliphilum were closely related in terms of their 16S rRNA gene phylogeny; however, they varied greatly in their genomic DNA G+C content (56 mol% versus 45 mol%) and cellular fatty acid compositions, as well as in many metabolic capabilities. Strain S5(T) represents a novel species for which the name Desulfurispirillum indicum sp. nov. is proposed; the type strain is S5(T) (=DSM 22839(T) =ATCC BAA-1389(T)).

Complete genome sequence of Desulfurispirillum indicum strain S5(T).

Desulfurispirillum indicum strain S5(T) is a strictly anaerobic bacterium isolated from river sediment in Chennai, India. D. indicum belongs to the deep branching phylum of Chrysiogenetes, which currently only includes three other cultured species. Strain S5(T) is the type strain of the species and it is capable of growth using selenate, selenite, arsenate, nitrate or nitrite as terminal electron acceptors. The 2,928,377 bp genome encodes 2,619 proteins and 49 RNA genes, and the information gained from its sequence will be relevant to the elucidation of microbially-mediated transformations of arsenic and selenium, in addition to deepening our knowledge of the underrepresented phylum of Chrysiogenetes.