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.

Identity and mechanisms of alkane-oxidizing metalloenzymes from deep-sea hydrothermal vents.

Six aerobic alkanotrophs (organism that can metabolize alkanes as their sole carbon source) isolated from deep-sea hydrothermal vents were characterized using the radical clock substrate norcarane to determine the metalloenzyme and reaction mechanism used to oxidize alkanes. The organisms studied were Alcanivorax sp. strains EPR7 and MAR14, Marinobacter sp. strain EPR21, Nocardioides sp. strains EPR26w, EPR28w, and Parvibaculum hydrocarbonoclasticum strain EPR92. Each organism was able to grow on n-alkanes as the sole carbon source and therefore must express genes encoding an alkane-oxidizing enzyme. Results from the oxidation of the radical-clock diagnostic substrate norcarane demonstrated that five of the six organisms (EPR7, MAR14, EPR21, EPR26w, and EPR28w) used an alkane hydroxylase functionally similar to AlkB to catalyze the oxidation of medium-chain alkanes, while the sixth organism (EPR92) used an alkane-oxidizing cytochrome P450 (CYP)-like protein to catalyze the oxidation. DNA sequencing indicated that EPR7 and EPR21 possess genes encoding AlkB proteins, while sequencing results from EPR92 confirmed the presence of a gene encoding CYP-like alkane hydroxylase, consistent with the results from the norcarane experiments.

Detection and phylogenetic analysis of the membrane-bound nitrate reductase (Nar) in pure cultures and microbial communities from deep-sea hydrothermal vents.

Over the past few years the relevance of nitrate respiration in microorganisms from deep-sea hydrothermal vents has become evident. In this study, we surveyed the membrane-bound nitrate reductase (Nar) encoding gene in three different deep-sea vent microbial communities from the East Pacific Rise and the Mid-Atlantic Ridge. Additionally, we tested pure cultures of vent strains for their ability to reduce nitrate and for the presence of the NarG-encoding gene in their genomes. By using the narG gene as a diagnostic marker for nitrate-reducing bacteria, we showed that nitrate reductases related to Gammaproteobacteria of the genus Marinobacter were numerically prevalent in the clone libraries derived from a black smoker and a diffuse flow vent. In contrast, NarG sequences retrieved from a community of filamentous bacteria located about 50 cm above a diffuse flow vent revealed the presence of a yet to be identified group of enzymes. 16S rRNA gene-inferred community compositions, in accordance with previous studies, showed a shift from Alpha- and Gammaproteobacteria to Epsilonproteobacteria as the vent fluids become warmer and more reducing. Based on these findings, we argue that Nar-catalyzed nitrate reduction is likely relevant in temperate and less reducing environments where Alpha- and Gammaproteobacteria are more abundant and where nitrate concentrations reflect that of background deep seawater.

Parvibaculum hydrocarboniclasticum sp. nov., a mesophilic, alkane-oxidizing alphaproteobacterium isolated from a deep-sea hydrothermal vent on the East Pacific Rise.

An aerobic, alkane-oxidizing bacterium, designated strain EPR92(T), was isolated from hydrothermal fluids that had been collected from a deep-sea vent on the East Pacific Rise (at 9° 50' N 104° 17' W). The cells of the novel strain were Gram-staining-negative rods that measured approximately 1.4 µm in length and 0.4 µm in width. Strain EPR92(T) grew at 20-40 °C (optimum 35 °C), with1.0-5.0% (w/v) NaCl (optimum 2.5%), and at pH 4.0-8.5 (optimum pH 7.5). The generation time under optimal conditions was 63 min. Strain EPR92(T) grew aerobically in artificial seawater minimal medium with n-alkanes as sole carbon and energy sources, and also in artificial seawater medium supplemented with peptone and yeast extract. The predominant fatty acids were C(18:1)ω7c, C(19:0) cyclo ω8c, 11-methyl C(18:1)ω7c and a putative C(12:0) aldehyde. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and four unidentified aminolipids. The major respiratory quinone was Q-10 and the genomic DNA G+C content was 60.7 mol%. Phylogenetic analyses of the 16S rRNA gene showed that strain EPR92(T) belongs in the class Alphaproteobacteria and the recognized species that were most closely related to the novel strain were identified as Parvibaculum indicum P-31(T) (98.7% sequence similarity) and Parvibaculum lavamentivorans DS-1(T) (95.8%). In DNA-DNA hybridizations, the level of DNA-DNA relatedness observed between strain EPR92(T) and P. indicum P-31(T) was 47.7%, indicating that the two strains do not belong to the same species. Based on the phylogenetic, physiological, chemotaxonomic and genetic evidence, strain EPR92(T) represents a novel species within the genus Parvibaculum, for which the name Parvibaculum hydrocarboniclasticum sp. nov. is proposed. The type strain is EPR92(T) ( = DSM 23209 = JCM 16666(T)).

Complete genome sequence of Thermovibrio ammonificans HB-1(T), a thermophilic, chemolithoautotrophic bacterium isolated from a deep-sea hydrothermal vent.

Thermovibrio ammonificans type strain HB-1(T) is a thermophilic (Topt: 75°C), strictly anaerobic, chemolithoautotrophic bacterium that was isolated from an active, high temperature deep-sea hydrothermal vent on the East Pacific Rise. This organism grows on mineral salts medium in the presence of CO2/H2, using NO3(-) or S(0) as electron acceptors, which are reduced to ammonium or hydrogen sulfide, respectively. T. ammonificans is one of only three species within the genus Thermovibrio, a member of the family Desulfurobacteriaceae, and it forms a deep branch within the phylum Aquificae. Here we report the main features of the genome of T. ammonificans strain HB-1(T) (DSM 15698(T)).