Moving a Journal Article-Based Upper-Level Microbiology Dry Lab from In-Person to Online Instruction.

During the spring semester of 2020, a journal article-based upper-level microbiology laboratory course was offered through Western New Mexico University at Glendale Community College in Glendale, AZ. Because most of the students had taken a lower-level microbiology class with a traditional wet laboratory, a dry lab format was used instead. In the first period of each 2-week cycle, a microbiology article selected by the instructor from the primary literature was discussed using a PowerPoint presentation and a detailed study sheet. Students then turned in answers to five specific questions about the article. In the second period of each 2-week cycle, students met to discuss possible research projects based on that article. They then turned in a two- to three-page research proposal describing their project. Before the COVID-19 pandemic became severe and the college moved to online instruction, there were active discussions between the instructor and the students in both class periods. After the campus was shut down, discussions of the journal articles and preparation of the research proposals were done online using Canvas as the learning platform. Students were provided with discussion sites, but no video instruction systems were used. In general, the answers to the journal article questions and the quality of the research proposals were better during in-person instruction. Instructors may be able to adapt this journal article-based lab approach to a fully online format, but it will require extensive training and the use of Zoom or other video instruction methods.

Genomic sequencing of Gracilibacillus dipsosauri reveals key properties of a salt-tolerant α-amylase.

Gracilibacillus dipsosauri is a moderately-halophilic Gram-positive bacterium which forms an extracellular α-amylase that is induced by starch, repressed by D-glucose, and active in 2.0 M KCl. Previous studies showed that while enzyme activity could be measured with the synthetic substrate 2-chloro-4-nitrophenyl-α-D-maltotrioside (CNPG3), other assays were inconsistent and the protein showed aberrant mobility during nondenaturing gel electrophoresis. To clarify the properties of this enzyme, the genome of G. dipsosauri was sequenced and was found to be 4.19 Mb in size with an overall G+C content of 36.9%. A gene encoding an α-amylase composed of 691 amino acids was identified. The protein was a member of the glycosyl hydrolase 13 family, which had a molecular mass of 77,396 daltons and a pI of 4.39 due to an unusually large number of aspartate and glutamate residues (95/691 or 13.7%). BLAST analysis of the amino acid sequence revealed significant matches to other proteins with cyclodextrin glycosyltransferase activity. Partial purification of the protein from G. dipsosauri showed that fractions catalyzing the hydrolysis of CNPG3 and p-nitrophenyl-D-maltoheptoside also catalyzed the formation of ß-cyclodextrin but not α-cyclodextrin or γ-cyclodextrin. Formation of ß-cyclodextrin was not stimulated by high salt concentrations but did occur with rice, potato, wheat, and corn starches and amylopectin. These studies explain the unusual features of the α-amylase from G. dipsosauri and indicate it should be classified as EC 2.4.1.19. The availability of the complete genomic sequence of G. dipsosauri will provide the basis for studies on other enzymes from this halophile which may be useful for biotechnology.

Inhibition of urease activity in the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis by dimethylsulfoxide (DMSO).

AIMS: Urease is a virulence factor for the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis. Dimethylsulfoxide (DMSO) is structurally similar to urea, used as a solvent for urease inhibitors, and an effective treatment for interstitial cystitis/bladder pain syndrome (IC/BPS). The aims of this study were to test DMSO as a urease inhibitor and determine its physiological effects on S. saprophyticus and P. mirabilis. METHODS AND RESULTS: Urease activity in extracts and whole cells was measured by the formation of ammonium ions. Urease was highly sensitive to noncompetitive inhibition by DMSO (Ki about 6 mmol l-1 ). DMSO inhibited urease activity in whole cells, limited bacterial growth in media containing urea, and slowed the increase in pH which occurred in artificial urine medium. CONCLUSIONS: DMSO should be used with caution as a solvent when testing plant extracts or other potential urease inhibitors. Because it can inhibit bacterial growth and delay an increase in pH, it may be an effective treatment for urinary tract infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed study of the inhibition of urease by DMSO. Dimethylsulfoxide may be used to treat urinary tract infections that are resistant to antibiotics or herbal remedies.

L-Proline catabolism by the high G + C Gram-positive bacterium Paenarthrobacter aurescens strain TC1.

Paenarthrobacter aurescens (formerly called Arthrobacter aurescens) strain TC1 is a high G + C Gram-positive aerobic bacterium that can degrade the herbicide atrazine. Analysis of its genome indicated strain TC1 has the potential to form a bifunctional PutA protein containing L-proline dehydrogenase and L-glutamate-γ-semialdehyde dehydrogenase (L-Δ1-pyrroline-5-carboxylate dehydrogenase) activities. P. aurescens strain TC1 grew well in minimal media with L-Proline as a supplemental nutrient, the nitrogen source, or the sole carbon and nitrogen source. Multicellular myceloids induced by NaCl or citrate also grew on L-proline. The specific activity of L-proline dehydrogenase in whole cells was higher whenever L-proline was added to the medium. Both L-proline dehydrogenase and L-glutamate-γ-semialdehyde dehydrogenase (L-Δ1-pyrroline-5-carboxylate dehydrogenase) activities were found primarily in a membrane fraction from exponential-phase cells. The two activities co-eluted from a Bio-Gel P-60 column after precipitation of proteins with ammonium sulfate and solubilization with 0.1% Tween 20. The PutA protein in the active fraction also oxidized 3,4-dehydro-DL-proline, but there was no activity with other L-proline analogues. When P. aurescens strain TC1 was grown in minimal media containing increasing concentrations of NaCl, there was a progressive decrease in the specific activity of L-proline dehydrogenase and a concomitant increase in the intracellular concentration of L-proline. These results indicate that P. aurescens strain TC1 can use L-proline as a nutrient in a regulated fashion. Because this bacterium also showed the ability to degrade most of the other common amino acids, it can serve as a useful model for the control of amino acid catabolism in the high G + C Actinobacteria.

Biochemistry laboratory reports: Filling in the introduction and discussion.

Laboratory reports written in the style of a standard scientific article are commonly used to assess student learning in biochemistry laboratory courses. While most students can complete the Materials and Methods or Results sections successfully, many have difficulty with the Introduction and Discussion. They fail to place their data in a larger experimental context or to compare their results to those previously published. To address this issue in a laboratory course focusing on l-lactate dehydrogenase, a new exercise was introduced that was designed 1) to provide more background information about l-lactate dehydrogenase; 2) to give students additional experience in using PubMed and Web of Science to locate specific papers about l-lactate dehydrogenase; 3) to introduce the major bioinformatics databases at the National Center for Biological Information, ExPASy (Swiss Institute of Bioinformatics), BRENDA (Braunschweig Enzyme Database), and the Protein Data Bank; 4) to allow students to recover detailed information about l-lactate dehydrogenases; and 5) to provide practice in reading a research article that is similar to what they do in the lab. The students completed a data sheet summarizing these activities and then prepared three laboratory reports. The lab reports improved over the course of the semester and were qualitatively better than in past years. The materials developed for this laboratory course can be adapted to similar projects that use another protein as a model system. They could also be modified for use in Course-based Undergraduate Research Experiences or research projects for undergraduate or graduate students. © 2018 International Union of Biochemistry and Molecular Biology, 46(6):619-622, 2018.

Browning in apples: Exploring the biochemical basis of an easily-observable phenotype.

Many fruits and vegetables undergo browning when they are cut and the tissue is exposed to the air. This is due to the activity of the enzyme polyphenol oxidase (PPO, EC 1.14.18.1) with endogenous substrates. In this laboratory experiment, students prepare slices of different varieties of apples and assess the rate of browning. They make a simple extract of the apple tissue and measure the activity of PPO using 3,4-dihydroxy-l-phenylalanine (l-DOPA) as substrate. They determine the protein concentration of the extract with the Bradford Coomassie Blue reagent and calculate the specific activity of PPO. Finally, the students measure the total concentration of the potential substrates for PPO with the Folin-Ciocalteau phenol reagent using a gallic acid standard curve. By comparing the tendency of the apples to turn brown, the specific activity of PPO, and the concentration of potential substrates, they can assess the biochemical basis of the browning phenotype. This experiment can be done as a series of weekly laboratory exercises, as an intensive 1-week laboratory project, or as the basis of an extended student research investigation. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):76-82, 2018.

Analysis of strains lacking known osmolyte accumulation mechanisms reveals contributions of osmolytes and transporters to protection against abiotic stress.

Osmolyte accumulation and release can protect cells from abiotic stresses. In Escherichia coli, known mechanisms mediate osmotic stress-induced accumulation of K(+) glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high- and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-N-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.

L-Malate dehydrogenase activity in the reductive arm of the incomplete citric acid cycle of Nitrosomonas europaea.

The autotrophic nitrifying bacterium Nitrosomonas europaea does not synthesize 2-oxoglutarate (α-ketoglutarate) dehydrogenase under aerobic conditions and so has an incomplete citric acid cycle. L-malate (S-malate) dehydrogenase (MDH) from N. europaea was predicted to show similarity to the NADP(+)-dependent enzymes from chloroplasts and was separated from the NAD(+)-dependent proteins from most other bacteria or mitochondria. MDH activity in a soluble fraction from N. europaea ATCC 19718 was measured spectrophotometrically and exhibited simple Michaelis-Menten kinetics. In the reductive direction, activity with NADH increased from pH 6.0 to 8.5 but activity with NADPH was consistently lower and decreased with pH. At pH 7.0, the K m for oxaloacetate was 20 µM; the K m for NADH was 22 µM but that for NADPH was at least 10 times higher. In the oxidative direction, activity with NAD(+) increased with pH but there was very little activity with NADP(+). At pH 7.0, the K m for L-malate was 5 mM and the K m for NAD(+) was 24 µM. The reductive activity was quite insensitive to inhibition by L-malate but the oxidative activity was very sensitive to oxaloacetate. MDH activity was not strongly activated or inhibited by glycolytic or citric acid cycle metabolites, adenine nucleotides, NaCl concentrations, or most metal ions, but increased with temperature up to about 55 °C. The reductive activity was consistently 10-20 times higher than the oxidative activity. These results indicate that the L-malate dehydrogenase in N. europaea is similar to other NAD(+)-dependent MDHs (EC 1.1.1.37) but physiologically adapted for its role in a reductive biosynthetic sequence.

L-Proline nutrition and catabolism in Staphylococcus saprophyticus.

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require L-proline but not L-arginine for growth in a defined culture medium. All three strains could utilize L-ornithine as a proline source and contained L-ornithine aminotransferase and Δ(1)-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use L-arginine as a proline source and had L-arginase activity. The proline requirement also could be met by L-prolinamide, L-proline methyl ester, and the dipeptides L-alanyl-L-proline and L-leucyl-L-proline. The bacteria exhibited L-proline degradative activity as measured by the formation of Δ(1)-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of L-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller-Hinton broth. A membrane fraction from this strain had L-proline dehydrogenase activity as detected both by reaction of Δ(1)-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min(-1) mg(-1)) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min(-1) mg(-1)). A soluble fraction from this strain had Δ(1)-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min(-1) mg(-1)) as determined by the NAD(+)-dependent oxidation of DL-Δ(1)-pyrroline-5-carboxylate. Addition of L-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with L: -ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-DL-proline, DL-thiazolidine-2-carboxylate, and L-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.

Oxidation of 3,4-dehydro-D-proline and other D-amino acid analogues by D-alanine dehydrogenase from Escherichia coli.

3,4-Dehydro-DL-proline is a toxic analogue of L-proline which has been useful in studying the uptake and metabolism of this key amino acid. When membrane fractions from Escherichia coli strain UMM5 (putA1::Tn5 proC24) lacking both L-proline dehydrogenase and L-Delta(1)-pyrroline-5-carboxylate reductase were incubated with 3,4-dehydro-DL-proline, pyrrole-2-carboxylate was formed. There was no enzyme activity with 3,4-dehydro-L-proline, but activity was restored after racemization of the substrate. Oxidation of 3,4-dehydro-DL-proline by membrane fractions from strain UMM5 was induced by growth in minimal medium containing D- or L-alanine, had a pH optimum of 9, and was competitively inhibited by D-alanine. An E. coli strain with no D-alanine dehydrogenase activity due to the dadA237 mutation was unable to oxidize either 3,4-dehydro-D-proline or D-alanine, as were spontaneous Dad(-) mutants of E. coli strain UMM5. Membrane fractions containing D-alanine dehydrogenase also catalyzed the oxidation of D-2-aminobutyrate, D-norvaline, D-norleucine, cis-4-hydroxy-D-proline, and DL-ethionine. These results indicate that d-alanine dehydrogenase is responsible for the residual 3,4-dehydro-DL-proline oxidation activity in putA proC mutants of E. coli and provide further evidence that this enzyme plays a general role in the metabolism of D-amino acids and their analogues.