Charles E. Deutch

Inhibition of urease activity in the urinary tract pathogen Staphylococcus saprophyticus

Urease is a virulence factor for the Gram‐positive urinary tract pathogen Staphylococcus saprophyticus. The susceptibility of this enzyme to chemical inhibition was determined using soluble extracts of Staph. saprophyticus strain ATCC 15305. Acetohydroxamic acid (Ki = 8·2 μg ml−1 = 0·106mmol l−1) and DL‐phenylalanine hydroxamic acid (Ki = 21 μg ml−1 = 0·116mmol l−1) inhibited urease activity competitively. The phosphorodiamidate fluorofamide also caused competitive inhibition (Ki = 0·12 μg ml−1= 0·553 μmol l−1 = 0·000553 mmol l−1), but the imidazole omeprazole had no effect. Two flavonoids found in green tea extract [(+)‐catechin hydrate (Ki = 357 μg ml−1 = 1·23 mmol l−1) and (−)‐epigallocatechin gallate (Ki= 210 μg ml−1 = 0·460 mmol l−1)] gave mixed inhibition. Acetohydroxamic acid, DL‐phenylalanine hydroxamic acid, fluorofamide, (+)‐catechin hydrate and (−)‐epigallocatechin gallate also inhibited urease activity in whole cells of strains ATCC 15305, ATCC 35552 and ATCC 49907 grown in a rich medium or an artificial urine medium. Addition of acetohydroxamic acid or fluorofamide to cultures of Staph. saprophyticus in an artificial urine medium delayed the increase in pH that normally occurs during growth. These results suggest that urease inhibitors may be useful for treating urinary tract infections caused by Staph. saprophyticus.

Susceptibility of Escherichia coli to L-selenaproline and other L-proline analogues in laboratory culture media and normal human urine

Aims:  The aims of this study were to identify analogues of L‐proline which inhibit the growth of Escherichia coli in both laboratory culture media and normal human urine and to study their mechanisms of uptake.

Microbial Contamination of Chicken Wings An Open-Ended Laboratory Project

Numerous studies indicate that investigative laboratory experiments are more successful than simple exercises in teaching students the process of science (Leonard 1989; Sundberg & Moncada 1994; Thornton 1972). Projects that extend over several weeks give students a chance to design their own experiments, to master one or more basic techniques, and to collect and analyze several related data sets. Although investigative experiments can be done individually, they most often are carried out by small groups, providing opportunities for teamwork and student interaction. The results of a laboratory investigation may be summarized and evaluated as an oral report, a poster presentation, or a lab report written in a scientific style (Billington 1997; Gratz 1990; Mulnix & Penhale 1997). In this article, I describe an open-ended project dealing with microbial contamination of chicken wings, which can be used by students at several different levels.

Seasonal Variations in Microbial Communities in the Nasal Passages of Captive Desert Tortoises

Abstract Populations of the desert tortoise, Gopherus agassizii, have been severely impacted by upper respiratory tract disease (URTD), a potentially fatal condition caused by Mycoplasma agassizii. Because natural communities of microorganisms in animals may serve as barriers to infection by potential pathogens or may influence the course of a disease, we characterized the bacteria in the nasal passages of captive desert tortoises over an entire season. Tortoises housed in outdoor pens at the Adobe Mountain Wildlife Center in Phoenix, AZ, were divided into four groups: three healthy tortoises that were sampled monthly, three tortoises with signs of URTD that were sampled monthly, three healthy tortoises that were sampled bimonthly, and three healthy tortoises that were sampled once at the end of the season. At each sampling time, the health of each tortoise was assessed and the nares were probed with moistened sterile swabs. The bacteria on the swabs were suspended in sterile saline, serially diluted, and plated on tryptic soy agar medium. Total bacterial counts varied among tortoises from about 104 to 107 per ml and were usually higher in tortoises with signs of URTD. The proportions of different colony types varied from month to month within each tortoise. Although the microbial communities were dominated by pigmented Gram-positive cocci, we also found Gram-positive bacilli, Gram-variable coryneforms, and Gram-negative rods. Many of the same bacteria were recovered from both healthy and URTD tortoises, but others were unique to the URTD tortoises. This study indicates that the nasal passages of desert tortoises contain large continuously changing communities of bacteria and suggests that further analysis of these microorganisms may be useful in assessing the health or stress of populations of desert tortoises and the susceptibility of individual tortoises to URTD.

Analysis of the RNA Content of the Yeast Saccharomyces cerevisiae

The principles involved in the storage and expression of genetic information are now well established and have been incorporated into biology curricula for elementary, secondary, and college students (National Research Council, 1996). Almost every biology textbook now describes the structure of DNA and the roles of messenger RNAs, transfer RNAs, and ribosomal RNAs in protein synthesis. Many different laboratory experiments have been developed in which students isolate genomic DNA (Dollard, 1994; Helms et al., 1998), subject fragments of DNA generated by restriction endonucleases or polymerase chain reactions to gel electrophoresis (Jenkins & Bielec, 2006; Kass, 2007), transform bacteria with DNA plasmids carrying genes for antibiotic resistance (Guifoile & Plum, 2000), or combine several of these methods to clone particular genes (Becker et al., 1996; Micklos et al., 2003; Winfrey et al., 1997). However, very few classroom experiments focus specifically on RNA. Bregman (2002) has described two experiments in which students study cellular RNA microscopically either by staining whole cells in a blood smear with a combination of methyl green and pyronin or by staining tissue culture cells with the ammoniacal silver method for ribosomal RNA. However, neither of these experiments is quantitative and both require microscopy skills beyond those of many beginning students. Direct measurement of RNA formation by transcription of a DNA template or analysis of RNA function during translation normally involves the use of chemiluminescent or radioactively-labeled nucleotides or amino acids (Ausubel et al., 2002; Grandi, 2007; Martin, 1998; Sambrook & Russell, 2001). Although kits for doing these types of studies are commercially available, most schools do not have either the liquid scintillation counters or X-ray film developers need to detect the products, and lack the support staff necessary to meet federal and state requirements for safely using radioactive compounds. These limitations are unfortunate in light of the growing body of scientific information about the pre-biotic RNA world. It now seems clear that the basic steps in protein synthesis were established before DNA became important as a way of storing genetic information in a stable way (Gesteland et al., 2006; Gilbert, 1986; Muller, 2006; Spirin, 2002; Woese, 2001). In addition to having self-catalytic activity, RNA molecules are involved in all the steps of translation, from the initial activation of amino acids by attaching them to transfer RNAs to the association of messenger RNAs and aminoacyltransfer RNAs with ribosomes to the actual polymerization of amino acids into polypeptide chains. Table 1 summarizes the characteristics and sizes of the major types of RNA commonly found in prokaryotic and eukaryotic cells. The Howard Hughes Medical Institute (2006) has produced a DVD from its Holliday Lectures on Science series which discusses the many roles of RNA and can be used to introduce students to this topic. In this article, we describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. The general goals of these experiments are to emphasize the importance of RNA in cell biology and to provide practice in basic biochemical and molecular analysis. Overview of Experiments This set of experiments focuses on RNAs from the yeast Saccharomyces cerevisiae, a unicellular budding microorganism that has served as a model of cellular and molecular processes in eukaryotes (Davis, 2003). S. cerevisiae can be grown easily in the laboratory, has a relatively small genome that has been completely sequenced, and is susceptible to genetic analysis using both classical and molecular techniques. In these experiments, students: 1. study yeast cells by light microscopy 2. …

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.

Using a Thematic Organism Across the Curriculum: Analysis of the Protein Composition in Desert Tree Lizard Tissue in Cell Biology and Genetics Laboratories

ABSTRACT The Division of Mathematical and Natural Sciences at the West campus of Arizona State University has adopted a new curriculum leading to a B.S. degree in the Life Sciences. An innovative feature of this curriculum is the inclusion of experimental work with the desert tree lizard (Urosaursus ornatus) in multiple courses to link them together thematically. The experiments conducted in the core courses in Cell Biology and Fundamentals of Genetics are designed to introduce students to techniques in modern cell and molecular biology within the context of the lizard theme. In the laboratory for the Cell Biology course, students perform a 3-week project using SDS-polyacrylamide gel electrophoresis to compare the proteins found in various lizard organs including heart and liver. In the laboratory for the Fundamentals of Genetics course, students carry out a 2-week project using nondenaturing gel electrophoresis in agarose gels to study the occurrence of allozymes of aconitate hydratase (EC 4.2.1.3) and NADP+-dependent malate dehydrogenase (EC 1.1.1.37) in different individuals in captive populations of desert tree lizards. In both cases, the students gain experience in casting and running gels, in staining and imaging gels digitally, and in analyzing the images quantitatively. By performing these two linked projects in separate courses, students can see the value of vertical and horizontal gel electrophoresis in different contexts and build on their technical skills as they move through the curriculum.

Using Data Analysis Problems in a Large General Microbiology Course.

A MAJOR goal of biological education should be to assist students in developing the ability to think critically (Moore 1993). They should be able to place new information in a broad scientific framework, use factual material they have learned in new contexts, interpret experimental data presented as graphs or tables, and draw appropriate conclusions from a series of observations. Student success in developing these skills is often evaluated on standardized tests through data analysis problems. A data analysis problem usually consists of an introductory paragraph describing a particular situation or experiment, one or more figures or tables of data, and a series of multiple-choice questions. Data analysis problems form a major part of the Science Test offered by the American College Testing (ACT) Program and the Scholastic Assessment Tests (SAT I and SAT II) offered by the Educational Testing Service. They also make up a large component of the Graduate Record Examination (GRE) and the Medical College Admissions Test (MCAT). Although data analysis problems play an important role in the admission of students to undergraduate, graduate or professional programs, they are still not widely used in introductory biology courses. This is particularly true in the area of microbiology. A survey of the test banks and student guides that accompany the most commonly used textbooks in this field reveals an almost total lack of data analysis material. The questions in these test banks and student guides are primarily designed to assess mastery of basic vocabulary and factual material. While this is certainly important in an introductory course, the inclusion of data analysis problems is essential as well. These problems provide a mechanism for helping students move beyond rote memorization. They give them practice in critical thinking and illustrate the process of scientific inquiry. Accordingly, I have made an effort in the last several years to include data analysis problems in a large General Microbiology course. This article describes my experience with this course and

Teaching the Principles That Govern the Structure & Function of Biomolecules.

All living organisms contain a common set of chemical compounds that range in size from simple ions containing a single element to large macromolecules composed of thousands of atoms covalently bonded together. General biology textbooks for high school or college students usually contain a long chapter that introduces students to the biochemistry of proteins, nucleic acids, carbohydrates, and lipids. This chapter is filled with diagrams showing the structures of the 20 amino acids found in proteins, the five purine and pyrimidine nucleotides that occur in DNA and RNA, some of the monosaccharides found in oligosaccharides and polysaccharides, and several examples of lipids. This introductory chapter may also include a discussion of the secondary, tertiary, and quaternary structures of proteins; the B and Z forms of DNA; the sheetlike structure of cellulose; and the organization of phospholipids in biological membranes. Many students have difficulty understanding this material, and so more advanced textbooks in biochemistry, cell biology, microbiology and physiology often contain chapters that repeat much of the same information. In teaching a large course in general microbiology, I have found that even if students have memorized the structures of some biologically important molecules, they have little understanding of the basic principles that govern their structure and function. Accordingly, I have tried to develop these principles more explicitly and to focus on general concepts rather than rote memorization. In this article, I describe four principles essential to understanding the properties of biological molecules. I then suggest some pedogogical strategies for teaching these principles using examples from my own area. While other teachers might use other strategies or examples, the principles I describe are quite general. By taking a more conceptual approach, I have found student mastery of this material can be increased dramatically.