A Simplified but Comprehensive Laboratory Curriculum for Microbiology in Nursing and Allied Health (MINAH) Courses.

Some U.S. nursing programs have considered or already have removed microbiology courses from their curriculum. In response to this, the American Society for Microbiology (ASM) published in 2018 the Microbiology in Nursing and Allied Health (MINAH) Undergraduate Curriculum Guidelines. Parts of these guidelines include competencies and skills, which are best taught in a laboratory setting. Recognizing that some programs face a burden of running nursing/allied health microbiology lab classes, we have designed a curriculum of seven laboratories that covers the concepts in the MINAH guidelines. These labs cover aseptic technique and proper specimen collection, microscopy, enumerating microorganisms, and diagnostic tests in identifying unknowns. In addition, we provide some tips and tools for keeping down the costs of offering a microbiology lab. It is our goal that these tips and the suggested guidelines will allow microbiology to remain in the nursing and allied health curriculum.

An Introductory Module and Experiments To Improve the Graphing Skills of Non-Science Majors.

Graphing allows for the succinct communication of scientific data and is therefore a critical learning objective in science curricula. Unfortunately, many students, particularly non-science majors, lack the necessary skills to prepare and interpret graphs. Many students are able to interpolate data and observe general trends but demonstrate only a cursory ability to contextualize their results. In this paper, we suggest an introductory module and graphing lessons to improve the quantitative skills of non-science majors. In each of these lessons, students go through four phases of data analysis: (a) collection; (b) graphing; (c) interpolation/trend detection (reading), and (d) determining the underlying mechanism resulting in the trends they observe (interpretation). By employing these activities, we are continuing to improve the scientific literacy of students.

Discovery of the Collaborative Nature of Science with Undergraduate Science Majors and Non-Science Majors through the Identification of Microorganisms Enriched in Winogradsky Columns.

Today's science classrooms are addressing the need for non-scientists to become scientifically literate. A key aspect includes the recognition of science as a process for discovery. This process relies upon interdisciplinary collaboration. We designed a semester-long collaborative exercise that allows science majors taking a general microbiology course and non-science majors taking an introductory environmental science course to experience collaboration in science by combining their differing skill sets to identify microorganisms enriched in Winogradsky columns. These columns are self-sufficient ecosystems that allow researchers to study bacterial populations under specified environmental conditions. Non-science majors identified phototrophic bacteria enriched in the column by analyzing the signature chlorophyll absorption spectra whereas science majors used 16S rRNA gene sequencing to identify the general bacterial diversity. Students then compiled their results and worked together to generate lab reports with their final conclusions identifying the microorganisms present in their column. Surveys and lab reports were utilized to evaluate the learning objectives of this activity. In pre-surveys, nonmajors' and majors' answers diverged considerably, with majors providing responses that were more accurate and more in line with the working definition of collaboration. In post-surveys, the answers between majors and nonmajors converged, with both groups providing accurate responses. Lab reports showed that students were able to successfully identify bacteria present in the columns. These results demonstrate that laboratory exercises designed to group students across disciplinary lines can be an important tool in promoting science education across disciplines.

A non-catalytic histidine residue influences the function of the metalloprotease of Listeria monocytogenes.

Mpl, a thermolysin-like metalloprotease, and PC-PLC, a phospholipase C, are synthesized as proenzymes by the intracellular bacterial pathogen Listeria monocytogenes. During intracellular growth, L. monocytogenes is temporarily confined in a membrane-bound vacuole whose acidification leads to Mpl autolysis and Mpl-mediated cleavage of the PC-PLC N-terminal propeptide. Mpl maturation also leads to the secretion of both Mpl and PC-PLC across the bacterial cell wall. Previously, we identified negatively charged and uncharged amino acid residues within the N terminus of the PC-PLC propeptide that influence the ability of Mpl to mediate the maturation of PC-PLC, suggesting that these residues promote the interaction of the PC-PLC propeptide with Mpl. In the present study, we identified a non-catalytic histidine residue (H226) that influences Mpl secretion across the cell wall and its ability to process PC-PLC. Our results suggest that a positive charge at position 226 is required for Mpl functions other than autolysis. Based on the charge requirement at this position, we hypothesize that this residue contributes to the interaction of Mpl with the PC-PLC propeptide.

Protein transport across the cell wall of monoderm Gram-positive bacteria.

In monoderm (single-membrane) Gram-positive bacteria, the majority of secreted proteins are first translocated across the cytoplasmic membrane into the inner wall zone. For a subset of these proteins, final destination is within the cell envelope as either membrane-anchored or cell wall-anchored proteins, whereas another subset of proteins is destined to be transported across the cell wall into the extracellular milieu. Although the cell wall is a porous structure, there is evidence that, for some proteins, transport is a regulated process. This review aims at describing what is known about the mechanisms that regulate the transport of proteins across the cell wall of monoderm Gram-positive bacteria.

Posttranslocation chaperone PrsA2 regulates the maturation and secretion of Listeria monocytogenes proprotein virulence factors.

PrsA2 is a conserved posttranslocation chaperone and a peptidyl prolyl cis-trans isomerase (PPIase) that contributes to the virulence of the Gram-positive intracellular pathogen Listeria monocytogenes. One of the phenotypes associated with a prsA2 mutant is decreased activity of the broad-range phospholipase C (PC-PLC). PC-PLC is made as a proenzyme whose maturation is mediated by a metalloprotease (Mpl). The proforms of PC-PLC and Mpl accumulate at the membrane-cell wall interface until a decrease in pH triggers their maturation and rapid secretion into the host cell. In this study, we examined the mechanism by which PrsA2 regulates the activity of PC-PLC. We observed that in the absence of PrsA2, the proenzymes are secreted at physiological pH and do not mature upon a decrease in pH. The sensitivity of the prsA2 mutant to cell wall hydrolases was modified. However, no apparent changes in cell wall porosity were detected. Interestingly, synthesis of PC-PLC in the absence of its propeptide lead to the secretion of a fully active enzyme in the cytosol of host cells independent of PrsA2, indicating that neither the propeptide of PC-PLC nor PrsA2 is required for native folding of the catalytic domain, although both influence secretion of the enzyme. Taken together, these results suggest that PrsA2 regulates compartmentalization of Mpl and PC-PLC, possibly by influencing cell wall properties and interacting with the PC-PLC propeptide. Moreover, the ability of these proproteins to respond to a decrease in pH during intracellular growth depends on their localization at the membrane-cell wall interface.

The metalloprotease of Listeria monocytogenes is regulated by pH.

Listeria monocytogenes is an intracytosolic bacterial pathogen. Among the factors contributing to escape from vacuoles are a phosphatidylcholine phospholipase C (PC-PLC) and a metalloprotease (Mpl). Both enzymes are translocated across the bacterial membrane as inactive proproteins, whose propeptides serve in part to maintain them in association with the bacterium. We have shown that PC-PLC maturation is regulated by Mpl and pH and that Mpl maturation occurs by autocatalysis. In this study, we tested the hypothesis that Mpl activity is pH regulated. To synchronize the effect of pH on bacteria, the cytosolic pH of infected cells was manipulated immediately after radiolabeling de novo-synthesized bacterial proteins. Immunoprecipitation of secreted Mpl from host cell lysates revealed the presence of the propeptide and catalytic domain in samples treated at pH 6.5 but not at pH 7.3. The zymogen was present in small amounts under all conditions. Since proteases often remain associated with their respective propeptide following autocatalysis, we aimed at determining whether pH regulates autocatalysis or secretion of the processed enzyme. For this purpose, we used an Mpl construct that contains a Flag tag at the N terminus of its catalytic domain and antibodies that can distinguish N-terminal and non-N-terminal Flag. By fluorescence microscopy, we observed the Mpl zymogen associated with the bacterium at physiological pH but not following acidification. Mature Mpl was not detected in association with the bacterium at either pH. Using purified proteins, we determined that processing of the PC-PLC propeptide by mature Mpl is also pH sensitive. These results indicate that pH regulates the activity of Mpl on itself and on PC-PLC.

The propeptide of the metalloprotease of Listeria monocytogenes controls compartmentalization of the zymogen during intracellular infection.

Integral to the virulence of the intracellular bacterial pathogen Listeria monocytogenes is its metalloprotease (Mpl). Mpl regulates the activity and compartmentalization of the bacterial broad-range phospholipase C (PC-PLC). Mpl is secreted as a proprotein that undergoes intramolecular autocatalysis to release its catalytic domain. In related proteases, the propeptide serves as a folding catalyst and can act either in cis or in trans. Propeptides can also influence protein compartmentalization and intracellular trafficking or decrease folding kinetics. In this study, we aimed to determine the role of the Mpl propeptide by monitoring the behavior of Mpl synthesized in the absence of its propeptide (MplDeltapro) and of two Mpl single-site mutants with unstable propeptides: Mpl(H75V) and Mpl(H95L). We observed that all three Mpl mutants mediate PC-PLC activation when bacteria are grown on semisolid medium, but to a lesser extent than wild-type Mpl, indicating that, although not essential, the propeptide enhances the production of active Mpl. However, the mutant proteins were not functional in infected cells, as determined by monitoring PC-PLC maturation and compartmentalization. This defect could not be rescued by providing the propeptide in trans to the mplDeltapro mutant. We tested the compartmentalization of Mpl during intracellular infection and observed that the mutant Mpl species were aberrantly secreted in the cytosol of infected cells. These data indicated that the propeptide of Mpl serves to maintain bacterium-associated Mpl and that this localization is essential to the function of Mpl during intracellular infection.

DNA damage differentially activates regional chromosomal loci for Tn7 transposition in Escherichia coli.

The bacterial transposon Tn7 recognizes replicating DNA as a target with a preference for the region where DNA replication terminates in the Escherichia coli chromosome. It was previously shown that DNA double-strand breaks in the chromosome stimulate Tn7 transposition where transposition events occur broadly around the point of the DNA break. We show that individual DNA breaks actually activate a series of small regional hotspots in the chromosome for Tn7 insertion. These hotspots are fixed and become active only when a DNA break occurs in the same region of the chromosome. We find that the distribution of insertions around the break is not explained by the exonuclease activity of RecBCD moving the position of the DNA break, and stimulation of Tn7 transposition is not dependent on RecBCD. We show that other forms of DNA damage, like exposure to UV light, mitomycin C, or phleomycin, also stimulate Tn7 transposition. However, inducing the SOS response does not stimulate transposition. Tn7 transposition is not dependent on any known specific pathway of replication fork reactivation as a means of recognizing DNA break repair. Our results are consistent with the idea that Tn7 recognizes DNA replication involved in DNA repair and reveals discrete regions of the chromosome that are differentially activated as transposition targets.