The penC mutation conferring antibiotic resistance in Neisseria gonorrhoeae arises from a mutation in the PilQ secretin that interferes with multimer stability: GonococcalpilQmutants with increased antibiotic resistance

The penC resistance gene was previously characterized in a FA19 penA mtrR penB gonococcal strain (PR100) as a spontaneous mutation that increased resistance to penicillin and tetracycline. We show here that antibiotic resistance mediated by penC is the result of a Glu-666 to Lys missense mutation in the pilQ gene that interferes with the formation of the SDS-resistant high-molecular-mass PilQ secretin complex, disrupts piliation, and decreases transformation frequency by 50-fold. Deletion of pilQ in PR100 confers the same level of antibiotic resistance as the penC mutation, but increased resistance was observed only in strains containing the mtrR and penB resistance determinants. Site-saturation mutagenesis of Glu-666 revealed that only acidic or amidated amino acids at this position preserved PilQ function. Consistent with early studies suggesting the importance of cysteine residues on stability of the PilQ multimer, mutation of either of the two cysteine residues in FA19 PilQ led to a similar phenotype as penC: increased antibiotic resistance, loss of piliation, intermediate levels of transformation competence, and absence of SDS-resistant PilQ oligomers. These data show that a functional secretin complex can enhance the entry of antibiotics into the cell and suggest that the PilQ oligomer forms a pore in the outer membrane through which antibiotics diffuse into the periplasm

Genome sequences of four cluster P mycobacteriophages

Four bacteriophages infecting Mycobacterium smegmatis mc2155 (three belonging to subcluster P1 and one belonging to subcluster P2) were isolated from soil and sequenced. All four phages are similar in the left arm of their genomes, but the P2 phage differs in the right arm. All four genomes contain features of temperate phages

Instructional Models for Course-Based Research Experience (CRE) Teaching

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching

Models of classroom assessment for course-based research experiences

Course-based research pedagogy involves positioning students as contributors to authentic research projects as part of an engaging educational experience that promotes their learning and persistence in science. To develop a model for assessing and grading students engaged in this type of learning experience, the assessment aims and practices of a community of experienced course-based research instructors were collected and analyzed. This approach defines four aims of course-based research assessment—(1) Assessing Laboratory Work and Scientific Thinking; (2) Evaluating Mastery of Concepts, Quantitative Thinking and Skills; (3) Appraising Forms of Scientific Communication; and (4) Metacognition of Learning—along with a set of practices for each aim. These aims and practices of assessment were then integrated with previously developed models of course-based research instruction to reveal an assessment program in which instructors provide extensive feedback to support productive student engagement in research while grading those aspects of research that are necessary for the student to succeed. Assessment conducted in this way delicately balances the need to facilitate students’ ongoing research with the requirement of a final grade without undercutting the important aims of a CRE education

The obligate human pathogen, Neisseria gonorrhoeae, is polyploid.

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts

Microscopic Analysis of Gc DNA Content

<p>(A) Fluorescence microscopy of Hoechst-stained cells. Phase contrast, fluorescence, and merged images are shown for examples of each cell type observed. (i) monococci, (ii) diplococci. (B) Histogram of fluorescence intensities for exponentially growing and Tet-treated Hoechst-stained cells as determined from fluorescent micrographs.</p

Model for Multiple Genomes per Monococcal Cell

<p>The proposed model has a monococcal cell undergoing DNA replication and cell division over time as indicated in min below each drawing. Segregation of chromosomes to promote homozygosity is demonstrated by the dotted chromosomal DNA.</p

Diagram of the FA1090 Chromosome

<p>Quantitative PCR target sequences are shown, as well as putative origin sites, genes associated with bacterial origins, and the <i>dif</i> site which is linked to the terminus. </p

Rapid Pivot of CURE Wet Lab to Online with the Help of Instructor Communities

The pivot to remote and hybrid learning during the Covid-19 pandemic presented a challenge for many in academia. Most institutions were not prepared to support this rapid change, and instructors were left with the burden of converting a traditional face-to-face course into multiple modalities with very limited preparation time. When institutional support is lacking, we posit that instructor communities of practice can help provide the resources needed to meet the instructional demands. Tiny Earth, a course-based-undergraduate research experience (CURE) and international network of instructors and students, responded to the instructional challenges of the pandemic by leveraging its large community of instructors to create several smaller working groups to form focused communities of practice. Using the pedagogical principles of backward design and scientific teaching, one working group, the Tiny Earth Pivot Group (Pivot Group) generated a course map of remote learning activities and simulated learning resources to fulfill the Tiny Earth learning objectives and maintain the essential tenets of a CURE. Additional working groups were created to disseminate the resources collated and created by the Pivot Group to the greater community. In terms of Tiny Earth, the community structure provided the means for instructors to rapidly pivot their course materials to multiple modalities while upholding the student CURE experience. Harnessing the hallmarks of communities of practice—collective workpower toward common purpose, diversity of perspectives, and ongoing evolution—coupled with high-structured course design allows instructors flexibility and adaptability in meeting the changing modalities of higher education