Lysogeny and Use of Mycobacteriophage Pita2

The CDC has classified antibiotic resistance as the biggest health challenge of our era; every year 2 million lives are impacted and even lost due to resistant bacteria. Bacteriophages provide an alternative route to fighting infections that does not further the development of antibiotic resistance among bacterial species. A bacteriophage replicates inside a bacterial cell and then causes that cell to lyse, an event that kills the bacterial host. However, some phage can integrate their genomes into the host chromosome without causing lysis. The HHMI SEA-PHAGES program has generated a collection of bacteriophage that infect Actinobacteria species. Over 13,000 phages have been collected thus far, but fewer than 3,000 have been sequenced and genetically analyzed. The purpose of research into the lysogeny of discovered, but unsequenced, bacteriophage is to classify them by immunity range; that is, closely related bacteriophage are unable to infect the lysogenic host, while more distantly related phage are. Initially, a lysogen had to be isolated. For Pita2, a phage isolated and analyzed at Bowling Green State University, the host is Mycobacterium smegmatis. A purified lysogen of Pita2 was analyzed against bacteriophage with known DNA sequences to confirm that it is immune to infection by closely related phage. The lysogeny was then examined for its ability to identify close vs. more distantly related phage among a set unknown bacteriophage. Another virus, namely SARS-CoV2, prevented the completion of this study. However, it was possible to generate preliminary data that established the ability to use immunity as a means to classify newly isolated phage based upon the degree to which they can successfully infect the Pita2 lysogen

Isolation of anoxygenic photosynthetic bacteria from Songkhla Lake for use in a two-staged biohydrogen production process from palm oil mill effluent

We are developing a process to produce biohydrogen from palm oil mill effluent. Part of this process will involve photohydrogen production from volatile fatty acids under low light conditions. We sought to isolate suitable bacteria for this purpose fromSongkhla Lake in Southern Thailand. Enrichment for phototrophic bacteria from 34 samples was conducted providing acetate as a major carbon source and applying culturing conditions of anaerobic-low light (3000 lux) at 30 C. Among the independent isolates from these enrichments 19 evolved hydrogen with productivities between 4 and 326 ml l 1 d 1. Isolate TN1 was the most efficient producer at a rate of 1.85 mol H2 mol acetate 1 with a light conversion efficiency of 1.07%. The maximum hydrogen production rate for TN1 was determined to be 43 ml l 1 h 1. Environmentally desirable features of photohydrogen production by TN1 included the absence of pH change in the cultures and no detectable residual CO

Compensatory changes in GroEL/Gp31 affinity as a mechanism for allele-specific genetic interaction

Previous work has shown that the GroEL-GroES interaction is primarily mediated by the GroES mobile loop. In bacteriophage T4 infection, GroES is substituted by the gene 31-encoded cochaperonin, Gp31. Using a genetic selection scheme, we have identified a new set of mutations in gene 31 that affect interaction with GroEL; all mutations result in changes in the mobile loop of Gp31. Biochemical analyses reveal that the mobile loop mutations alter the affinity between Gp31 and GroEL, most likely by modulating the stability of the GroEL-bound hairpin conformation of the mobile loop. Surprisingly, mutations in groEL that display allele-specific interactions with mutations in gene 31 alter residues in the GroEL intermediate domain, distantly located from the mobile loop binding site. The observed patterns of genetic and biochemical interaction between GroES or Gp31 and GroEL point to a mechanism of genetic allele specificity based on compensatory changes in affinity of the protein-protein interaction. Mutations studied in this work indirectly alter affinity by modulating a folding transition in the Gp31 mobile loop or by modulating a hinged conformational change in GroEL

Reconstruction of the Core and Extended Regulons of Global Transcription Factors

The processes underlying the evolution of regulatory networks are unclear. To address this question, we used a comparative genomics approach that takes advantage of the large number of sequenced bacterial genomes to predict conserved and variable members of transcriptional regulatory networks across phylogenetically related organisms. Specifically, we developed a computational method to predict the conserved regulons of transcription factors across α-proteobacteria. We focused on the CRP/FNR super-family of transcription factors because it contains several well-characterized members, such as FNR, FixK, and DNR. While FNR, FixK, and DNR are each proposed to regulate different aspects of anaerobic metabolism, they are predicted to recognize very similar DNA target sequences, and they occur in various combinations among individual α-proteobacterial species. In this study, the composition of the respective FNR, FixK, or DNR conserved regulons across 87 α-proteobacterial species was predicted by comparing the phylogenetic profiles of the regulators with the profiles of putative target genes. The utility of our predictions was evaluated by experimentally characterizing the FnrL regulon (a FNR-type regulator) in the α-proteobacterium Rhodobacter sphaeroides. Our results show that this approach correctly predicted many regulon members, provided new insights into the biological functions of the respective regulons for these regulators, and suggested models for the evolution of the corresponding transcriptional networks. Our findings also predict that, at least for the FNR-type regulators, there is a core set of target genes conserved across many species. In addition, the members of the so-called extended regulons for the FNR-type regulators vary even among closely related species, possibly reflecting species-specific adaptation to environmental and other factors. The comparative genomics approach we developed is readily applicable to other regulatory networks

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

Construction and characterization oftrpR-based chimaeric genes and their application to the analysis of regulatory mechanisms

A plasmid has been constructed to express and secrete Trp repressor in yeast. It was demonstrated that Trp repressor is secreted into the culture media to a final concentration of approximately 1 mg/l. With modifications, this system should provide experimental material for investigating in detail which E. coli proteins interact with Trp repressor. An unusual feature of the trpR gene, a G/C-rich palindrome located in the region upstream of the trpR coding sequence, has long been the subject of speculation regarding its potential contribution to the level of expression of trpR. Using the products of in vivo and in vitro DNA manipulations, it has been determined that this palindrome does not significantly affect the level of expression; rather, it appears that the Shine-Dalgarno sequence plays a significant role in determining the intracellular levels of Trp repressor. Using a subset of the biological tools developed for the investigation of regulatory features of the trpR gene, an in vivo structural analysis of donor peptide in the $\alpha$-complementation reaction of $\beta$-galactosidase was executed. The $\alpha$-donors that were analyzed consisted of truncated derivatives of chimaeric Trp repressor\sp\prime-\sp\primeLacZ polypeptides. It was found that chimaeric $\alpha$-peptides containing greater amounts of \sp\primelacZ$\alpha$ coding sequence (residues 9-146) have more latitude with respect to the nature of the N-terminus than homologous $\alpha$-peptide chimaeras with lesser amounts of \sp\prime lacZ\alpha coding sequence (residues 9-51). It has also been possible to demonstrate, in vivo, an interaction between the chimaeric $\alpha$-donor peptides and the $\alpha$-acceptor protein that takes place independently of the formation of catalytically active $\beta$-galactosidase. A genetic system designed to obtain trpR mutants having mutations in residues critical to the formation of Trp repressor dimers was developed. This system involved, inter alia, the construction of a trpR\sp\prime-\sp\primetetA fusion and a trpR\sp\prime-\sp\primenptII fusions. The Trp repressor\sp\prime-\sp\primeTetA chimaeric protein has operator binding activity. The Trp repressor\sp\prime-\sp\primeNptII protein is less well-characterized owing to the fact that the protein undergoes post-translational processing. Investigation of the properties of these chimaeric proteins has helped define certain novel aspects of the structure-function relationships applicable to Trp repressor, for example the degree of sequence flexibility tolerated within the DNA specificity helix (Schevitz et al., 1985), that many now be investigated by genetic and biochemical approaches

Role of the fnrL Gene in Photosystem Gene Expression and Photosynthetic Growth of Rhodobacter sphaeroides 2.4.1

Anoxygenic photosynthetic growth of Rhodobacter sphaeroides 2.4.1 requires a functional fnrL gene, which encodes the anaerobic regulator, FnrL. Using transcriptional fusions to the puc operon in which the upstream FNR consensus-like sequence is either present or absent, we obtained results that suggest that FnrL has both a direct and an indirect role in puc operon expression. In addition to FnrL, several other factors, including the two-component Prr regulatory system and the transcriptional repressor PpsR, are known to mediate oxygen control of photosynthesis gene expression in this organism. Therefore, we examined the relationship between FnrL and these other regulatory elements. Our results indicate that while mutations of prr or ppsR can lead to an increase in expression of some photosynthesis genes under aerobic and anaerobic conditions, regardless of the presence or absence of FnrL, there remains an absolute requirement for a functional fnrL gene for photosynthetic growth. We examined the potential role(s) of FnrL in photosynthetic growth by considering several target genes which may be required for this growth mode