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

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: regulation through alterations in the cellular redox state.

Rhodobacter sphaeroides 2.4.1 has the ability to synthesize a variety of tetrapyrroles, reflecting the metabolic versatility of this organism and making it capable of aerobic, anaerobic, photosynthetic, and diazotrophic growth. The hemA and hemT genes encode isozymes that catalyze the formation of 5-aminolevulinic acid, the first step in the biosynthesis of all tetrapyrroles present in R. sphaeroides 2.4.1. As part of our studies of the regulation and expression of these genes, we developed a genetic selection that uses transposon mutagenesis to identify loci affecting the aerobic expression of the hemA gene. In developing this selection, we found that sequences constituting an open reading frame immediately upstream of hemA positively affect hemA transcription. Using a transposon-based selection for increased hemA expression in the absence of the upstream open reading frame, we isolated three independent mutants. We have determined that the transposon insertions in these strains map to three different loci located on chromosome 1. One of the transposition sites mapped in the vicinity of the recently identified R. sphaeroides 2.4.1 homolog of the anaerobic regulatory gene fnr. By marker rescue and DNA sequence analysis, we found that the transposition site was located between the first two genes of the cco operon in R. sphaeroides 2.4.1, which encodes a cytochrome c terminal oxidase. Examination of the phenotype of the mutant strain revealed that, in addition to increased aerobic expression of hemA, the transposition event also conferred an oxygen-insensitive development of the photosynthetic membranes. We propose that the insertion of the transposon in cells grown in the presence of high oxygen levels has led to the generation of a cellular redox state resembling either reduced oxygen or anaerobiosis, thereby resulting in increased expression of hemA, as well as the accumulation of spectral complex formation. Several models are presented to explain these findings

Two classes of extragenic suppressor mutations identify functionally distinct regions of the GroEL chaperone of Escherichia coli.

The GroES and GroEL proteins of Escherichia coli function together as the GroE molecular chaperone machine to (i) prevent denaturation and aggregation and (ii) assist the folding and oligomerization of other proteins without being part of the final structure. Previous genetic and biochemical analyses have determined that this activity requires interactions of the GroES 7-mer with the GroEL 14-mer. Recently, we have identified a region of the GroES protein that interacts with the GroEL protein. To identify those residues of the GroEL protein that interact with GroES, we have exploited the thermosensitive phenotype of strains bearing mutations at one or the other of two GroEL-interacting residues of GroES. We have isolated, cloned, and sequenced six suppressor mutations in groEL, three independent isolates for each groES mutant. Changes of only three different amino acid substitutions in GroEL protein were found among these six groEL suppressor mutations. On the basis of a number of in vivo analyses of the chaperone activity of various combinations of groES mutant alleles and groEL suppressor alleles, we propose that an amino-proximal region of the GroEL protein which includes amino acid residues 174 and 190 interacts with GroES and that a carboxyl-proximal region which includes residue 375 interacts with substrate proteins

Sequence analysis and phenotypic characterization of groEL mutations that block lambda and T4 bacteriophage growth.

The groES and groEL genes of Escherichia coli have been shown previously to belong to a single operon under heat shock regulation. Both proteins have been universally conserved in nature, as judged by the presence of similar proteins throughout evolution. The GroEL protein has been shown to bind promiscuously to many unfolded proteins, thus preventing their aggregation. ATP hydrolysis by GroEL results in the release of the bound polypeptides, a process that often requires the action of GroES. In an effort to understand GroEL and GroES structure and function, we have determined the nucleotide changes of nine mutant alleles of groEL. All of these mutant alleles were isolated because they block bacteriophage lambda growth. Our sequencing results demonstrate that (i) many of these alleles are identical, in spite of the fact that they were independently isolated, and (ii) most of the different alleles are clustered in the same region of the gene. One of the mutant alleles was shown to possess two nucleotide alterations in the groEL coding phase, one of which is located in a putative ATP-binding domain. The two nucleotide changes were separated by genetic engineering, and each individual change was shown to exert an effect on bacteriophage growth. But, using genetic analyses, we demonstrate that the restriction on bacterial growth at elevated temperatures is conferred only by the mutation within the putative ATP-binding domain. We have cloned the mutant alleles on multicopy plasmids and overexpressed their products. By testing for the ability of bacteriophage either to propagate or to form colonies at 43 degrees C, we have been able to divide the mutant proteins into those with no activity and those with residual activity under the various conditions tested

In Vitro and In Vivo Analysis of the Role of PrrA in Rhodobacter sphaeroides 2.4.1 hemA Gene Expression

The hemA gene codes for one of two synthases in Rhodobacter sphaeroides 2.4.1 which catalyze the formation of 5-aminolevulinic acid. We have examined the role of PrrA, a DNA binding protein that is associated with the metabolic switch between aerobic growth and anoxygenic photosynthetic growth, in hemA expression and found that hemA transcription is directly activated by PrrA. Using electrophoretic mobility shift assays and DNase I protection assays, we have mapped two binding sites for PrrA within the hemA upstream sequences, each of which contains an identical 9-bp motif. Using lacZ transcription reporter plasmids in wild-type strain 2.4.1 and PrrA(−) mutant strain PRRA2, we showed that PrrA was required for maximal expression. We also found that the relative impacts of altering DNA sequences within the two binding sites are different depending on whether cells are growing aerobically or anaerobically. This reveals a greater level of complexity associated with PrrA-mediated regulation of transcription than has been heretofore described. Our findings are of particular importance with respect to those genes regulated by PrrA having more than one upstream binding site. In the case of the hemA gene, we discuss possibilities as to how these new insights can be accommodated within the context of what has already been established for hemA transcription regulation in R. sphaeroides