Paul E. March

Evolution of a molecular switch: universal bacterial GTPases regulate ribosome function

The GTPases comprise a protein superfamily of highly conserved molecular switches adapted to many diverse functions. These proteins are found in all domains of life and often perform essential roles in fundamental cellular processes. Analysis of data from genome sequencing projects demonstrates that bacteria possess a core of 11 universally conserved GTPases (elongation factor G and Tu, initiation factor 2, LepA, Era, Obg, ThdF/TrmE, Ffh, FtsY, EngA and YchF). Investigations aimed at understanding the function of GTPases indicate that a second conserved feature of these proteins is that they elicit their function through interaction with RNA and/or ribosomes. An emerging concept suggests that the 11 universal GTPases are either necessary for ribosome function or transmitting information from the ribosome to downstream targets for the purpose of generating specific cellular responses. Furthermore, it is suggested that progenitor GTPases were early regulators of RNA function and may have existed in precursors of cellular systems driven by catalytic RNA. If this is the case, then a corollary of this hypothesis is that GTPases that do not bind RNA arose at a later time from an RNA‐binding progenitor that lost the capability to bind RNA.

Function of the universally conserved bacterial GTPases

The GTPase superfamily of cellular regulators is well represented in bacteria. A small number are universally conserved over the entire range of bacterial species. Such a pervasive taxonomic distribution suggests that these enzymes play important roles in bacterial cellular systems. Recent advances have demonstrated that bacterial GTPases are important regulators of ribosome function, and important for the distribution of DNA to daughter cells following cell division. In addition, the atomic structure of a unique GTPase, EngA, has recently been established. Unlike any other GTPase, EngA contains tandem GTP-binding domains. This structural study suggests that the GTPase cycles of the domains are regulated differentially in a manner that remains to be elucidated.

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome.

The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.

Molecular genetics of bacterial attachment and biofouling

Microbial adhesion to animate or inert surfaces is potentially mediated by nonspecific physical or specific ligand-receptor interactions. Growth and survival of the microbial community or biofilm then depends on adaptation to a series of changing environmental milieux. Within the realm of cell-cell interaction, recent advances suggest that flagella, fimbriae and other protein receptors are essential for bacterial attachment to surfaces. There has also been profound progress in the elucidation of genes and molecules necessary for bacterial attachments to surfaces and subsequent biofilm formation.

Expression and secretion of foreign proteins in Escherichia coli.

Publisher Summary This chapter discusses the development of a set of expression and secretion vectors designed to overcome several of the problems encountered when expressing a given gene in Escherichia coli . These vectors have been designed for use at specific steps during the cloning and expression of proteins as well as for the particular needs that might come with the nature of the protein to be produced, such as the necessity to secrete products that are toxic for cell growth. The chapter discusses the different vectors that are constructed and the way they can best be utilized for cloning, expression, and secretion. The first step in achieving expression of a protein is to clone the DNA fragment in generalized expression vectors. The chapter discusses the utilization of the pIN-H and pIN-III expression vector, secretion vectors in E. coli , and pIN-III-OmpA secretion vectors. As β-lactamase is an E. coli protein normally secreted into the periplasmic space, pIN-III-OmpA was tested for the production and secretion of an extracellular protein from gram-positive bacteria.

Site-directed, Ligase-Independent Mutagenesis (SLIM) for highly efficient mutagenesis of plasmids greater than 8kb.

Modifying the Site-directed, Ligase-Independent Mutagenesis (SLIM) protocol from a single reaction mode to a two-reaction mode enables highly efficient mutagenesis of plasmid constructs that exceed 8kb. This modified approach reduces the complexity of the PCR step and is optimised for generation of heteroduplexes from long PCR products. The two-reaction mode SLIM has 92% efficiency.

The DNA sequence of the gene (rnc) encoding ribonuclease III of Escherichia coli

The DNA sequence of a 1,076 base pair BglI-BamHI fragment containing the entire rnc gene for ribonuclease III (RNase III) was determined. An open reading frame of 681 base pairs was found in this region which encodes a protein of 227 amino acid residues (calculated molecular weight = 25,218). When this open reading frame was cloned into a high expression vector, pIN-III, a protein of apparent molecular weight of 26,000 was produced upon induction of the cloned gene. This product accounted for up to 5% of the total cellular protein, and comigrated with purified RNase III. RNase III enzyme activity was induced in parallel with the production of the 26,000 molecular weight protein. A putative promoter was found 170 base pairs upstream from the initiation codon. In the long leader region a very stable stem-bulge-stem structure was found which closely resembles typical RNase III cleavage sites. This structure may be cleaved by RNase III to auto-regulate the expression of the rnc gene.

Identification and functional analysis of RNase E of Vibrio angustum S14 and two-hybrid analysis of its interaction partners

RNase E is an essential enzyme that catalyses RNA processing. Microdomains which mediate interactions between RNase E and other members of the degradosome have been defined. To further elucidate the role of these microdomains in molecular interactions, we studied RNase E from Vibrio angustum S14. Protein sequence analysis revealed that its C-terminal half is less conserved and structured than its N-terminal half. Within this structural disorder, however, exist five small regions of predicted structural propensity. Four are similar to interaction-mediating microdomains identified in other RNase E proteins; the fifth did not correspond to any known functional motif. The function of the V. angustum S14 enolase-binding microdomain was confirmed using bacterial two-hybrid analysis, demonstrating the conserved function of this microdomain for the first time in a species other than Escherichia coli. Further, PNPase in V. angustum S14 was shown to interact with the last 80 amino acids of the C-terminal region of RNase E. This raises the possibility that PNPase interacts with the small ordered region at residues 1026-1041. The role of RNase E as a hub protein and the implications of microdomain-mediated interactions in relation to specificity and function are discussed.

A biological assay for detection of heterogeneities in the surface hydrophobicity of polymer coatings exposed to the marine environment.

Minimally adhesive polymers are being developed as potential coatings for use in the marine environment. A ‘bioprobe’, the bacterium Psychrobacter sp. strain SW5, was employed to detect heterogeneities in substratum hydrophobicity at a micrometer level, rather than the millimeter level detected by traditional contact angle measurements. This novel assay was based on substratum‐induced shifts in bacterial morphology and was used to demonstrate that characteristics of these surfaces can be evaluated for maintenance of parameters such as low surface free energy as well as temporal stability when immersed in water. Immersion of developmental substrata in artificial seawater for up to 90d prior to testing with the bioprobe potentially affects the stability of the designed characteristics of the polymers. It is proposed that the shifts in cell and biofilm morphology results from changes influencing the surface hydrophobicity of the polymers. An unpredicted outcome of this testing was the detection of modifications to coatings inferred by the addition of filler particles. Exposure of coatings to the natural microbial community of seawater revealed colonization characteristics that substantiate the results obtained by using the bioindicator.

Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene.

The translocation stage of protein synthesis is a highly conserved process in all cells. Although the components necessary for translocation have been delineated, the mechanism of this activity has not been well defined. Ribosome movement on template mRNA must allow for displacement of tRNA-mRNA complexes from the ribosomal A to P sites and P to E sites, while ensuring rigid maintenance of the correct reading frame. In Escherichia coli, translocation of the ribosome is promoted by elongation factor G (EF-G). To examine the role of EF-G and rRNA in translocation we have characterized mutations in rRNA genes that can suppress a temperature-sensitive (ts) allele of fusA, the gene in E. coli that encodes EF-G. This analysis was performed using the ts E. coli strain PEM100, which contains a point mutation within fusA. The ts phenotype of PEM100 can be suppressed by either of two mutations in the decoding region of the 16S rRNA when present in combination with a mutation at position 2058 in the peptidyltransferase domain of the 23S rRNA. Communication between these ribosomal domains is essential for coordinating the events of the elongation cycle. We propose a model in which EF-G promotes translocation by modulating this communication, thereby increasing the efficiency of this fundamental process.