John P. Morrissey

The 5' end of yeast 5.8S rRNA is generated by exonucleases from an upstream cleavage site.

We have developed techniques for the detailed analysis of cis‐acting sequences in the pre‐rRNA of Saccharomyces cerevisiae and used these to study the processing of internal transcribed spacer 1 (ITS1) leading to the synthesis of 5.8S rRNA. As is the case for many eukaryotes, the 5′ end of yeast 5.8S rRNA is heterogeneous; we designate the major, short form 5.8S(S), and the minor form (which is seven or eight nucleotides longer) 5.8S(L). These RNAs do not have a precursor/product relationship, but result from the use of alternative processing pathways. In the major pathway, a previously unidentified processing site in ITS1, designated A3, is cleaved. A 10 nucleotide deletion at site A3 strongly inhibits processing of A3 and the synthesis of 5.8S(S); processing is predominantly transferred to the alternative 5.8S(L) pathway. Site A3 lies 76 nucleotides 5′ to the end of 5.8S(S), and acts as an entry site for 5′‐‐>3′ exonuclease digestion which generates the 5′ end of 5.8S(S). This pathway is inhibited in strains mutant for XRN1p and RAT1p. Both of these proteins have been reported to have 5′‐‐>3′ exonuclease activity in vitro. Formation of 5.8S(L) is increased by mutations at A3 in cis or in RAT1p and XRN1p in trans, and is kinetically faster than 5.8S(S) synthesis.

Pseudomonas for biocontrol of phytopathogens: from functional genomics to commercial exploitation.

Pseudomonas spp. that can colonise the roots of crop plants and produce antifungal metabolites represent a real alternative to the application of chemical fungicides. Presently, much research is aimed at understanding, at the molecular level, the mechanisms that enable Pseudomonas strains to act as efficient biological control agents. This approach is facilitating the development of novel strains with modified traits for enhanced biocontrol efficacy. However, without solving some inherent problems associated with the effective delivery of microbial inoculants to seeds and without knowledge on the biosafety aspects of novel biocontrol agents, the commercial potential of Pseudomonas spp. for plant disease control will not be realised.

Marine Metagenomics: New Tools for the Study and Exploitation of Marine Microbial Metabolism

The marine environment is extremely diverse, with huge variations in pressure and temperature. Nevertheless, life, especially microbial life, thrives throughout the marine biosphere and microbes have adapted to all the divergent environments present. Large scale DNA sequence based approaches have recently been used to investigate the marine environment and these studies have revealed that the oceans harbor unprecedented microbial diversity. Novel gene families with representatives only within such metagenomic datasets represent a large proportion of the ocean metagenome. The presence of so many new gene families from these uncultured and highly diverse microbial populations represents a challenge for the understanding of and exploitation of the biology and biochemistry of the ocean environment. The application of new metagenomic and single cell genomics tools offers new ways to explore the complete metabolic diversity of the marine biome.

Are microbes at the root of a solution to world food production

Rational exploitation of interactions between microbes and plants can help to transform agriculture

Signal-mediated interactions between Pseudomonas aeruginosa and Candida albicans

Pseudomonas aeruginosa causes infections in a wide variety of hosts and is the leading cause of mortality in cystic fibrosis (CF) patients. Although most clinical isolates of P. aeruginosa share common virulence determinants, it is known that strains evolve and change phenotypically during CF lung infections. These changes can include alterations in the levels of N-acyl homoserine lactones (HSLs), which are secreted signal molecules. In the CF lung, fungi, especially Candida albicans and Aspergillus fumigatus, may coexist with P. aeruginosa but the implications for disease are not known. Recent studies have established that signalling can occur between P. aeruginosa and C. albicans, with the bacterial molecule 3-oxo-C12HSL affecting Candida morphology, and the fungal metabolite farnesol reducing levels of the Pseudomonas quinolone signal and pyocyanin in Pseudomonas. Whether these interactions are common and typical in clinical strains of P. aeruginosa was addressed using CF isolates that produced varied levels of HSLs. It was found that, whereas some clinical P. aeruginosa strains affected C. albicans morphology, others did not. This correlated closely with the amounts of 3-oxo-C12HSL produced by the isolates. Furthermore, it was established that signalling is bidirectional and that the C. albicans molecule farnesol inhibits swarming motility in P. aeruginosa CF strains. This work demonstrates that clinical isolates of these opportunistic pathogens can interact in strain-specific ways via secreted signals and illustrates the importance of studying these interactions to fully understand the microbial contribution to disease in polymicrobial infections.

Birth of the snoRNPs: the evolution of RNase MRP and the eukaryotic pre-rRNA-processing system

The ribonucleoprotein particle RNase MRP is required for the processing of yeast pre-ribosomal RNA (pre-rRNA). A structurally related particle, RNase P, is universally required for processing of pre-tRNA, but in bacteria and archaea also cleaves a site in the pre-rRNA. This suggests that RNase MRP may have arisen in eukaryotes as a form of RNase P specialized for pre-rRNA processing. Other eukaryotic small nucleolar RNAs may have arisen as trans-acting factors that functionally replace cis-acting pre-rRNA interactions in bacteria and archaea.

Genome Diversity of Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients and the Hospital Environment

ABSTRACT Pseudomonas aeruginosa is a gram-negative rod that is ubiquitous in nature. P. aeruginosa is also the quintessential opportunistic pathogen, causing a wide variety of infections in compromised hosts. In cystic fibrosis patients, P. aeruginosa is the leading cause of death. In this study, the evolutionary genetic relationships among 17 P. aeruginosa isolates were examined by comparative sequence analysis of the housekeeping gene encoding malate dehydrogenase and the chaperone groEL. The P. aeruginosa isolates examined included the sequenced strain PAO1, 11 strains recovered from cystic fibrosis patients in Ireland, 4 environmental isolates recovered from a hospital environment, and 1 isolate recovered from a plant rhizosphere. Phylogenetically, clinical and environmental isolates clustered together with one another on the mdh gene tree. At the groEL locus, among the 17 isolates examined, only two polymorphic sites were observed, highlighting the close genetic relationship between isolates from these different environments. Phenotypic analysis of 12 traits among our isolates, however, found that only clinical isolates produced phenazines and elastase. Furthermore, molecular analysis of the distribution of 15 regions associated with virulence showed that two of the environmental isolates examined lacked the majority of regions. Among the clinical isolates examined, the 15 virulence regions were variably present. The distribution of two prophages (Bacto1, Pf1) was also determined, with most isolates encoding both these regions. Of the four genomic islands (the flagellum island and PAGI-1, -2, and -3) examined, only two isolates contained the flagellum island, and PAGI-1, -2, and -3 were absent from all isolates tested. Our data demonstrate the significant role horizontal gene transfer and recombination, together with gene loss, play in the evolution of this important human pathogen.

Functional genomics analysis of plant growth-promoting rhizobacterial traits involved in rhizosphere competence

In soil, some specific bacterial populations, called plant growth-promoting rhizobacteria are able to promote plant growth and/or reduce the incidence of soil-borne diseases. Rhizosphere competence is an important prerequisite for the efficacy of these biocontrol strains. Therefore, over decades, multiple approaches have been combined to understand the molecular basis of bacterial traits involved in rhizosphere competence. This review addresses the bacterial genes expressed during bacterial–plant interactions in the rhizosphere of different plant species. The distribution of these key genes in natural populations of rhizobacteria is also discussed.

Dual Effects of Plant Steroidal Alkaloids on Saccharomyces cerevisiae

ABSTRACT Many plant species accumulate sterols and triterpenes as antimicrobial glycosides. These secondary metabolites (saponins) provide built-in chemical protection against pest and pathogen attack and can also influence induced defense responses. In addition, they have a variety of important pharmacological properties, including anticancer activity. The biological mechanisms underpinning the varied and diverse effects of saponins on microbes, plants, and animals are only poorly understood despite the ecological and pharmaceutical importance of this major class of plant secondary metabolites. Here we have exploited budding yeast (Saccharomyces cerevisiae) to investigate the effects of saponins on eukaryotic cells. The tomato steroidal glycoalkaloidα -tomatine has antifungal activity towards yeast, and this activity is associated with membrane permeabilization. Removal of a single sugar from the tetrasaccharide chain of α-tomatine results in a substantial reduction in antimicrobial activity. Surprisingly, the complete loss of sugars leads to enhanced antifungal activity. Experiments with α-tomatine and its aglycone tomatidine indicate that the mode of action of tomatidine towards yeast is distinct from that of α-tomatine and does not involve membrane permeabilization. Investigation of the effects of tomatidine on yeast by gene expression and sterol analysis indicate that tomatidine inhibits ergosterol biosynthesis. Tomatidine-treated cells accumulate zymosterol rather than ergosterol, which is consistent with inhibition of the sterol C24 methyltransferase Erg6p. However, erg6 and erg3 mutants (but not erg2 mutants) have enhanced resistance to tomatidine, suggesting a complex interaction of erg mutations, sterol content, and tomatidine resistance.

Functional metagenomic strategies for the discovery of novel enzymes and biosurfactants with biotechnological applications from marine ecosystems.

Marine ecosystems are home to bacteria which are exposed to a wide variety of environmental conditions, such as extremes in temperature, salinity, nutrient availability and pressure. Survival under these conditions must have necessitated the adaptation and the development of unique cellular biochemistry and metabolism by these microbes. Thus, enzymes isolated from these microbes have the potential to possess quite unique physiological and biochemical properties. This review outlines a number of function‐based metagenomic approaches which are available to screen metagenomic libraries constructed from marine ecosystems to facilitate the exploitation of some of these potentially novel biocatalysts. Functional screens to isolate novel cellulases, lipases and esterases, proteases, laccases, oxidoreductases and biosurfactants are described, together with approaches which can be employed to help overcome some of the typical problems encountered with functional metagenomic‐based screens.