Joanne Hothersall

Bio: Joanne Hothersall is an academic researcher from University of Birmingham. The author has contributed to research in topics: Pseudomonas fluorescens & Mupirocin. The author has an hindex of 17, co-authored 27 publications receiving 1431 citations.

Genomic and genetic analyses of diversity and plant interactions of Pseudomonas fluorescens

Abstract: Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species. Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome. P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.

Resistance to and synthesis of the antibiotic mupirocin

Abstract: Mupirocin, a polyketide antibiotic produced by Pseudomonas fluorescens, is used to control the carriage of methicillin-resistant Staphylococcus aureus on skin and in nasal passages as well as for various skin infections. Low-level resistance to the antibiotic arises by mutation of the mupirocin target, isoleucyl-tRNA synthetase, whereas high-level resistance is due to the presence of an isoleucyl-tRNA synthetase with many similarities to eukaryotic enzymes. Mupirocin biosynthesis is carried out by a combination of type I multifunctional polyketide synthases and tailoring enzymes encoded in a 75 kb gene cluster. Chemical synthesis has also been achieved. This knowledge should allow the synthesis of new and modified antibiotics for the future.

Quorum-sensing-dependent regulation of biosynthesis of the polyketide antibiotic mupirocin in Pseudomonas fluorescens NCIMB 10586.

Abstract: Mupirocin (pseudomonic acid) is a polyketide antibiotic, targeting isoleucyl-tRNA synthase, and produced by Pseudomonas fluorescens NCIMB 10586. It is used clinically as a topical treatment for staphylococcal infections, particularly in contexts where there is a problem with methicillin-resistant Staphylococcus aureus (MRSA). In studying the mupirocin biosynthetic cluster the authors identified two putative regulatory genes, mupR and mupI, whose predicted amino acid sequences showed significant identity to proteins involved in quorum-sensing-dependent regulatory systems such as LasR/LuxR (transcriptional activators) and LasI/LuxI (synthases for N-acylhomoserine lactones – AHLs – that activate LasR/LuxR). Inactivation by deletion mutations using a suicide vector strategy confirmed the requirement for both genes in mupirocin biosynthesis. Cross-feeding experiments between bacterial strains as well as solvent extraction showed that, as predicted, wild-type P. fluorescens NCIMB 10586 produces a diffusible substance that overcomes the defect of a mupI mutant. Use of biosensor strains showed that the MupI product can activate the Pseudomonas aeruginosa lasRlasI system and that P. aeruginosa produces one or more compounds that can replace the MupI product. Insertion of a xylE reporter gene into mupA, the first ORF of the mupirocin biosynthetic operon, showed that together mupR/mupI control expression of the operon in such a way that the cluster is switched on late in exponential phase and in stationary phase.

Assembly-line enzymology for polyketide and nonribosomal Peptide antibiotics: logic, machinery, and mechanisms.

Abstract: biotics of the penicillin and cephalosporin families, 3,4 as well as the glycopeptides of the vancomycin family 5 (Figure 1a). * To whom correspondence should be addressed: christopher_walsh@ hms.harvard.edu. † Harvard Medical School. ‡ Harvard University. Christopher T. Walsh is the Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology (BCMP) at Harvard Medical School. He has had extensive experience in academic administration, including Chairmanship of the MIT Chemistry Department (1982−1987) and the HMS Biological Chemistry & Molecular Pharmacology Department (1987−1995) as well as serving as President and CEO of the Dana Farber Cancer Institute (1992−1995). His research has focused on enzymes and enzyme inhibitors, with recent specialization on antibiotics. He and his group have authored over 590 research papers, a text (Enzymatic Reaction Mechanisms), and two books (Antibiotics: Origins, Actions, Resistance and Posttranslational Modification of Proteins: Expanding Nature’s Inventory). He is a member of the National Academy of Sciences, the Institute of Medicine, and the American Philosophical Society.

The Biosynthetic Logic of Polyketide Diversity

Abstract: Molecular Lego: Polyketides represent a highly diverse group of natural products with structurally intriguing carbon skeletons (see picture) which are assembled from simple acyl building blocks. A combination of chemical, biochemical, and genetics studies have provided exciting new insights into the programming of polyketide assembly and the sophisticated enzymatic machineries involved. This review highlights recent developments in the field.Polyketides constitute one of the major classes of natural products. Many of these compounds or derivatives thereof have become important therapeutics for clinical use; in contrast, various polyketides are infamous food-spoiling toxins or virulence factors. What is particularly remarkable about this heterogeneous group of compounds comprising of polyethers, polyenes, polyphenols, macrolides, and enediynes is that they are mainly derived from one of the simplest building blocks available in nature: acetic acid. Investigations at the chemical, genetic, and biochemical levels have shed light on the biosynthetic programs that lead to the large structural diversity of polyketides .This review highlights recently unveiled biosynthetic mechanisms to generate highly diverse and complex molecules.

Regulation of antibiotic production in root-colonizing pseudomonas spp. and relevance for biological control of plant disease

Abstract: Certain strains of fluorescent pseudomonads are important biological components of agricultural soils that are suppressive to diseases caused by pathogenic fungi on crop plants. The biocontrol abilities of such strains depend essentially on aggressive root colonization, induction of systemic resistance in the plant, and the production of diffusible or volatile antifungal antibiotics. Evidence that these compounds are produced in situ is based on their chemical extraction from the rhizosphere and on the expression of antibiotic biosynthetic genes in the producer strains colonizing plant roots. Well-characterized antibiotics with biocontrol properties include phenazines, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, lipopeptides, and hydrogen cyanide. In vitro, optimal production of these compounds occurs at high cell densities and during conditions of restricted growth, involving (i) a number of transcriptional regulators, which are mostly pathway-specific, and (ii) the GacS/GacA two-component system, which globally exerts a positive effect on the production of extracellular metabolites at a posttranscriptional level. Small untranslated RNAs have important roles in the GacS/GacA signal transduction pathway. One challenge in future biocontrol research involves development of new strategies to overcome the broad toxicity and lack of antifungal specificity displayed by most biocontrol antibiotics studied so far.

Pseudomonas genomes: diverse and adaptable

Abstract: Members of the genus Pseudomonas inhabit a wide variety of environments, which is reflected in their versatile metabolic capacity and broad potential for adaptation to fluctuating environmental conditions. Here, we examine and compare the genomes of a range of Pseudomonas spp. encompassing plant, insect and human pathogens, and environmental saprophytes. In addition to a large number of allelic differences of common genes that confer regulatory and metabolic flexibility, genome analysis suggests that many other factors contribute to the diversity and adaptability of Pseudomonas spp. Horizontal gene transfer has impacted the capability of pathogenic Pseudomonas spp. in terms of disease severity (Pseudomonas aeruginosa) and specificity (Pseudomonas syringae). Genome rearrangements likely contribute to adaptation, and a considerable complement of unique genes undoubtedly contributes to strain- and species-specific activities by as yet unknown mechanisms. Because of the lack of conserved phenotypic differences, the classification of the genus has long been contentious. DNA hybridization and genome-based analyses show close relationships among members of P. aeruginosa, but that isolates within the Pseudomonas fluorescens and P. syringae species are less closely related and may constitute different species. Collectively, genome sequences of Pseudomonas spp. have provided insights into pathogenesis and the genetic basis for diversity and adaptation.

Human commensals producing a novel antibiotic impair pathogen colonization

Abstract: The nasal commensal bacterium Staphylococcus lugdunensis produces a novel cyclic peptide antibiotic, lugdunin, that inhibits colonization by S. aureus in animal models and is associated with a significantly reduced S. aureus carriage rate in humans, suggesting that human commensal bacteria could be a valuable resource for the discovery of new antibiotics. The vast majority of systemic bacterial infections are caused by facultative, often antibiotic-resistant, pathogens colonizing human body surfaces. Nasal carriage of Staphylococcus aureus predisposes to invasive infection, but the mechanisms that permit or interfere with pathogen colonization are largely unknown. Whereas soil microbes are known to compete by production of antibiotics, such processes have rarely been reported for human microbiota. We show that nasal Staphylococcus lugdunensis strains produce lugdunin, a novel thiazolidine-containing cyclic peptide antibiotic that prohibits colonization by S. aureus, and a rare example of a non-ribosomally synthesized bioactive compound from human-associated bacteria. Lugdunin is bactericidal against major pathogens, effective in animal models, and not prone to causing development of resistance in S. aureus. Notably, human nasal colonization by S. lugdunensis was associated with a significantly reduced S. aureus carriage rate, suggesting that lugdunin or lugdunin-producing commensal bacteria could be valuable for preventing staphylococcal infections. Moreover, human microbiota should be considered as a source for new antibiotics. The majority of systemic bacterial infections are caused by endogenous pathogens from human microbiota, and the opportunistic pathogen Staphylococcus aureus, commonly found in the external opening of the nostrils, is one of the most clinically important because of the prevalence of multi-drug resistant strains. The mechanisms that permit or interfere with pathogen colonization have remained unclear. This study shows that S. lugdunensis, a commensal bacterium that shares the nasal niche with S. aureus and is associated with a reduced S. aureus carriage rate in humans, produces a novel cyclic peptide antibiotic (lugdunin) that inhibits colonization by S. aureus in animal models. Lugdunin is bactericidal against major pathogens and not prone to causing development of resistance in S. aureus, suggesting that lugdunin or lugdunin-producing commensals could be valuable for preventing staphylococcal infections.