RESUMO
Competitive behaviours of plant growth promoting rhizobacteria (PGPR) are integral to their ability to colonize and persist on plant roots and outcompete phytopathogenic fungi, oomycetes and bacteria. PGPR engage in a range of antagonistic behaviours that have been studied in detail, such as the production and secretion of compounds inhibitory to other microbes. In contrast, their defensive activities that enable them to tolerate exposure to inhibitory compounds produced by their neighbours are less well understood. In this study, the genes involved in the Pseudomonas protegens Pf-5 response to metabolites from eight diverse rhizosphere competitor organisms, Fusarium oxysporum, Rhizoctonia solani, Gaeumannomyces graminis var. tritici, Pythium spinosum, Bacillus subtilis QST713, Pseudomonas sp. Q2-87, Streptomyces griseus and Streptomyces bikiniensis subspecies bikiniensi, were examined. Proximity induced excreted metabolite responses were confirmed for Pf-5 with all partner organisms through HPLC before culturing a dense Pf-5 transposon mutant library adjacent to each of these microbes. This was followed by transposon-directed insertion site sequencing (TraDIS), which identified genes that influence Pf-5 fitness during these competitive interactions. A set of 148 genes was identified that were associated with increased fitness during competition, including cell surface modification, electron transport, nucleotide metabolism, as well as regulatory genes. In addition, 51 genes were identified for which loss of function resulted in fitness gains during competition. These included genes involved in flagella biosynthesis and cell division. Considerable overlap was observed in the set of genes observed to provide a fitness benefit during competition with all eight test organisms, indicating commonalities in the competitive response to phylogenetically diverse micro-organisms and providing new insight into competitive processes likely to take place in the rhizosphere.
Assuntos
Oomicetos , Rizosfera , Bacillus subtilis , Pseudomonas/genéticaRESUMO
Gene essentiality studies have been performed on numerous bacterial pathogens, but essential gene sets have been determined for only a few plant-associated bacteria. Pseudomonas protegens Pf-5 is a plant-commensal, biocontrol bacterium that can control disease-causing pathogens on a wide range of crops. Work on Pf-5 has mostly focused on secondary metabolism and biocontrol genes, but genome-wide approaches such as high-throughput transposon mutagenesis have not yet been used for this species. In this study, we generated a dense P. protegens Pf-5 transposon mutant library and used transposon-directed insertion site sequencing (TraDIS) to identify 446 genes essential for growth on rich media. Genes required for fundamental cellular machinery were enriched in the essential gene set, while genes related to nutrient biosynthesis, stress responses, and transport were underrepresented. The majority of Pf-5 essential genes were part of the P. protegens core genome. Comparison of the essential gene set of Pf-5 with those of two plant-associated pseudomonads, P. simiae and P. syringae, and the well-studied opportunistic human pathogen P. aeruginosa PA14 showed that the four species share a large number of essential genes, but each species also had uniquely essential genes. Comparison of the Pf-5 in silico-predicted and in vitro-determined essential gene sets highlighted the essential cellular functions that are over- and underestimated by each method. Expanding essentiality studies into bacteria with a range of lifestyles may improve our understanding of the biological processes important for bacterial survival and growth.IMPORTANCE Essential genes are those crucial for survival or normal growth rates in an organism. Essential gene sets have been identified in numerous bacterial pathogens but only a few plant-associated bacteria. Employing genome-wide approaches, such as transposon insertion sequencing, allows for the concurrent analyses of all genes of a bacterial species and rapid determination of essential gene sets. We have used transposon insertion sequencing to systematically analyze thousands of Pseudomonas protegens Pf-5 genes and gain insights into gene functions and interactions that are not readily available using traditional methods. Comparing Pf-5 essential genes with those of three other pseudomonads highlights how gene essentiality varies between closely related species.
Assuntos
Proteínas de Bactérias/genética , Genes Essenciais , Pseudomonas/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis , Biblioteca Gênica , Genoma Bacteriano , Mutagênese Insercional , Plantas/microbiologia , Pseudomonas/metabolismoRESUMO
Class 1 integrons have played a major role in the global dissemination of antibiotic resistance. Reconstructing the history of class 1 integrons might help us control further spread of antibiotic resistance by understanding how human activities influence microbial evolution. Here we describe a class 1 integron that represents an intermediate stage in the evolutionary history of clinical integrons. It was embedded in a series of nested transposons, carried on an IncP plasmid resident in Enterobacter, isolated from the surface of baby spinach leaves. Based on the structure of this integron, we present a modified hypothesis for integron assembly, where the ancestral clinical class 1 integron was captured from a betaproteobacterial chromosome to form a Tn402-like transposon. This transposon then inserted into a plasmid-borne Tn21-like ancestor while in an environmental setting, possibly a bacterium resident in the phyllosphere. We suggest that the qacE gene cassette, conferring resistance to biocides, together with the mercury resistance operon carried by Tn21, provided a selective advantage when this bacterium made its way into the human commensal flora via food. The integron characterized here was located in Tn6007, which along with Tn6008, forms part of the larger Tn6006 transposon, itself inserted into another transposable element to form the Tn21-like transposon, Tn6005. This element has previously been described from the human microbiota, but with a promoter mutation that upregulates integron cassette expression. This element we describe here is from an environmental bacterium, and supports the hypothesis that the ancestral class 1 integron migrated into anthropogenic settings via foodstuffs. Selection pressures brought about by early antimicrobial agents, including mercury, arsenic and disinfectants, promoted its initial fixation, the acquisition of promoter mutations, and subsequent dissemination into various species and pathogens.
Assuntos
Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana/genética , Doenças Transmitidas por Alimentos/genética , Integrons/genética , Enterobacter/efeitos dos fármacos , Enterobacter/genética , Enterobacter/patogenicidade , Evolução Molecular , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Mutação , Plasmídeos/genética , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
Giardiasis is a communicable gastrointestinal disease caused by Giardia duodenalis and two genetic assemblages, A and B, cause human infection. In remote Indigenous communities of Australia, giardiasis is highly prevalent among children but disease transmission is poorly understood. This study investigated the prevalence of Giardia and genetic subtypes contributing to human disease in a remote Indigenous community, in the Northern Territory of Australia. Eighty-seven faecal samples were collected from 74 children (<15 years) over an 18 month period, and the distribution of positive cases relative to participant age and gender were examined. Screening by microscopy and 18S rRNA PCR amplification showed 66.7% (58/87) of faecal samples were positive for Giardia. Both males and females were equally affected and high detection rates were obtained for participants aged 0-<5 years and 5-<10 years (66.0 and 60.0% respectively). For 58.6% of the positive samples, Giardia was only detected by 18S rRNA PCR. Approximately 75% of cases were assemblage B, and subassemblage analyses using terminal restriction fragment length polymorphism of the glutamate dehydrogenase gene demonstrated that a variety of genetic variants were present. The high proportion of positive cases that were not detectable by microscopy, and dominance of assemblage B cases highlights the need for further research in this community, to assess the contribution of Giardia to chronic gastrointestinal disease among children, and to understand conditions conductive to assemblage B transmission.
Assuntos
Giardia lamblia/classificação , Giardia lamblia/genética , Giardíase/epidemiologia , Giardíase/parasitologia , Adolescente , Austrália/etnologia , Criança , Pré-Escolar , DNA de Protozoário/análise , Fezes/parasitologia , Feminino , Variação Genética , Giardia lamblia/isolamento & purificação , Humanos , Lactente , Masculino , RNA Ribossômico 18S/análiseRESUMO
Giardia and Cryptosporidium are amongst the most common protozoan parasites identified as causing enteric disease in pinnipeds. A number of Giardia assemblages and Cryptosporidium species and genotypes are common in humans and terrestrial mammals and have also been identified in marine mammals. To investigate the occurrence of these parasites in an endangered marine mammal, the Australian sea lion (Neophoca cinerea), genomic DNA was extracted from faecal samples collected from wild populations (n = 271) in Southern and Western Australia and three Australian captive populations (n = 19). These were screened using PCR targeting the 18S rRNA of Giardia and Cryptosporidium. Giardia duodenalis was detected in 28 wild sea lions and in seven captive individuals. Successful sequencing of the 18S rRNA gene assigned 27 Giardia isolates to assemblage B and one to assemblage A, both assemblages commonly found in humans. Subsequent screening at the gdh and ß-giardin loci resulted in amplification of only one of the 35 18S rRNA positive samples at the ß-giardin locus. Sequencing at the ß-giardin locus assigned the assemblage B 18S rRNA confirmed isolate to assemblage AI. The geographic distribution of sea lion populations sampled in relation to human settlements indicated that Giardia presence in sea lions was highest in populations less than 25 km from humans. Cryptosporidium was not detected by PCR screening in either wild colonies or captive sea lion populations. These data suggest that the presence of G. duodenalis in the endangered Australian sea lion is likely the result of dispersal from human sources. Multilocus molecular analyses are essential for the determination of G. duodenalis assemblages and subsequent inferences on transmission routes to endangered marine mammal populations.
RESUMO
Giardia intestinalis is a protozoan parasite and a human pathogen. It is a leading cause of human diarrheal disease and a significant cause of morbidity worldwide. At the molecular level, G. intestinalis is a species complex, consisting of genetic assemblages (A to G) and sub-assemblage strains. The genotypes that cause human disease have been characterised to assemblages A and B, and include strains AI, AII, BIII and BIV. PCR amplification of diagnostic loci is used to genotype samples and is required to understand different transmission cycles within communities. A multi-locus approach is required for validation of Giardia genotyping and molecular diagnostic techniques that are efficient across numerous loci have not been established. This study evaluated several published protocols for the 18S small subunit ribosomal RNA (18S rRNA) and glutamate dehydrogenase genes (gdh) genes. Assays were compared using spiked faecal samples and by measuring the concentration of DNA generated following DNA extraction and PCR amplification. An optimal molecular method for G. intestinalis identification was established from direct DNA extraction of faecal material and GC-rich PCR chemistry. The protocol was applied to 50 clinical samples and produced PCR success rates of 90% and 94% at the 18S rRNA and gdh loci. Cyst concentration prior to DNA extraction was not necessary, and the optimal protocol was highly sensitive and an efficient method for testing clinical samples.