Genomic islands in Photorhabdus

Genomic islands are responsible for unique aspects of bacterial behavior such as symbiosis and pathogenicity. Photorhabdus luminescens is a pathogen of insects that spends part of its lifecycle in symbiosis with a nematode. Here, we describe novel genomic islands from Photorhabdus that are involved in symbiosis and pathogenicity, and discuss the inter-relationship between virulence factors used against invertebrates and vertebrates.     

Insect pathogenicity islands in the insect pathogenic bacterium Photorhabdus

Abstract. Genomic islands are regions of the bacterial genome responsible for unique aspects of bacterial behaviour, such as host symbiosis and pathogenicity. Where such regions are involved in pathogenesis, they are termed pathogenicity islands (PAIs). Photorhabdus luminescens is an insect pathogen that spends part of its life in symbiosis with a nematode and part of its life as an insect pathogen. Here, several novel PAIs from P. luminescens ssp. akhurstii strain W14 are described that encode factors involved apparently in both nematode symbiosis and insect pathogenicity. The structures of these islands are compared with those found in mammalian pathogens, and the potential cross‐talk between virulence factors used against invertebrates and those used against vertebrates is discussed.     

PROGRESSIVE LOSS OF ANT-RELATED TRAITS OF CECROPIA PELTATA ON SELECTED CARIBBEAN ISLANDS

Cecropia of South and Central America, and Trinidad and Tobago, maintain a symbiotic relationship with Azteca ants. The plant provides a domicile and food supply and in return the ants defend the plant from insect attack and/or vine overgrowth. Cecropia is present on certain Caribbean islands, north of Trinidad and Tobago, but does not maintain a relationship with ants. The Cecropia on Puerto Rico have lost all structures normally associated with the production of the ant food. On the islands between Trinidad and Puerto Rico, stages in the loss of traits related to the ant symbiosis are present.     

Photorhabdus: towards a functional genomic analysis of a symbiont and pathogen

Pathogenicity and symbiosis are central to bacteria-host interactions. Although several human pathogens have been subjected to functional genomic analysis, we still understand little about bacteria-invertebrate interactions despite their ecological prevalence. Advances in our knowledge of this area are often hindered by the difficulty of isolating and working with invertebrate pathogenic bacteria and their hosts. Here we review studies on pathogenicity and symbiosis in an insect pathogenic bacterium Photorhabdus and its entomopathogenic nematode vector and model insect hosts. Whilst switching between these hosts, Photorhabdus changes from a state of symbiosis with its nematode vector to one of pathogenicity towards its new insect host and both the bacteria and the nematode then cooperatively exploit the dying insect. We examine candidate genes involved in symbiosis and pathogenicity, their secretion and expression patterns in culture and in the host, and begin to dissect the extent of their genetic coregulation. We describe the presence of several large genomic islands, putatively involved in pathogenicity or symbiosis, within the otherwise Yersinia-like backbone of the Photorhabdus genome. Finally, we examine the emerging comparative genomics of the Photorhabdus group and begin to describe the interrelationship between anti-invertebrate virulence factors and those used against vertebrates.     

Virulotyping of <Emphasis Type="Italic">Salmonella enterica</Emphasis> subsp. <Emphasis Type="Italic">enterica</Emphasis> isolated from indigenous vegetables and poultry meat in Malaysia using multiplex-PCR

The increased occurrence of Salmonella occurrence in local indigenous vegetables and poultry meat can be a potential health hazards. This study is aimed to detect the prevalence of twenty different virulence factors among Salmonella enterica strains isolated from poultry and local indigenous vegetables in Malaysia via an optimized, rapid and specific multiplex PCR assay. The assay encompasses a total of 19 Salmonella pathogenicity islands genes and a quorum sensing gene (sdiA) in three multiplex reaction sets. A total of 114 Salmonella enterica isolates belonging to 38 different serovars were tested. Each isolate in under this study was found to possess up to 70% of the virulence genes tested and exhibited variable pathogenicity gene patterns. Reproducibility of the multiplex PCR assay was found to be 100% and the detection limit of the optimized multiplex PCR was tested with lowest detectable concentration of DNA 0.8 pg μl⁻¹. This study demonstrated various Salmonella pathogenicity island virulence gene patterns even within the same serovar. This sets of multiplex PCR system provide a fast and reliable typing approach based on Salmonella pathogenicity islands, thus enabling an effective monitoring of emerging pathogenic Salmonella strains as an additional tool in Salmonella surveillance studies.     

Experimental Evolution of a Plant Pathogen into a Legume Symbiont

Rhizobia are phylogenetically disparate α- and β-proteobacteria that have achieved the environmentally essential function of fixing atmospheric nitrogen in symbiosis with legumes. Ample evidence indicates that horizontal transfer of symbiotic plasmids/islands has played a crucial role in rhizobia evolution. However, adaptive mechanisms that allow the recipient genomes to express symbiotic traits are unknown. Here, we report on the experimental evolution of a pathogenic Ralstonia solanacearum chimera carrying the symbiotic plasmid of the rhizobium Cupriavidus taiwanensis into Mimosa nodulating and infecting symbionts. Two types of adaptive mutations in the hrpG-controlled virulence pathway of R. solanacearum were identified that are crucial for the transition from pathogenicity towards mutualism. Inactivation of the hrcV structural gene of the type III secretion system allowed nodulation and early infection to take place, whereas inactivation of the master virulence regulator hrpG allowed intracellular infection of nodule cells. Our findings predict that natural selection of adaptive changes in the legume environment following horizontal transfer has been a major driving force in rhizobia evolution and diversification and show the potential of experimental evolution to decipher the mechanisms leading to symbiosis.     

Bacterial Genomic Islands: Organization,Function, and Evolutionary Role

Data on the structural organization and evolutionary role of specific bacterial DNA regions known as genomic islands are reviewed. Emphasis is placed on the most extensively studied genomic islands, pathogenicity islands (PAIs), which are present in the chromosome of Gram-negative and Gram-positive pathogenic bacteria and absent from related nonpathogenic strains. PAIs are long DNA regions that harbor virulence genes and often differ in GC content from the remainder of the bacterial genome. Many PAI occur in the tRNA gene loci, which provide a convenient target for foreign gene insertion. Some PAI are highly homologous to each other and contain sequences similar to ISs, phage att sites, and plasmid ori sites, along with functional or defective integrase and transposase genes, suggesting horizontal transfer of PAI among bacteria.     

Genetic and phenotypic diversity in <Emphasis Type="Italic">Burkholderia</Emphasis>: contributions by prophage and phage-like elements

Background  

Burkholderia species exhibit enormous phenotypic diversity, ranging from the nonpathogenic, soil- and water-inhabiting Burkholderia thailandensis to the virulent, host-adapted mammalian pathogen B. mallei. Genomic diversity is evident within Burkholderia species as well. Individual isolates of Burkholderia pseudomallei and B. thailandensis, for example, carry a variety of strain-specific genomic islands (GIs), including putative pathogenicity and metabolic islands, prophage-like islands, and prophages. These GIs may provide some strains with a competitive advantage in the environment and/or in the host relative to other strains.     

Mosaic Structure of Pathogenicity Islands in <Emphasis Type="Italic">Legionella pneumophila</Emphasis>

A gene complex, dot/icm, located in two independent chromosomal loci of L. pneumophila, the causative agent of Legionnaires' disease, is related to virulence. To investigate the evolutionary pattern of these pathogenicity islands of L. pneumophila, portions of four genes in the dot/icm complex, namely, dotA, dotB, icmB, and icmT, were amplified, sequenced, and phylogenetically analyzed, in addition to rpoB, which encodes an RNA polymerase -subunit. The nucleotide sequences and phylogenetic analyses of these five genes of 96 L. pneumophila strains revealed that several subgroups of L. pneumophila proliferated clonally. However, incongruent gene tree topologies and the results of statistical testing (Templeton Willcoxon signed-ranked and incongruence length differences tests) indicated that the evolutionary histories of these genes within the pathogenicity islands are not uniform, and that they constitute a mosaic structure. In addition, the non-uniform grouping of some reference strains suggests that intraspecific recombination might be still occurring in nature or in the laboratory.     

Legume growth-promoting rhizobia: An overview on the Mesorhizobium genus

The need for sustainable agricultural practices is revitalizing the interest in biological nitrogen fixation and rhizobia-legumes symbioses, particularly those involving economically important legume crops in terms of food and forage. The genus Mesorhizobium includes species with high geographical dispersion and able to nodulate a wide variety of legumes, including important crop species, like chickpea or biserrula. Some cases of legume-mesorhizobia inoculant introduction represent exceptional opportunities to study the rhizobia genomes evolution and the evolutionary relationships among species. Complete genome sequences revealed that mesorhizobia typically harbour chromosomal symbiosis islands. The phylogenies of symbiosis genes, such as nodC, are not congruent with the phylogenies based on core genes, reflecting rhizobial host range, rather than species affiliation. This agrees with studies showing that Mesorhizobium species are able to exchange symbiosis genes through lateral transfer of chromosomal symbiosis islands, thus acquiring the ability to nodulate new hosts. Phylogenetic analyses of the Mesorhizobium genus based on core and accessory genes reveal complex evolutionary relationships and a high genomic plasticity, rendering the Mesorhizobium genus as a good model to investigate rhizobia genome evolution and adaptation to different host plants. Further investigation of symbiosis genes as well as stress response genes will certainly contribute to understand mesorhizobia-legume symbiosis and to develop more effective mesorhizobia inoculants.     

Functional analysis of pyochelin-/enantiopyochelin-related genes from a pathogenicity island of Pseudomonas aeruginosa strain PA14

Genomic islands are foreign DNA blocks inserted in so-called regions of genomic plasticity (RGP). Depending on their gene content, they are classified as pathogenicity, symbiosis, metabolic, fitness or resistance islands, although a detailed functional analysis is often lacking. Here we focused on a 34-kb pathogenicity island of Pseudomonas aeruginosa PA14 (PA14GI-6), which is inserted at RGP5 and carries genes related to those for pyochelin/enantiopyochelin biosynthesis. These enantiomeric siderophores of P. aeruginosa and certain strains of Pseudomonas protegens are assembled by a thiotemplate mechanism from salicylate and two molecules of cysteine. The biochemical function of several proteins encoded by PA14GI-6 was investigated by a series of complementation analyses using mutants affected in potential homologs. We found that PA14_54940 codes for a bifunctional salicylate synthase/salicyl-AMP ligase (for generation and activation of salicylate), that PA14_54930 specifies a dihydroaeruginoic acid (Dha) synthetase (for coupling salicylate with a cysteine-derived thiazoline ring), that PA14_54910 produces a type II thioesterase (for quality control), and that PA14_54880 encodes a serine O-acetyltransferase (for increased cysteine availability). The structure of the PA14GI-6-specified metabolite was determined by mass spectrometry, thin-layer chromatography, and HPLC as (R)-Dha, an iron chelator with antibacterial, antifungal and antitumor activity. The conservation of this genomic island in many clinical and environmental P. aeruginosa isolates of different geographical origin suggests that the ability for Dha production may confer a selective advantage to its host.     

LEEways: tales of EPEC,ATEC and EHEC

Intestinal pathogenic Escherichia coli are a major cause of worldwide morbidity and mortality. Currently seven intestinal pathovars are recognized causing a wide range of intestinal disorders that are sometimes associated with severe and even lethal complications. The arsenal of virulence factors is used to subvert cellular functions of the host thereby enhancing adaptation, virulence and pathogenicity. Virulence factor profiles are largely the result of the acquisition of mobile genetic elements such as prophages and pathogenicity islands. A group of highly adapted intestinal pathogenic E. coli that are characterized by the induction of ‘attaching‐and‐effacing (A/E) lesions’ have acquired a decisive pathogenicity island, the ‘locus of enterocyte effacement – LEE’ by horizontal gene transfer. This review focuses on recent advances in our understanding of A/E E. coli. It highlights novel functions of effector proteins, addresses the LEE flanking regions where additional genetic elements such as the LifA/Efa1 region have been identified, and points to implications for diagnostics and therapy due to the putative interconversion of A/E E. coli during infection.     

Preliminary report on the study of postharvest fruit rot bacteria and yeasts in Lanzhou Lily (Lilium davidii var. unicolor) in China

Fruit rot is one of the most important factors affecting the postharvest quality and shelf life of Lanzhou Lily fruits. The aims of this study were to characterize different isolates from Lanzhou Lily using morphological and molecular approaches and to test their pathogenicity in Lanzhou Lily fruit. Different isolates were collected from decayed Lanzhou Lily fruits in Lanzhou in Gansu Province in China during 2016 and 2017. In total, four isolates were obtained and identified based on their DNA sequences of the 16S rDNA and 26S rDNA genes in combination with the morphological characteristics of the cultures and sporulation. Of the four isolates, one was identified as Bacillus safensis, one was Stenotrophomonas maltophilia and the other two isolates were Metschnikowia pulcherrima. The pathogenicity tests showed that all four isolates were pathogenic in Lanzhou Lily fruit. Previously Fusarium tricinctum has been reported to be the primary cause of fruit rot in Lanzhou Lily throughout the world. Our report is the first to show results that indicate that B. safensis, S. maltophilia and M. pulcherrima are pathogenic in Lanzhou Lily in China. This is the first report on the bacteria and yeast that infect Lanzhou Lily fruits.     

Acanthamoeba stevensoni N. Sp. (Protozoa: Amoebida) from Sewage-Contaminated Shellfish Beds in Raritan Bay, New York

Marine sediments from 12 shallow water stations in Raritan Bay, New York were tested for the presence of Acanthamoeba. Eight stations were positive for one or more species of Acanthamoeba, A. castellanii, A. comandoni, A. hatchetti, A. lenticulata, A. polyphaga, A. rhysodes, and Acanthamoeba spp. Isolates that grew at 38–40° C were found at four stations (A. comandoni, A. lenticulata, and two unidentified strains). The two unknown strains were characterized on the basis of morphological features, isoenzyme profiles, and mouse pathogenicity tests. One of the two strains was determined to be a new species and is designated herein as Acanthamoeba stevensoni n. sp., ATCC 50388. Mature cysts were most similar to those of morphological Group II of Pussard & Pons (1977). Acanthamoeba stevensoni n. sp. was isolated from inshore coastal sediments where seawater ranged from 20–30%‰ (ppt.). The sediments supported commercially valuable populations of hard clams, Mercenaria mercenaria, that required depuration prior to sale because of contamination by sewage-associated bacteria.     

Heteromeric transposase elements: generators of genomic islands across diverse bacteria

Horizontally acquired genetic information in bacterial chromosomes accumulates in blocks termed genomic islands. Tn7‐like transposons form genomic islands at a programmed insertion site in bacterial chromosomes, attTn7. Transposition involves five transposon‐encoded genes (tnsABCDE) including an atypical heteromeric transposase. One transposase subunit, TnsB, is from the large family of bacterial transposases, the second, TnsA, is related to endonucleases. A regulator protein, TnsC, functions with different target site selecting proteins to recognize different targets. TnsD directs transposition into attTn7, while TnsE encourages horizontal transmission by targeting mobile plasmids. Recent work suggests that distantly related elements with heteromeric transposases exist with alternate targeting pathways that also facilitate the formation of genomic islands. Tn6230 and related elements can be found at a single position in a gene of unknown function (yhiN) in various bacteria as well as in mobile plasmids. Another group we term Tn6022‐like elements form pathogenicity islands in the Acinetobacter baumannii comM gene. We find that Tn6022‐like elements also appear to have an uncharacterized mechanism for provoking internal transposition and deletion events that serve as a conduit for evolving new elements. As a group, heteromeric transposase elements utilize diverse target site selection mechanisms adapted to the spread and rearrangement of genomic islands.     

Evolutionary perspective on the origin of Haitian cholera outbreak strain

Cholera epidemic has not been reported in Haiti for at least 100?years, although cholera has been present in Latin America since 1991. Surprisingly, the recent cholera epidemic in Haiti (October 2010) recorded more than 250,000 cases and 4000 deaths in the first 6?months and became one of the most explosive and deadly cholera outbreak in recent history. In the present study, we conducted genomic analyses of pathogenicity islands of three Haitian Vibrio cholerae strains and compared them with nine different V. cholerae O1 El Tor genomes. Although CIRS101 is evolutionarily most similar to the Haitian strains, our study also provides some important differences in the genetic organization of pathogenicity islands of Haitian strains with CIRS101. Evolutionary analysis suggests that unusual functional constraints have been imposed on the Haitian strains and we hypothesize that amino acid substitution is more deleterious in Haitian strains than in nonHaitian strains.     

Presence of Quorum-Sensing-Mediated Gene Regulation in Pathogenic Ice-Nucleation-Active (INA) Bacteria

Nine out of a total of 20 pathogenic ice-nucleation-active bacteria, with different levels of inducible INA, were tested and found positive for their ability to synthesize quorum-sensing (QS) signals. The bacteria were isolated from willow plants and belonged to the genera Bacillus, Erwinia, Pseudomonas and Sphingomonas. As reporter bacteria, to detect the homoserine lactone (HSL) autoinducer, Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeroginosa, Aeromonas hydrophila and Vibrio fischeri strains were used. We thus provide evidence that pathogenic ice-nucleation bacteria with inducible INA produce QS signals that in other bacteria have been shown to be in the control of genes of importance for pathogenicity.     

The past,present and future of secondary metabolite research in the Dothideomycetes

The Dothideomycetes represents a large and diverse array of fungi in which prominent plant pathogens are over‐represented. Species within the Cochliobolus, Alternaria, Pyrenophora and Mycosphaerella (amongst others) all cause diseases that threaten food security in many parts of the world. Significant progress has been made over the past decade in understanding how some of these pathogens cause disease at a molecular level. It is reasonable to suggest that much of this progress can be attributed to the increased availability of genome sequences. However, together with revealing mechanisms of pathogenicity, these genome sequences have also highlighted the capacity of the Dothideomycetes to produce an extensive array of secondary metabolites, far greater than originally thought. Indeed, it is now clear that we appear to have only scratched the surface to date in terms of the identification of secondary metabolites produced by these fungi. In the first half of this review, we examine the current status of secondary metabolite research in the Dothideomycetes and highlight the diversity of the molecules discovered thus far, in terms of both structure and biological activity. In the second part of this review, we survey the emerging techniques and technologies that will be required to shed light on the vast array of secondary metabolite potential that is encoded within these genomes. Experimental design, analytical chemistry and synthetic biology are all discussed in the context of how they will contribute to this field.