Roslyn G. Mourant

DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov.

The levels of DNA relatedness for a broad sample of Xenorhabdus strains isolated from different species of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) and from different geographical sources were estimated by the hydroxyapatite method. The level of DNA-DNA relatedness for the two phases of each isolate tested was not significantly different from 100%, demonstrating unequivocally that the phase variation demonstrated by all Xenorhabdus spp. is not due to contamination. The isolates of the described Xenorhabdus species coalesced into different DNA relatedness groups, confirming that Xenorhabdus nematophilus, Xenorhabdus bovienii, Xenorhabdus poinarii, and Xenorhabdus beddingii, defined on the basis of phenotypic differences, are valid species. The symbiont of Steinernema intermedia also coalesced with the X. bovienii isolates. This was the only symbiont of seven recently described and unamed Steinernema spp. (including Steinernema ritteri, Steinernema rara, and Steinernema anomali) that formed a group with any of the previously described Xenorhabdus species; new species descriptions are required to accommodate the other taxa, but too few isolates were available to allow satisfactory descriptions of them. The DNA relatedness data also showed that the bacteria currently classified as Xenorhabdus luminescens are significantly different from all other Xenorhabdus strains. These data strongly support indications from previous studies of phenotypic characteristics, cellular fatty acids, and DNA relatedness that X. luminescens should be classified as a separate genus. A new genus, Photorhabdus, with an amended description of the type species, Photorhabdus luminescens, is proposed.

Phenotypic and DNA Relatedness between Nematode Symbionts and Clinical Strains of the Genus Photorhabdus (Enterobacteriaceae)

Bacterial strains isolated from wide ranges of nematode hosts and geographic sources and strains isolated from human clinical specimens were used to assess the taxonomic structure of the genus Photorhabdus. The following two methods were used: DNA relatedness and phenotypic characterization. Analysis of the DNA relatedness data revealed that all of the strains studied were congeneric and that the genus Photorhabdus is, on the basis of DNA relatedness data, more homogeneous than the other genus of nematode-symbiotic bacteria, the genus Xenorhabdus. In contrast to previous reports, only two DNA relatedness groups were identified in the genus Photorhabdus. These groups corresponded to the symbiotic strains and the clinical strains. There appeared to be some subgroups within the symbiotic strain group on the basis of the interactions of the strains with nematodes, which corresponded to some extent with the DNA relatedness data. However, there were significant ambiguities in the DNA relatedness data, and this group could not be subdivided on the basis of DNA relatedness data or phenotypic data. The distinct functional differences within and between the DNA relatedness groups of symbiotic Photorhabdus strains indicated that there are biologically significant sub-groups within the genus Photorhabdus that cannot be defined at this time. Further investigation of the taxonomy of Photorhabdus by using different approaches and a suitably wide range of strains is recommended. However, it is clear that the clinical strains form a recognizable subgroup within the genus even though no formal subtaxon can be defined at this time.

Catalytic improvement and evolution of atrazine chlorohydrolase

ABSTRACT The atrazine chlorohydrolase AtzA has evolved within the past 50 years to catalyze the hydrolytic dechlorination of the herbicide atrazine. It is of wide research interest for two reasons: first, catalytic improvement of the enzyme would facilitate its application in bioremediation, and second, because of its recent evolution, it presents a rare opportunity to examine the early stages in the acquisition of new catalytic activities. Using a structural model of the AtzA-atrazine complex, a region of the substrate-binding pocket was targeted for combinatorial randomization. Identification of improved variants through this process informed the construction of a variant AtzA enzyme with 20-fold improvement in its kcat/Km value compared with that of the wild-type enzyme. The reduction in Km observed in the AtzA variants has allowed the full kinetic profile for the AtzA-catalyzed dechlorination of atrazine to be determined for the first time, revealing the hitherto-unreported substrate cooperativity in AtzA. Since substrate cooperativity is common among deaminases, which are the closest structural homologs of AtzA, it is possible that this phenomenon is a remnant of the catalytic activity of the evolutionary progenitor of AtzA. A catalytic mechanism that suggests a plausible mechanistic route for the evolution of dechlorinase activity in AtzA from an ancestral deaminase is proposed.

Degradation of dichloroaniline isomers by a newly isolated strain, Bacillus megaterium IMT21

An efficient 3,4-dichloroaniline (3,4-DCA)-mineralizing bacterium has been isolated from enrichment cultures originating from a soil sample with a history of repeated exposure to diuron, a major metabolite of which is 3,4-DCA. This bacterium, Bacillus megaterium IMT21, also mineralized 2,3-, 2,4-, 2,5- and 3,5-DCA as sole sources of carbon and energy. These five DCA isomers were degraded via two different routes. 2,3-, 2,4- and 2,5-DCA were degraded via previously unknown dichloroaminophenol metabolites, whereas 3,4- and 3,5-DCA were degraded via dichloroacetanilide.

Restriction Analysis of Phase Variation in Xenorhabdus spp. (Enterobacteriaceae), Entomopathogenic Bacteria Associated with Nematodes

Summary As part of an effort to elucidate the molecular basis of phase variation in Xenorhabdus , the two phases of X. nematophilus were examined for evidence of genomic rearrangements. No DNA sequence rearrangement was detected by RFLP analysis or Southern Cross hybridization of total DNA. Differential digestion eficiency by various restriction enzymes strongly indicated that the DNA of each of the Xenorhabdus species is highly methylated at both adenine and cytosine residues. The differential response to Sma I digestion by European and American isolates of X. nematophilus suggests a geographic variation in this species.

Detection of RHDV2 in European brown hares (Lepus europaeus) in Australia

RABBIT haemorrhagic disease virus 2 (RHDV2) belongs to the family Caliciviridae, genus Lagovirus , along with RHDV , European brown hare syndrome virus (EBHSV) and other unassigned rabbit caliciviruses (RCVs). RHDV2 was first detected in European rabbits ( Oryctolagus cuniculus ) in France in 2010 (Le Gall-Recule and others 2011). It spread rapidly throughout Europe (Dalton and others 2012, Abrantes and others 2013, Le Gall-Recule and others 2013, Baily and others 2014, Westcott and others 2014) and was detected in Australia in May 2015 (Hall and others 2015). In contrast to RHDV and EBHSV, which are strictly species-specific and restricted to Oryctolagus (rabbit) and Lepus (hare) genera, respectively (Lavazza and others 1996), RHDV2 causes a fatal hepatitis in European rabbits (Le Gall-Recule and others 2011), Sardinian Cape hares ( Lepus capensis mediterraneus ; Puggioni and others 2013), and in Italian hares ( Lepus corsicanus ) (Camarda and others 2014). Recently, RHDV2 has also been detected in European brown hares ( Lepus europaeus ) in Italy and Spain (Velarde and others, 2016). Australia has only two species of lagomorphs, O cuniculus and L europaeus , both introduced as game species in the mid-nineteenth century. The distribution of hares is limited to the south-east of the continent, mostly sympatric with rabbits, while rabbits inhabit an area covering 70 per cent of …

Rabbit Hemorrhagic Disease Virus 2 (RHDV2; GI.2) Is Replacing Endemic Strains of RHDV in the Australian Landscape within 18 Months of Its Arrival

ABSTRACT Rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) is a pathogenic calicivirus that affects European rabbits (Oryctolagus cuniculus) and various hare (Lepus) species. GI.2 was first detected in France in 2010 and subsequently caused epidemics in wild and domestic lagomorph populations throughout Europe. In May 2015, GI.2 was detected in Australia. Within 18 months of its initial detection, GI.2 had spread to all Australian states and territories and rapidly became the dominant circulating strain, replacing Rabbit hemorrhagic disease virus (RHDV/GI.1) in mainland Australia. Reconstruction of the evolutionary history of 127 Australian GI.2 isolates revealed that the virus arrived in Australia at least several months before its initial description and likely circulated unnoticed in wild rabbit populations in the east of the continent prior to its detection. GI.2 sequences isolated from five hares clustered with sequences from sympatric rabbit populations sampled contemporaneously, indicating multiple spillover events into hares rather than an adaptation of the Australian GI.2 to a new host. Since the presence of GI.2 in Australia may have wide-ranging consequences for rabbit biocontrol, particularly with the release of the novel biocontrol agent GI.1a/RHDVa-K5 in March 2017, ongoing surveillance is critical to understanding the interactions of the various lagoviruses in Australia and their impact on host populations. IMPORTANCE This study describes the spread and distribution of Rabbit hemorrhagic disease virus 2 (GI.2) in Australia since its first detection in May 2015. Within the first 18 months following its detection, RHDV2 spread from east to west across the continent and became the dominant strain in all mainland states of Australia. This has important implications for pest animal management and for owners of pet and farmed rabbits, as there currently is no effective vaccine available in Australia for GI.2. The closely related RHDV (GI.1) is used to control overabundant wild rabbits, a serious environmental and agricultural pest in this country, and it is currently unclear how the widespread circulation of GI.2 will impact ongoing targeted wild rabbit management operations.

Use of a Cry1Ac-Resistant Line of Helicoverpa armigera (Lepidoptera: Noctuidae) to Detect Novel Insecticidal Toxin Genes in Bacillus thuringiensis

This paper describes a screening strategy incorporating resistant insect lines for discovery of new Bacillus thuringiensis toxins against a background of known genes that would normally mask the activity of additional genes and the application of that strategy. A line of Helicoverpa armigera with resistance to Cry1Ac (line ISOC) was used to screen Cry1Ac-expressing strains of B. thuringiensis for additional toxins with activity against H. armigera. Using this approach, a number of Cry1Ac-producing strains with significant toxicity toward Cry1Ac-resistant H. armigera were identified. When the insecticidal protein complement of one of these strains, C81, was examined in detail, a novel cry2 gene (cry2Af1) was detected.

A strain-specific multiplex RT-PCR for Australian rabbit haemorrhagic disease viruses uncovers a new recombinant virus variant in rabbits and hares

Rabbit haemorrhagic disease virus (RHDV, or GI.1) is a calicivirus in the genus Lagovirus that has been widely utilized in Australia as a biological control agent for the management of overabundant wild European rabbit (Oryctolagus cuniculus) populations since 1996. Recently, two exotic incursions of pathogenic lagoviruses have been reported in Australia; GI.1a-Aus, previously called RHDVa-Aus, is a GI.1a virus detected in January 2014, and the novel lagovirus GI.2 (previously known as RHDV2). Furthermore, an additional GI.1a strain, GI.1a-K5 (also known as 08Q712), was released nationwide in March 2017 as a supplementary tool for wild rabbit management. To discriminate between these lagoviruses, a highly sensitive strain-specific multiplex RT-PCR assay was developed, which allows fast, cost-effective and sensitive detection of the four pathogenic lagoviruses currently known to be circulating in Australia. In addition, we developed a universal RT-qPCR assay to be used in conjunction with the multiplex assay that broadly detects all four viruses and facilitates quantification of viral RNA load in samples. These assays enable rapid detection, identification and quantification of pathogenic lagoviruses in the Australian context. Using these assays, a novel recombinant lagovirus was detected in rabbit tissue samples, which contained the non-structural genes of GI.1a-Aus and the structural genes of GI.2. This variant was also recovered from the liver of a European brown hare (Lepus europaeus). The impact of this novel recombinant on Australian wild lagomorph populations and its competitiveness in relation to circulating field strains, particularly GI.2, requires further studies.

Detection and Circulation of a Novel Rabbit Hemorrhagic Disease Virus in Australia

The highly virulent rabbit hemorrhagic disease virus (RHDV) has been widely used in Australia and New Zealand since the mid-1990s to control wild rabbits, an invasive vertebrate pest in these countries. In January 2014, an exotic RHDV was detected in Australia, and 8 additional outbreaks were reported in both domestic and wild rabbits in the 15 months following its detection. Full-length genomic analysis revealed that this virus is a recombinant containing an RHDVa capsid gene and nonstructural genes most closely related to nonpathogenic rabbit caliciviruses. Nationwide monitoring efforts need to be expanded to assess if the increasing number of different RHDV variants circulating in the Australian environment will affect biological control of rabbits. At the same time, updated vaccines and vaccination protocols are urgently needed to protect pet and farmed rabbits from these novel rabbit caliciviruses.