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Photorhabdus luminescens is an entomopathogenic bacterium found in symbiosis with the nematode Heterorhabditis. Dam DNA methylation is involved in the pathogenicity of many bacteria, including P. luminescens, whereas studies about the role of bacterial DNA methylation during symbiosis are scarce. The aim of this study was to determine the role of Dam DNA methylation in P. luminescens during the whole bacterial life cycle including during symbiosis with H. bacteriophora. We constructed a strain overexpressing dam by inserting an additional copy of the dam gene under the control of a constitutive promoter in the chromosome of P. luminescens and then achieved association between this recombinant strain and nematodes. The dam overexpressing strain was able to feed the nematode in vitro and in vivo similarly as a control strain, and to re-associate with Infective Juvenile (IJ) stages in the insect. No difference in the amount of emerging IJs from the cadaver was observed between the two strains. Compared to the nematode in symbiosis with the control strain, a significant increase in LT50 was observed during insect infestation with the nematode associated with the dam overexpressing strain. These results suggest that during the life cycle of P. luminescens, Dam is not involved the bacterial symbiosis with the nematode H. bacteriophora, but it contributes to the pathogenicity of the nemato-bacterial complex. [ABSTRACT FROM AUTHOR]

Photorhabdus is a bacterium that forms a mutualistic relationship with nematodes (Heterorhabditidae) and is responsible for insect mortality during nematode infection. The purpose of this study was to investigate virulence modulation (vmo) in Photorhabdus spp. where individual colonies exhibit different levels of virulence. Despite in-depth studies on culturing Photorhabdus spp. and its nematode partner, little is known about ideal growth conditions prior to virulence assays. Accordingly, eight Photorhabdus strains with representatives from each species were grown in four media types. All strains grew best in either Luria-Bertani broth + 0.1% pyruvate or tryptic soy broth + 0.5% yeast extract. However, on agar the only media on which all strains grew well were agar plates supplemented with pyruvate. To investigate vmo, virulence of individual colonies from three species was examined. Vmo was exhibited in two out of the three tested strains. Results may aid in the design of Photorhabdus virulence assays. [ABSTRACT FROM AUTHOR]

Biological control agents including a wide range of organisms such as predators, parasitoids, and entomopathogens (bacteria, fungi, nematodes, and viruses) are frequently used to control insect pests. Despite commercial availability of these biocontrol agents their widespread use is limited due to biological and economic difficulties. Efficient mass-production relies heavily on the environment in which the agent is grown. Nutrition can play a significant role in important biocontrol traits such as colonization and survival, tolerance to environmental stress, reproduction, and longevity. Therefore, to increase biocontrol potential nutritional aspects should be considered prior to commercial production. This review aims to explore the role nutrition plays in the production and efficacy of biocontrol agents by summarizing the effect nutrition has on important biocontrol traits, specifically traits in organisms that target insect pests including predators, parasitoids, and microbial agents. [ABSTRACT FROM AUTHOR]

Biological control agents have become increasingly important in integrated pest management programs. However, certain traits of these agents that are needed for efficient biocontrol often decrease or are lost during in vitro rearing. Entomopathogenic nematodes (EPNs) often exhibit trait deterioration when reared under laboratory conditions. EPN trait deterioration has been attributed (at least in part) to genetic causes; however, the underlying causes of trait deterioration in its bacterial endosymbiont have not been explored. In this study the EPN symbiont Photorhabdus luminescens was monitored for the deterioration of three traits; inclusion body production, reproductive potential, and virulence, in three different nutritional environments; lipid liquid medium (LLM), nutrient broth (NB), and tryptic soy broth + yeast extract (TSY). Significant trait deterioration did not occur for any of the traits in any environment. There was an increase in inclusion body production in TSY. Additionally, there was variation in growth within NB and TSY sub-cultured population lines and one TSY sub-population line was less virulent than the other two. However, returning bacteria to LLM restored all traits to wild-type levels. We infer the observed trait deterioration in Photorhabdus was minimal and appeared to be driven by environmental conditions as opposed to stable genetic changes. Our data suggest that variation among traits of in vitro cultures of Photorhabdus is more likely due to environmental variation than inadvertent laboratory selection or other genetic processes. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is an opportunistic pathogen frequently associated with nosocomially acquired infections. host cell adherence and biofilm formation of K. pneumoniae isolates is mediated by type 1 (T1p) and type 3 (MR/K) pili whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. The E. coli common pilus (ECP) is an adhesive structure produced by all E. coli pathogroups and a homolog of the ecpABCDE operon is present in the K. pneumoniae genome. In this study, we aimed to determine the prevalence of these three fimbrial genes among a collection of 69 clinical and environmental K. pneumoniae strains and to establish a correlation with fmbrial production during cell adherence and bioflm formation. The PCR-based survey demonstrated that 96% of the K. pneumoniae strains contained ecpA and 94% of these strains produced ECP during adhesion to cultured epithelial cells. eighty percent of the strains forming biofilms on glass produced ECP, suggesting that ECP is required, at least in vitro, for expression of these phenotypes. The fim operon was found in 100% of the strains and T1p was detected in 96% of these strains. While all the strains examined contained mrkA, only 57% of them produced MR/K fmbriae, alone or together with ECP. In summary, this study highlights the ability of K. pneumoniae strains to produce ECP, which may represent a new important adhesive structure of this organism. Further, it defines the multi-fimbrial nature of the interaction of this nosocomial pathogen with host epithelial cells and inert surfaces. [ABSTRACT FROM AUTHOR]

The free-living nematode Caenorhabditis elegans remains one of the most robust and flexible genetic systems for interrogating the complexities of animal biology. Targeted genetic manipulations, such as RNA interference (RNAi), CRISPR/Cas9- or array-based transgenesis, all depend on initial delivery of nucleic acids. Delivery of dsRNA by feeding can be effective, but the expression in Escherichia coli is not conducive to experiments intended to remain sterile or with defined microbial communities. Soaking-based delivery requires prolonged exposure of animals to high-material concentrations without a food source and is of limited throughput. Last, microinjection of individual animals can precisely deliver materials to animals' germlines, but is limited by the need to target and inject each animal one-by-one. Thus, we sought to address some of these challenges in nucleic acid delivery by developing a population-scale delivery method. We demonstrate efficient electroporation-mediated delivery of dsRNA throughout the worm and effective RNAi-based silencing, including in the germline. Finally, we show that guide RNA delivered by electroporation can be utilized by transgenic Cas9 expressing worms for population-scale genetic targeting. Together, these methods expand the scale and scope of genetic methodologies that can be applied to the C. elegans system. [ABSTRACT FROM AUTHOR]

Abstract: Our objective was to estimate the biocontrol potential of the recently discovered entomopathogenic nematode species Heterorhabditis georgiana (Kesha strain). Additionally, we conducted a phylogenetic characterization of the nematode’s symbiotic bacterium. In laboratory experiments, we compared H. georgiana to other entomopathogenic nematodes for virulence, environmental tolerance (to heat, desiccation, and cold), and host seeking ability. Virulence assays targeted Acheta domesticus, Agrotis ipsilon, Diaprepes abbreviatus, Musca domestica, Plodia interpunctella, Solenopsis invicta, and Tenebrio molitor. Each assay included H. georgiana and five or six of the following species: Heterorhabditis floridensis, Heterorhabditis indica, Heterorhabditis mexicana, Steinernema carpocapsae, Steinernema feltiae, Steinernema rarum, and Steinernema riobrave. Environmental tolerance assays included Heterorhabditis bacteriophora, H. georgiana, H. indica, S. carpocapsae, S. feltiae, and S. riobrave (except cold tolerance did not include S. carpocapsae or S. riobrave). Host seeking ability was assessed in H. bacteriophora, H. georgiana, S. carpocapsae, and Steinernema glaseri, all of which showed positive orientation to the host with S. glaseri having greater movement toward the host than S. carpocapsae (and the heterorhabditids being intermediate). Temperature range data (tested at 10, 13, 17, 25, 30 and 35°C) indicated that H. georgiana can infect Galleria mellonella between 13 and 35°C (with higher infection at 17–30°C), and could reproduce between 17 and 30°C (with higher nematode yields at 25°C). Compared with other nematode species, H. georgiana expressed low or intermediate capabilities in all virulence and environmental tolerance assays indicating a relatively low biocontrol potential. Some novel observations resulted from comparisons among other species tested. In virulence assays, H. indica caused the highest mortality in P. interpunctella followed by S. riobrave; S. carpocapsae caused the highest mortality in A. domesticus followed by H. indica; and S. riobrave was the most virulent nematode to S. invicta. In cold tolerance, S. feltiae exhibited superior ability to cause mortality in G. mellonella (100%) at 10°C, yet H. bacteriophora and H. georgiana exhibited the ability to produce attenuated infections at 10°C, i.e., the infections resumed and produced mortality at 25°C. In contrast, H. indica did not show an ability to cause attenuated infections. Based on the phylogenetic analysis, the bacterium associated with H. georgiana was identified as Photorhabdus luminescens akhurstii. [Copyright &y& Elsevier]