Genetics and physiology of motility by Photorhabdus spp

Photorhabdus is a bacterial symbiont of soil nematodes and a lethal pathogen of insects. Many pathogenic or symbiotic bacteria utilize various methods of motility to reach favorable conditions, colonize a host, or have motility genes that also regulate virulence expression. It is not known how motility is regulated, or how it may confer an advantage, in the complex life cycle of Photorhabdus.. We characterized motility in Photorhabdus and found that the bacterium was motile both by swimming (movement in liquid) and swarming (movement on surfaces) under appropriate conditions. Both types of motility utilized the same peritrichous flagella and shared genetic components. However, unlike swimming, swarming behavior was a social form of movement in which the cells coordinately formed intricate channels that covered a surface. The optimal conditions for motility were established including a Na + or K+ requirement. Interestingly, microarray experiments imply that NaCl and KCl regulate motility posttranscriptionally and not at the gene expression level. This ionic salt posttranscriptional regulation of motility has not been observed in other bacteria. We suggest that this form of regulation may be beneficial for an organism that must adapt quickly to changing environments. To identify the genes involved in motility, P. temperata mutants with altered motility behavior were generated with random transposon mutagenesis. An rssB mutant that displayed a hyperswarming phenotype was isolated, suggesting that RssB acts as a negative regulator of swarming behavior. A yidA mutant, whose function remains unknown, had inhibited swimming behavior and dramatically attenuated virulence. A plu3723 mutant (a luxR homolog) was isolated, that unlike the wild-type, was able to swim without NaCl or KCl. All together 86 motility mutants were isolated and physiologically characterized. Since many of the motility mutants had concomitant changes in expression of antibiotics, hemolysins, proteases, and insect virulence, expression of motility genes may be co-regulated with expression of virulence enzymes in Photorhabdus. The mutants isolated in this study will be useful long-term tools for additional experiments. The ability of Photorhabdus to swarm could provide a rapid and coordinated colonization of either nematode or insect host, or in traveling from one host to another. The nematode environment is low in nutrients, ionic salts, and amino acids, while the insect hemocoel is high in these solutes. When the nematodes release their bacterial symbionts into the insect hemocoel, the bacteria are exposed to the ionic salts that would induce the flagella regulon. Ecologically, it would be beneficial for the bacteria to be motile upon entering the insect to rapidly colonize the hemocoel. If aspects of virulence expression are co-regulated with motility genes as this research suggests, expression of virulence factors would also be induced upon exposure to the insect. The data presented in this study are the first steps for elucidating a model of motility in the life cycle of this insect pathogen and nematode symbiont

Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis

The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions

Swarming motility by Photorhabdus temperata is influenced by environmental conditions and uses the same flagella as that used in swimming motility

Photorhabdus temperata, an insect pathogen and nematode symbiont, is motile in liquid medium by swimming. We found that P. temperata was capable of surface movement, termed swarming behavior. Several lines of evidence indicate that P. temperata use the same flagella for both swimming and swarming motility. Both motility types required additional NaCl or KCl in the medium and had peritrichous flagella, which were composed of the same flagellin as detected by immunoblotting experiments. Mutants defective in flagellar structural proteins were nonmotile for both motility types. Unlike swimming, we observed swarming behavior to be a social form of movement in which the cells coordinately formed intricate channels covering a surface. The constituents of the swarm media affected motility. Swarming was optimal on low agar concentrations; as agar concentrations increased, swarm ring diameters decreased

Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis

The entomopathogenic nematode Heterorhabditis bacteriophora forms a specific mutualistic association with its bacterial partner Photorhabdus temperata. The microbial symbiont is required for nematode growth and development, and symbiont recognition is strain specific. The aim of this study was to sequence the genome of P. temperata and identify genes that plays a role in the pathogenesis of the Photorhabdus-Heterorhabditis symbiosis. A draft genome sequence of P. temperata strain NC19 was generated. The 5.2-Mb genome was organized into 17 scaffolds and contained 4,808 coding sequences (CDS). A genetic approach was also pursued to identify mutants with altered motility. A bank of 10,000 P. temperata transposon mutants was generated and screened for altered motility patterns. Five classes of motility mutants were identified: (i) nonmotile mutants, (ii) mutants with defective or aberrant swimming motility, (iii) mutant swimmers that do not require NaCl or KCl, (iv) hyperswimmer mutants that swim at an accelerated rate, and (v) hyperswarmer mutants that are able to swarm on the surface of 1.25% agar. The transposon insertion sites for these mutants were identified and used to investigate other physiological properties, including insect pathogenesis. The motility-defective mutant P13-7 had an insertion in the RNase II gene and showed reduced virulence and production of extracellular factors. Genetic complementation of this mutant restored wild-type activity. These results demonstrate a role for RNA turnover in insect pathogenesis and other physiological functions

Considerations for Optimization of High-Throughput Sequencing Bioinformatics Pipelines for Virus Detection

High-throughput sequencing (HTS) has demonstrated capabilities for broad virus detection based upon discovery of known and novel viruses in a variety of samples, including clinical, environmental, and biological. An important goal for HTS applications in biologics is to establish parameter settings that can afford adequate sensitivity at an acceptable computational cost (computation time, computer memory, storage, expense or/and efficiency), at critical steps in the bioinformatics pipeline, including initial data quality assessment, trimming/cleaning, and assembly (to reduce data volume and increase likelihood of appropriate sequence identification). Additionally, the quality and reliability of the results depend on the availability of a complete and curated viral database for obtaining accurate results; selection of sequence alignment programs and their configuration, that retains specificity for broad virus detection with reduced false-positive signals; removal of host sequences without loss of endogenous viral sequences of interest; and use of a meaningful reporting format, which can retain critical information of the analysis for presentation of readily interpretable data and actionable results. Furthermore, after alignment, both automated and manual evaluation may be needed to verify the results and help assign a potential risk level to residual, unmapped reads. We hope that the collective considerations discussed in this paper aid toward optimization of data analysis pipelines for virus detection by HTS