Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 570243, 10 pages http://dx.doi.org/10.1155/2015/570243 Research Article Identification of a New Alcaligenes faecalis Strain MOR02 and Assessment of Its Toxicity and Pathogenicity to Insects Rosa Estela Quiroz-Castañeda,1 Ared Mendoza-Mejía,1 Verónica Obregón-Barboza,1 Fernando Martínez-Ocampo,1 Armando Hernández-Mendoza,2 Felipe Martínez-Garduño,1 Gabriel Guillén-Solís,3 Federico Sánchez-Rodríguez,3 Guadalupe Peña-Chora,4 Laura Ortíz-Hernández,1 Paul Gaytán-Colín,3 and Edgar Dantán-González1 1 Centro de Investigacion´ en Biotecnolog´ıa, Universidad Autonoma´ del Estado de Morelos, 62210 Cuernavaca, MOR, Mexico 2Facultad de Ciencias, Universidad Autonoma´ del Estado de Morelos, 62210 Cuernavaca, MOR, Mexico 3Instituto de Biotecnolog´ıa, Universidad Nacional Autonoma´ de Mexico,´ 62210 Cuernavaca, MOR, Mexico 4Centro de Investigaciones Biologicas,´ Universidad Autonoma´ del Estado de Morelos, 62210 Cuernavaca, MOR, Mexico Correspondence should be addressed to Edgar Dantan-Gonz´ alez;´ [email protected] Received 14 November 2014; Revised 16 December 2014; Accepted 17 December 2014 Academic Editor: Yudong Cai Copyright © 2015 Rosa Estela Quiroz-Castaneda˜ et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We report the isolation of a bacterium from Galleria mellonella larva and its identification using genome sequencing and phylogenomic analysis. This bacterium was named Alcaligenes faecalis strain MOR02. Microscopic analyses revealed that the bacteria are located in the esophagus and intestine of the nematodes Steinernema feltiae, S. carpocapsae,andH. bacteriophora. Using G. mellonella larvae as a model, when the larvae were injected with 24,000 CFU in their hemocoel, more than 96% mortality was achieved after 24 h. Additionally, toxicity assays determined that 1 g of supernatant extract from A. faecalis MOR02 killed more than 70% G. mellonella larvae 96 h after injection. A correlation of experimental data with sequence genome analyses was also performed. We discovered genes that encode proteins and enzymes that are related to pathogenicity, toxicity, and host/environment interactions that may be responsible for the observed phenotypic characteristics. Our data demonstrates that the bacteria are able to use different strategies to colonize nematodes and kill insects to their own benefit. However, there remains an extensive group of unidentified microorganisms that could be participating in the infection process. Additionally, a nematode-bacterium association could be established probably as a strategy of dispersion and colonization. 1. Introduction the nematode provides the bacteria with nutrients, protec- tion, and environmental dispersion. Meanwhile, the bacteria Nematodes have a global impact on ecosystems and econo- providenutritiontothenematodethroughavailableinsect- mies; however, parasitic nematodes also can have many derived nutrients as well as antimicrobial compounds that beneficial impacts on human interests and health [1]. For prevent the development of bacteria, fungi, and yeast in the example, entomopathogenic nematodes (EPNs) that belong insect. For example, Photorhabdus produces bacteriocins and to the families Steinernematidae and Heterorhabditidae are lumicins [4, 5]. commercially used as biological control agents for crop Both nematodes and bacteria can infect and kill insects. pests [2]. The EPNs Heterorhabditis and Steinernema have Once the infective juvenile reaches the insect hemocoel, unique mechanisms to associate with and transmit bacteria they release the symbiotic bacteria into a rich medium that to insect hosts; specifically, these nematodes have a symbiotic grows the bacterial cells. The bacterial cells then release association with pathogenic bacteria from the Photorhabdus antimicrobial compounds, toxins, and exoenzymes causing and Xenorhabdus genera, respectively [3]. In this relationship, the insect death usually within 24–48 h [6, 7]. 2 BioMed Research International Although it has been postulated that the nematode- tRNA genes, respectively. Clusters of Orthologous Groups bacteria interaction is unique some nonsymbiotic bacteria of proteins (COG) [14] and Gene Ontology (GO) [15, 16] that are able to coinhabit or colonize the insect cadaver annotations were performed using a BLAST search against and the nematode have been reported. Lysenko and Weiser the downloaded databases. KEGG Orthology (KO) [17, 18] [8] isolated bacteria associated with S. carpocapsae,suchas annotation was performed at the KEGG Automatic Annota- Alcaligenes, Pseudomonas,andAcinetobacter spp., which also tion Server (KAAS) with the Bidirectional Best Hits (BBH) are pathogenic to Galleria mellonella larvae. More recently, method (http://www.genome.jp/kegg/kaas/). PFAM annota- the bacteria Flavobacterium sp., Providencia vermicola,and tion was performed at the EMBL-EBI batch search server Alcaligenes faecalis were isolated from the nematode Rhabdi- (http://pfam.xfam.org/search). tis blumi [9]. Several bacterial species have also been identified from 2.2.3. Phylogeny and Identification. The isolated 16S rRNA hemolymph of insect cadavers infected with EPNs as well as sequence was identified using the RNAmmer version 1.2 frominfectivejuvenileEPNs[4]. server [11](http://www.cbs.dtu.dk/services/RNAmmer/)on In this study, we report the isolation and identification contig1 (722,086 bp). This sequence was used for a of the bacteria A. faecalis strain MOR02 from G. mellonella BLASTN search (http://blast.ncbi.nlm.nih.gov/)usingthe dead larva recovered from soil samples in Tenango, Morelos, MEGABLAST algorithm to search for nonredundant (nr) Mexico. The pathogenicity and toxicity of this bacterium were nucleotide [19] databases. The higher significant alignments also analyzed and we determined that A. faecalis MOR02 reported were downloaded for phylogenetic analysis. A causes mortality in G. mellonella larvae 24 h after injection. full tree with 165 sequences and a representative tree with Additionally, the protein extract from a supernatant culture of 25 sequences of the genera Alcaligenes and closely related the bacteria is toxic in G. mellonella larvae 96 h after injection. bacteria were constructed using all 16S rRNA sequences. We also microscopically observed the association of A. These sequences were first aligned using MUSCLE server faecalis MOR02 and nematodes from the genera Steinernema version 3.7 (http://phylogeny.lirmm.fr/phylo cgi/one task and Heterorhabditis. We performed genome sequencing on A. .cgi?task type=muscle)[20] and the resulting alignment was faecalis MOR02, and the bioinformatic data analysis supports analyzed using the phylogenetic analysis program MEGA the phenotypic characteristics of the bacteria. version 6.0 [21]. A maximum likelihood method was used to calculate distances and the neighbor-joining method was 2. Materials and Methods used to infer the evolutionary tree and bootstrap values were also calculated using 1000 replicates. 2.1. Bacterial Isolation from the Nematode. The bacteria were isolated from the hemolymph of a G. mellonella larvae 2.2.4. Phenotypic Characterization. Three tests were per- cadaver found in the soil of Tenango (Santa Ana), Morelos, formed as part of phenotypic characterization: the swarming Mexico, by Guadalupe Pena.˜ G. mellonella larvae were disin- motility test, chitinase activity assay, and esterase/lipase fected with ethanol 70% and the contents were streaked on LB hydrolysis test. media plates for bacterial growth. The isolated bacteria were ∘ To assess swarming motility, we slightly modified a previ- grown at 30 Cat250rpmovernightandthenweregrownon ously reported method [22].Briefly,aplateoftrypticasesoy LB media plates. broth (TSB) (Bioxon) and 0.5% agar (Bioxon) was inoculated Bacterial genomic DNA extraction was performed using in the center with a drop of a bacteria enriched liquid culture ∘ the UltraClean Microbial DNA Isolation kit (MOBIO). that was incubated at 25 Cfor7days.E. coli DH5 that have reportedly poor motility were used as a control [23]. 2.2. Bioinformatic Methods For the chitinase activity assay, a sterile solution con- taining colloidal chitin was prepared as follows: 2 g colloidal 2.2.1. Assembly. For the sequencing of the A. faecalis MOR02 chitin was dissolved in 100 mL of a solution prepared contain- genome, sequencing on the Genome Analyzer IIx (GAIIx) ing 0.5 g casein peptone (Bioxon), 0.5 g yeast extract (Bioxon), Illumina platform was performed by the UUSMD (Unidad 0.1 g KH2PO4 (High Purity) 0.01 g MgSO4⋅7H2O(J.T.Baker), Universitaria de Secuenciacion´ Masiva de DNA, Instituto 1.5 g agar (Bioxon), and pH adjusted to 6.5. A drop of an de Biotecnolog´ıa, UNAM). The 19,250,362 paired-end reads enriched culture of A. faecalis MOR02 was inoculated on the ∘ wereassembleddenovousingtheSPAdesprogram(version plates and incubated at 37 C until a white halo of degradation 3.1.1) and 23 contigs were generated with a 315-fold median was observed in the center of the plate. E. coli DH5 cells were coverage depth. used as a negative control. The streptococcus thermophiles activity of the bacteria were assessed using the Tween 80 2.2.2. Annotation. The RAST (Rapid Annotation using hydrolysis test that was performed as previously reported Subsystem Technology) version 2.0 (http://rast.nmpdr.org/) [24]. [10],