Xenorhabdus nematophila Requires an Intact iscRSUA-hscBA-fdx Operon To Colonize Steinernema carpocapsae Nematodes

Abstract

An insertion between iscA and hscB of the Xenorhabdus nematophila iscRSUA-hscBA-fdx locus, predicted to encode Fe-S assembly machinery, prevented colonization of Steinernema carpocapsae nematodes. The insertion disrupted cotranscription of iscA and hscB, but did not reduce hscBA expression, suggesting that X. nematophila requires coordinated expression of the isc-hsc-fdx locus for colonization.The intestines of Steinernema carpocapsae infective juvenilestage (IJ) nematodes are mutualistically colonized by Xenorhabdus nematophila bacteria (4). Germfree S. carpocapsae nematode eggs applied to lawns of X. nematophila will develop through juvenile and reproductive stages (32) until high nematode population density and low nutrient concentrations result in formation of progeny IJ nematodes colonized by X. nematophila (13,17). Our lab is investigating molecular mechanisms mediating X. nematophila-S. carpocapsae interactions by identifying X. nematophila genes required for IJ nematode colonization.Identification of a colonization-defective X. nematophila mutant. X. nematophila HGB081 (Table 1) was mutagenized with mini-Tn10, using plasmid pKV124 (31) transferred by conjugation from S17-1 (pir) (7). Exconjugants selected on rifampin (100 g/ml) and chloramphenicol (30 g/ml) were individually cultivated with S. carpocapsae (Strain All) nematodes. Progeny IJ nematodes were harvested from each coculture and microscopically examined for the presence or absence of X. nematophila colonizers (32). One of 692 bacterial mutants screened was deficient in colonization and was designated HGB166. This frequency (0.16%) is within the range found in an independent Tn5 screen (8) and suggests that colonization genes comprise a small mutagenesis target.In a quantitative colonization assay (8), HGB166 exhibited a severe colonization defect ( (Fig. 1 and Table 1). Plasmid isolation, sequencing, and sequence analysis were carried out as previously described (8).The HGB166 colonization defect is caused by Tn10 insertion in an isc-hsc-fdx locus. The transposon insertion of HGB166 is in a conserved locus with the gene order iscRSUA-hscBA-fdx, 3 nucleotides downstream of the predicted iscA stop codon and 56 nucleotides upstream of the putative hscB start codon (19,34) (Fig. 1). In Escherichia coli, this locus encodes iron-sulfur center assembly machinery (12,19,26,28,29). Iron-sulfur centers are components of many cellular proteins with redox, regulatory, or catalytic function (3), and the mechanism of their assembly by isc-hsc-fdx-encoded proteins has begun to be elucidated. IscS, a cysteine desulfurase, donates sulfur to a nascent cluster (6, 29, 34) forming on the scaffolding protein IscU (1). hscA and hscB encode Hsc66 and Hsc20, respectively (10,21,30), which interact with IscU, resulting in increased Hsc66 ATPase activity (9,23). IscA is proposed to be an alternative scaffold for cluster formation (12) or an iron donor for iron-sulfur assembly on Fdx, an electron-transferring ferredoxin (15).To determine if the HGB166 colonization defe...

nilR is necessary for co‐ordinate repression of Xenorhabdus nematophila mutualism genes

“…Xenorhabdus nematophila colonization of S. carpocapsae nematodes includes an initiation stage and an outgrowth stage (Martens et al ., 2003a) and genes required for each have been identified (Vivas and Goodrich‐Blair, 2001; Heungens et al ., 2002; Martens et al ., 2003b; 2005). The nilA , B and C genes are each necessary for colonization initiation, but their function in this process remains obscure.…”

Common trends in mutualism revealed by model associations between invertebrates and bacteria: Table 1

“…Much progress has been made in both systems toward identifying and characterizing bacterial virulence and mutualism determinants. Mutualism determinants include factors involved in nutrition (Heungens et al , 2002; Martens et al , 2003b; Martens et al , 2005; Orchard & Goodrich-Blair, 2005; Watson et al , 2005), surface structure (Bennett & Clarke, 2005), gene regulation (Heungens et al , 2002; Cowles & Goodrich-Blair, 2006; Joyce et al , 2006; Cowles et al , 2007; Herbert et al , 2007), and stress response (Vivas & Goodrich-Blair, 2001; Heungens et al , 2002), as well as factors of unknown function (Heungens et al , 2002; Cowles & Goodrich-Blair, 2004; Cowles & Goodrich-Blair, 2008). Additionally, RNA interference has been successfully adapted for use in Heterorhabditis bacteriophora nematodes (Ciche & Sternberg, 2007) and the genome of this nematode is being sequenced (Ciche, 2007).…”

Visualizing Bacteria in Nematodes using Fluorescent Microscopy

“…In addition, this approach can be used to conduct a high throughput mutant screens to identify bacterial factors involved in the symbiosis, using fluorescence to detect colonization 17,18 . Other methods such as signature tagged mutagenesis require the use of radioactively labeled probes and are more time consuming 22,28 . Therefore, the use of fluorescence microscopy for bacterial symbiont visualization in nematodes is an effective and efficient screening tool for investigating bacterial interactions with a nematode host.…”

“…Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes 21,24 . Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization 17,18 , and is less laborious than other methods, including sonication 22,[25][26][27] and individual nematode dissection 28,29 . …”