Nitrosomonas europaea strain Schmidt produces at least three acyl homoserine lactone (AHL) signal molecules: C 6 -homoserine lactone (HSL), C 8 -HSL, and C 10 -HSL. These compounds were identified in extracts of chemostat culture effluent by three independent methods. The concentrations of AHL in effluent were low (0.4 to 2.2 nM) but within the range known to induce AHL-responsive systems. The absence of LuxI and LuxM homologs from the genome of N. europaea strain Schmidt suggested that AHL synthesis occurs by an alternate pathway, possibly mediated by an HdtS homolog. To the best of our knowledge, the present report is the first to document the types and levels of AHLs produced by N. europaea.Nitrosomonas europaea and other ammonia-oxidizing bacteria (AOB) play a pivotal role in affecting the fate and behavior of nitrogen in the environment. Terrestrial and freshwater AOB grow predominantly in biofilms (1,7,13,18), but little is known about how conditions unique to the biofilm environment affect their biology and, consequently, nitrogen cycling. In other members of the class Proteobacteria, a major process affecting cellular structure and function in biofilms is quorum sensing, which is mediated by acyl-homoserine lactone (AHL) signal molecules (6). Much is known about AHL production by a diversity of heterotrophic proteobacteria, but to date, AHL production by the chemolithotrophic AOB has not been conclusively documented. The objectives of this study were to determine if N. europaea strain Schmidt produces AHL and, if so, to identify the types and levels of these molecules.N. europaea strain Schmidt was obtained from the American Type Culture Collection (ATCC strain 19718). Batch cultures were grown in ATCC medium 2265 (2) at 25°C in lightshielded flasks to which aliquots of sterile 30% (wt/vol) K 2 CO 3 were periodically added. Continuous culture of N. europaea was done in a BioFlo 110 modular benchtop fermentor (New Brunswick Scientific Co., Edison, NJ). Cells were grown in ATCC medium 2265 (lacking cresol red) in a light-shielded vessel at a specific growth rate of 0.025/h (71% of the maximum specific growth rate reported for N. europaea [9]). The temperature was held at 25°C, the dissolved oxygen concentration was maintained at 5 mg/liter (60% of the saturation level at 25°C), and the pH was maintained at 7.1 Ϯ 0.1. A nearneutral pH was used to minimize the potential for AHL degradation by lactonolysis (20). At steady state, the culture optical density at 600 nm was 0.11 (6 ϫ 10 6 cells/ml, 45 g biomass dry weight/ml). Aliquots (100 l) of the culture were regularly plated on 1/10 nutrient broth to verify the absence of heterotrophic contaminants.Extracts were prepared from supernatants of N. europaea batch cultures (5 liters total) and chemostat effluent. Batch cultures were harvested when the cultures had twice acidified their medium, after which accumulation of nitrite-limited growth (final optical density at 600 nm ϭ 0.08). For the chemostat culture, 7 liters of effluent was collected, the cells were r...

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...