The genomes of many bacteria that participate in nitrogen cycling through the 
process of nitrification contain putative genes associated with acyl-homoserine 
lactone (AHL) quorum sensing (QS). AHL QS or bacterial cell-cell signaling is a 
method of bacterial communication and gene regulation and may be involved in 
nitrogen oxide fluxes or other important phenotypes in nitrifying bacteria. 
Here, we carried out a broad survey of AHL production in nitrifying bacteria in 
three steps. First, we analyzed the evolutionary history of AHL synthase and AHL 
receptor homologs in sequenced genomes and metagenomes of nitrifying bacteria to 
identify AHL synthase homologs in ammonia-oxidizing bacteria (AOB) of the genus 
Nitrosospira and nitrite-oxidizing bacteria (NOB) of the genera Nitrococcus, 
Nitrobacter, and Nitrospira Next, we screened cultures of both AOB and NOB with 
uncharacterized AHL synthase genes and AHL synthase-negative nitrifiers by a 
bioassay. Our results suggest that an AHL synthase gene is required for, but 
does not guarantee, cell density-dependent AHL production under the conditions 
tested. Finally, we utilized mass spectrometry to identify the AHLs produced by 
the AOB Nitrosospira multiformis and Nitrosospira briensis and the NOB 
Nitrobacter vulgaris and Nitrospira moscoviensis as N-decanoyl-l-homoserine 
lactone (C10-HSL), N-3-hydroxy-tetradecanoyl-l-homoserine lactone 
(3-OH-C14-HSL), a monounsaturated AHL (C10:1-HSL), and N-octanoyl-l-homoserine 
lactone (C8-HSL), respectively. Our survey expands the list of AHL-producing 
nitrifiers to include a representative of Nitrospira lineage II and suggests 
that AHL production is widespread in nitrifying bacteria.IMPORTANCE 
Nitrification, the aerobic oxidation of ammonia to nitrate via nitrite by 
nitrifying microorganisms, plays an important role in environmental nitrogen 
cycling from agricultural fertilization to wastewater treatment. The genomes of 
many nitrifying bacteria contain genes associated with bacterial cell-cell 
signaling or quorum sensing (QS). QS is a method of bacterial communication and 
gene regulation that is well studied in bacterial pathogens, but less is known 
about QS in environmental systems. Our previous work suggested that QS might be 
involved in the regulation of nitrogen oxide gas production during nitrite 
metabolism. This study characterized putative QS signals produced by different 
genera and species of nitrifiers. Our work lays the foundation for future 
experiments investigating communication between nitrifying bacteria, the purpose 
of QS in these microorganisms, and the manipulation of QS during nitrification.