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  • An N-Acyl Homoserine Lactone Synthase in the Ammonia-OxidizingBacterium Nitrosospira multiformis

    Jie Gao, Anzhou Ma, Xuliang Zhuang, Guoqiang Zhuang

    Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China

    The chemolithoautotrophic bacterium Nitrosospira multiformis is involved in affecting the process of nitrogen cycling. Here wereport the existence and characterization of a functional quorum sensing signal synthase in N. multiformis. One gene (nmuI)playing a role in generating a protein with high levels of similarity to N-acyl homoserine lactone (AHL) synthase protein familieswas identified. Two AHLs (C14-AHL and 3-oxo-C14-AHL) were detected using an AHL biosensor and liquid chromatography-mass spectrometry (LC-MS) when nmuI, producing a LuxI homologue, was introduced into Escherichia coli. However, by ex-tracting N. multiformis culture supernatants with acidified ethyl acetate, no AHL product was obtained that was capable of acti-vating the biosensor or being detected by LC-MS. According to reverse transcription-PCR, the nmuI gene is transcribed in N.multiformis, and a LuxR homolog (NmuR) in this ammonia-oxidizing strain showed great sensitivity to long-chain AHL signalsby solubility assay. A degradation experiment demonstrated that the absence of AHL signals might be attributed to the possibleAHL-inactivating activities of this strain. To summarize, an AHL synthase gene (nmuI) acting as a long-chain AHL producer hasbeen found in a chemolithotrophic ammonia-oxidizing microorganism, and the results provide an opportunity to complete theknowledge of the regulatory networks in N. multiformis.

    Quorum sensing (QS) is a form of cell-cell communicationthat regulates gene expression in response to fluctuations incell density. QS bacteria can alter their behavior through produc-ing, releasing, and responding to autoinducing signaling moleculesthat accumulate in the environment (1). A variety of physiologicalfunctions, including biofilm formation, bioluminescence, virulence,swarming, plasmid transfer, and antibiotic biosynthesis, are subjectto QS regulation (13). In Gram-negative bacteria, several signalmolecules have now been identified, such as N-acyl homoserine lac-tones (AHLs), quinolone, p-coumarate, and 3-OH palmitic acidmethyl ester (3-OH PAME), and the AHLs have probably been themost intensively investigated of these (3, 4). In the currently acceptedLuxI/LuxR-type regulatory system of QS, AHL biosynthesis dependsprimarily on a synthase protein (I protein), and target genes are thenactivated via the interaction between the signal molecules and a re-sponse regulator protein (R protein) (1, 3, 5).

    Nitrosospira multiformis is a chemolithoautotrophic bacteriumthat is capable of oxidizing ammonia to obtain energy for growth(6). The ecological importance of N. multiformis and other am-monia-oxidizing bacteria (AOB) is that they affect the biologicaloxidation of inorganic nitrogen compounds in the environment.During the ammonia oxidation process in water or soil, biofilmformed by AOB can greatly affect the nitrification efficiency andecological behavior of nitrifying bacteria (79). In many Gram-negative bacteria, the QS process controls exopolysaccharide pro-duction and biofilm formation, which is mediated by an AHLautoinducer (10). However, only a few studies have shown thetypes of AHL signal molecules produced by Nitrosomonas euro-paea (11) and AOB biofilm activity regulated by an AHL-basedcommunication system (12, 13). Knowledge of the QS regulatorysystem directly involved in AOB biofilm processes and other phys-iological functions is limited. Furthermore, the functional QS sys-tem of AOB has not been proved conclusively to date.

    In the current study, we describe an AHL synthase (NmuI) ofN. multiformis and the signal molecules produced by NmuI intro-duced into Escherichia coli. Two different types of AHLs were

    identified in the heterogeneous expression system by using a sig-nal biosensor and liquid chromatography-mass spectrometry(LC-MS). By evaluating the QS activity in N. multiformis, ourresults suggest that the nmuI gene is functional in N. multiformisand that long-chain AHLs solubilized the R protein (NmuR) of N.multiformis in extracts of recombinant E. coli. Moreover, the ab-sence of AHL signals in this strain might be attributed to possibleAHL-inactivating activities.

    MATERIALS AND METHODSBacterial strains and growth media. N. multiformis (ATCC 25196) wasgrown in ATCC medium 929 at 26C in the dark as described previously(14). N. multiformis cultures were subcultured into fresh medium after thepH had been adjusted three times, and cells were also harvested from a5-liter fermentor in fed-batch cultivation. (NH4)2SO4 was supplied in afed-batch manner from stock solution so that its concentration was no lessthan 10 mM. The pH and aeration of the reactor were further controlled at7.5 and 0.1 liter/min, respectively. E. coli strain BL21(DE3)/pLysS andderivatives of this strain were grown in Luria-Bertani (LB) medium and,where required, ampicillin (100 g/ml) or kanamycin (50 g/ml). Agro-bacterium tumefaciens A136 was incubated on LB medium plus tetracy-cline (4.5 g/ml) and spectinomycin (50 g/ml) at 30C.

    Bioinformatic analyses. To identify genes encoding AHL synthasesand transcriptional activators, the genome of N. multiformis, which wasmade available by the BLAST program (,was searched for genes with similarity to all QS-related genes. Withthe SWISS-MODEL server (, Clustal W2(, and the Swiss Protein DataBank (PDB) viewer (, amino acid sequence

    Received 9 October 2013 Accepted 17 November 2013

    Published ahead of print 22 November 2013

    Address correspondence to Xuliang Zhuang,, or GuoqiangZhuang,

    Copyright 2014, American Society for Microbiology. All Rights Reserved.


    February 2014 Volume 80 Number 3 Applied and Environmental Microbiology p. 951958 951

    on July 20, 2019 by guesthttp://aem


    nloaded from

  • alignments were achieved, and a three-dimensional homology model ofNmuI was automatically created by using the LasI structure as a template.

    DNA manipulations. Genomic DNA isolated from N. multiformis wasused as the template for nmuI gene amplification with the primers 5=-CAGGATCCATGCTTGCACAACATGGCA-3= (5= end) and 5=-CCGCTCGAGTCATGCGGCCTTCCTTTG-3= (3= end). The 5=-end primer includeda BamHI restriction site, and the 3=-end primer included an XhoI restric-tion site (underlined). PCR was performed by incubation for 2 min at95C followed by 30 cycles of 30 s at 95C, 30 s at 51C, and 1 min at 72C,with a final step of 10 min at 72C. Amplified nmuI was cloned intopGEX-4T-1 (GE) as described by the manufacturer, and the resultingrecombinant plasmid was termed pGEX-nmuI. We also created the LuxRhomolog protein (NmuR) expression plasmid pET-R. The NmuR genewas amplified with the primers 5=-CCGGAATTCATGGATAACTTGACCTTATTC-3= (5= end) and 5=-CCGCTCGAGCTAGCGAATCGTACGGGG-3= (3= end). The 5=-end primer included an EcoRI restriction site, andthe 3=-end primer included an XhoI restriction site (underlined). PCR wasperformed by the procedure described above. The amplified DNA frag-ment was cloned into pET-30a () (Novagen) as described by the sup-plier, and the resulting recombinant plasmid was termed pET-R. pGEX-nmuI and pET-R were transformed into E. coli BL21(DE3)/pLysS, and thetransformants were grown on LB medium containing ampicillin (100g/ml) and kanamycin (50 g/ml), respectively, at 37C. Details of thecharacteristics of bacterial strains and plasmids used in this study areshown in Table 1.

    AHL reporter plate assays and LC-MS analysis. The bacterial cellsand supernatants were extracted three times with half a volume of acidi-fied ethyl acetate (EtAc) containing 0.2% glacial acetic acid. Supernatantwas dried by N2 gas and the residue reconstituted in high-pressure liquidchromatography (HPLC)-grade acetonitrile. The extracts were spottedonto overlaid LB agar plates (containing 80 g/ml 5-bromo-4-chloro-3-indolyl--D-galactopyranoside [X-Gal]) mixed with the AHL reporterstrain A. tumefaciens A136 (15) and incubated overnight at 30C. AHLprofiling was confirmed by use of a Waters Micromass Q-TOF micromassspectrometer (LC-MS) system using a C18 reverse-phase column (5 mby 250 mm by 4.6 mm) (Zorbax Eclipse XDB-C18; Agilent) coupled withpositive-ion electrospray ionization (ESI) mass spectrometry (16). Theelution procedure consisted of an isocratic profile of acetonitrile-water(60:40 [vol/vol]) for 10 min followed by a linear gradient from 60 to 100%acetonitrile in water over 18 min and an isocratic profile over 22 min. ESIspectra (m/z range, 50 to 400) containing a fragment product at m/z 102were compared for retention time and spectral properties to the corre-sponding synthetic AHL standards. All detected AHL standards were ob-tained from Sigma-Aldrich.

    To determine whether AHLs were inactivated by N. multiformis, thestrain and medium solution were incubated with 10 M long-chain AHLs(C14-AHL and 3-oxo-C14-AHL) for 0 h, 4 h, or 8 h, after which the culturesupernatants were spotted onto AHL reporter plates (see above). Theresidual AHLs were detected by use of A. tumefaciens A136.

    RNA manipulations. To study if nmuI and nmuR were functional inN. multiformis, gene expression was analyzed by reverse transcription-PCR (RT-PCR). Total RNA was extracted from the bacterial culture dur-ing exponential growth by using TRIzol reagent (Life


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