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Applied and Environmental Microbiology, April 2004, p. 2520-2524, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2520-2524.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vibrio fischeri {sigma}54 Controls Motility, Biofilm Formation, Luminescence, and Colonization

Alan J. Wolfe,1 Deborah S. Millikan,2 Joy M. Campbell,1 and Karen L. Visick1*

Department of Microbiology and Immunology, Loyola University Chicago, Maywood, Illinois 60153,1 Pacific Biomedical Research Center, University of Hawaii, Honolulu, Hawaii 968132

Received 19 September 2003/ Accepted 29 December 2003


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ABSTRACT
 
In this study, we demonstrated that the putative Vibrio fischeri rpoN gene, which encodes {sigma}54, controls flagellar biogenesis, biofilm development, and bioluminescence. We also show that rpoN plays a requisite role initiating the symbiotic association of V. fischeri with juveniles of the squid Euprymna scolopes.


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INTRODUCTION
 
The sigma factor {sigma}54, encoded by rpoN, is distributed widely among bacteria. The ability of {sigma}54 to regulate nitrogen metabolism is well established (reviewed in reference 16). In Escherichia coli, about half of all {sigma}54-dependent operons function in the assimilation and metabolism of nitrogen (28). In other organisms, {sigma}54 regulates diverse functions in addition to controlling nitrogen metabolism. In Vibrio parahaemolyticus, it controls the biogenesis of the polar flagella required for swimming and the lateral flagella necessary for swarming (31). In Vibrio harveyi, it regulates motility and bioluminescence (14). In Pseudomonas aeruginosa, it activates transcription of both the flagellin and pilin genes, negatively affects quorum-sensing genes, and promotes virulence (8-10, 32). In Vibrio cholerae, it regulates flagellin gene transcription and influences mouse colonization. Its role in colonization, however, appears distinct from the requirement for motility and nitrogen assimilation (13).

The symbiosis between the bioluminescent marine bacterium Vibrio fischeri and the Hawaiian squid Euprymna scolopes has become established as a model system for investigating mutualistic associations (reviewed in references 18 and 35). Colonization of newly hatched juvenile E. scolopes occurs rapidly and requires motility: nonmotile bacteria fail to colonize (5), while hypermotile mutants exhibit delays in colonization (21). A recent report characterized the symbiotic phenotype of a mutant defective for FlrA, the putative {sigma}54-dependent transcriptional activator likely to be at the top of the flagellar hierarchy in V. fischeri (22). The flrA mutant was defective for both motility and colonization. This mutant was fully complemented for motility but only partially complemented for colonization. Thus, the flrA colonization defect may result from more than a lack of flagella (22).

In this study, we identified a nonmotile mutant of V. fischeri defective for the putative rpoN gene, which encodes the sigma factor {sigma}54. Given the scope of its regulatory control in other bacteria and the importance of the putative {sigma}54-dependent transcriptional activator FlrA in establishing the V. fischeri-E. scolopes symbiotic relationship, we examined the role of rpoN in culture and during colonization. We asked whether rpoN regulates traits known to be or potentially associated with symbiotic colonization, including flagellar biogenesis (5, 21, 22), nitrogen metabolism, iron uptake (6), and bioluminescence (34).


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Role for the rpoN gene in motility.
 
In a search for flagellar mutants of V. fischeri, we identified a transposon insertion mutant, KV618 (Table 1), that failed to migrate in tryptone-based soft agar plates. We complemented the defect in this strain by introducing a plasmid library of BglII-digested chromosomal DNA and screening for motility in soft agar plates (38). From a motile clone, we isolated plasmid pES2-2 (Table 1), which carried a 2.5-kb chromosomal insert resembling the rpoN locus from a number of other organisms, including E. coli, V. harveyi, and V. cholerae (11, 13, 14). It contained putative genes for an ABC-type transporter (yhbG, open reading frame 1 [ORF1]), {sigma}54 (rpoN), a {sigma}54 regulatory gene (yhbH, ORF 95), and a nitrogen regulatory phosphotransferase component (ptsN) (Table 2). We cloned the DNA flanking the transposon insertion and determined that the transposon had inserted into codon 10 of the putative rpoN gene.


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  		TABLE 1. List of strains and plasmids used in this study


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  		TABLE 2. Sequence analysis of the rpoN locus

To characterize the rpoN gene further, we first constructed a null mutation in the symbiosis-competent wild-type V. fischeri strain ES114. We introduced plasmid pLMS71 (Table 1) into ES114 by conjugation and isolated a nonmotile recombinant, designated KV1513 (Table 1). We confirmed the identity of the mutant with Southern analysis. The mutation introduces a KpnI site; therefore, we probed KpnI-digested chromosomal DNA with pES2-2 and found, as expected, that the mutant contained two smaller bands in place of the single larger band of the wild-type strain.

Examination by transmission electron microscopy revealed that KV1513 lacked flagella (data not shown). Introduction of pES2-2 restored motility to KV1513 (data not shown), although migration of the complemented strain on soft agar plates was delayed relative to that of the plasmid-bearing wild-type counterpart. These data suggest that complementation with rpoN on a low-copy-number plasmid is not optimal for the motility of V. fischeri. Prolonged incubation of the rpoN null mutant KV1513 on soft agar plates failed to yield motile revertants (data not shown), an observation similar to that made with null mutants of V. cholerae rpoN (13) and V. fischeri flrA (22). These results suggest that V. fischeri motility absolutely requires {sigma}54, which likely operates in conjunction with FlrA.

In V. cholerae, {sigma}54 controls flagellar gene transcription on at least two levels (13, 27). In conjunction with the transcriptional activator FlrA, {sigma}54 induces transcription of the class II genes flrB and flrC, which encode a two-component signaling pathway. With FlrC, {sigma}54 then activates class III gene transcription, including the major flagellin subunit, flaA. Since V. fischeri motility also requires flrA and flaA homologs (22; D.S. Millikan and E.G. Ruby, unpublished data), we determined whether rpoN controls transcription of the V. fischeri flaA gene. Into the rpoN mutant KV1513 and its wild-type parent we introduced the plasmid pDM104-6 (Table 1), which carries a lacZ reporter gene fused downstream of the flaA promoter. We grew the resultant transconjugants in SWT medium (2) and measured ß-galactosidase activity when the cells reached the mid-exponential phase of growth (A600 = 1.1 to 1.3). Transcription of the reporter gene was reduced by 10-fold in the rpoN mutant, a result similar to that achieved by the flrA mutant (Table 3). These data confirm that {sigma}54 plays a role in motility by activating transcription of at least one flagellar gene.


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  		TABLE 3. Transcription of flaA in wild-type and mutant strains


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Role for the rpoN gene in biofilm formation.
 
In some organisms, biofilm formation is enhanced by the presence of flagella (25, 26, 33, 37). Therefore, we asked whether the ability of V. fischeri to form biofilms in culture was enhanced by the presence of the rpoN gene. To assay biofilm formation, we pregrew cells in SWT at 28°C with shaking and then transferred the cultures to glass test tubes. The cells were incubated without shaking for 10 h at 28°C and then exposed to a solution of 1% crystal violet to visualize cells that had formed a biofilm on the test tube (25). After further incubation for 15 min, the tubes were rinsed with distilled H2O. Biofilms formed at the air-liquid interface were stained purple. Under these conditions (Fig. 1A) and others (data not shown), the rpoN mutant formed a biofilm that differed from that of the wild type: it was consistently broader and stained less intensely. Since levels of growth of the rpoN mutant and its wild-type parent were similar (Fig. 1B), these data support a role for rpoN in biofilm formation. To control for the role of flagella in biofilm formation, we examined the biofilm-forming ability of another mutant, KV661 (Table 1), that cannot form flagella because of a disruption in the key biosynthetic flgB operon. Since this mutant produced biofilms similar to those of its wild-type parent (data not shown), the role of rpoN in biofilm formation must be independent of its role in flagellar gene expression.



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  		FIG. 1. Biofilm and static growth of the rpoN mutant and its parent. (A) Wild-type V. fischeri and rpoN mutant KV1513 were grown for 10 h in static culture in glass test tubes and then stained with 1% crystal violet. Representative biofilm bands are shown. (B) Growth of the wild type (filled squares) and rpoN mutant (open circles) strains in static culture is shown in triplicate.


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Nitrogen metabolism and iron sequestration by the rpoN mutant.
 
In a number of organisms, a functional copy of the rpoN gene is required for the assimilation of nitrogen from nonammonia sources, e.g., glutamine and serine (19, 28). Thus, we examined growth of the V. fischeri rpoN mutant in the presence of a variety of nitrogen sources. The rpoN mutant was not defective for growth in rich media, such as SWT, at a variety of temperatures (data not shown), nor was it defective for growth in a glucose minimal medium (MM-G) (29) containing ammonium chloride and Casamino Acids (Fig. 2A), ammonium chloride (Fig. 2B), or glutamine (data not shown). In contrast to its parent, the rpoN mutant grew poorly in MM-G containing serine as the sole nitrogen source (Fig. 2C). Thus, the V. fischeri rpoN gene controls assimilation of nitrogen from serine.



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  		FIG. 2. Growth of the rpoN mutant in minimal media containing various nitrogen sources. Wild-type strain ES114 (filled squares) and the rpoN mutant KV1513 (open circles) were pregrown in MM-G containing NH4Cl and Casamino Acids, washed with MM-G without a nitrogen source, and then inoculated into MM-G containing NH4Cl and Casamino Acids (A), NH4Cl (B), and Ser (C). ES114 (open squares) and KV1513 (filled circles) were also inoculated into MM-G lacking a nitrogen source as a control (C). Growth was measured using a spectrophotometer at 600 nm. The experiment was performed in triplicate, and the error bars represent the standard deviations. In panel C, growth of KV1513 in the presence of serine is indistinguishable from its growth in media lacking a nitrogen source. In some experiments, growth of KV1513 in MM-G with serine was delayed but reached an optical density measurement similar to that of the wild-type strain at the 24-h time point.

In V. fischeri, siderophore production and thus iron uptake depend upon GlnD (6), a protein that modulates the E. coli and Salmonella enterica response to nitrogen availability (reviewed in reference 16). The pathway by which V. fischeri GlnD regulates siderophores remains unknown. However, in E. coli, GlnD (the uridylyltransferase/uridylyl-removing enzyme) modulates PII, a protein whose activity ultimately affects activation of the {sigma}54-dependent transcriptional activator NtrC. We anticipated, therefore, that a defect in {sigma}54 might affect iron uptake in a manner similar to that of the V. fischeri glnD mutant. Instead, the rpoN mutant retained a significant ability to sequester iron from the environment, in contrast to the isogenic glnD mutant (data not shown). Although the rpoN mutant grew more slowly than did its wild-type parent on chrome azurol S siderophore assay plates, its growth was not significantly different from that of wild-type cells in the presence of the iron chelator ethylenediamine-di(o-hydroxyphenyl-acetic acid) (data not shown). Thus, GlnD likely controls siderophore production by a pathway that does not involve rpoN.


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Bioluminescence emission by the rpoN mutant.
 
Luminescence regulation in V. fischeri requires the prototypical quorum-sensing system, which consists of the transcriptional activator LuxR (a member of the TetR family) and the autoinducer synthase LuxI (4). Regulation of V. fischeri luminescence also involves homologs of the V. harveyi luxO and luxR (distinct from the V. fischeri luxR) genes (3, 15, 23). Inactivation of the V. fischeri luxR homolog, litR, delayed and reduced light emission in culture (3), while inactivation of the luxO homolog in V. fischeri strains MJ1 and ES114 resulted in increased luminescence (15, 23). The latter phenotype is identical to that of a V. harveyi luxO mutant (1).

Because the V. harveyi LuxO protein functions as a {sigma}54-dependent transcriptional activator (14), we asked whether regulation of bioluminescence in V. fischeri involves the rpoN gene. We monitored the light emission of the rpoN mutant and its wild-type parent during growth of the cells in SWT medium. Under a variety of aeration, medium, and temperature conditions, the rpoN mutant consistently achieved higher bioluminescence levels, such as the three- to fourfold difference at the peak of luminescence shown in Fig. 3. This result is similar to that seen with a V. fischeri luxO mutant (15). This observation supports a role for rpoN in repressing luminescence in V. fischeri, likely in conjunction with the luxO homolog. The target for these regulators remains to be determined.



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  		FIG. 3. Bioluminescence of the rpoN mutant and its parent in culture. The rpoN mutant KV1513 (open circles) and its parent ES114 (filled squares) were grown at 24°C in 125-ml flasks containing 25 ml of SWT (2). Luminescence was measured over time using a Turner 20/20 Biosystems luminometer at the factory settings. Specific luminescence was determined by dividing the relative luminescence per milliliter by the optical density at each time point. The experiment was repeated multiple times under a variety of temperature and aeration conditions. The rpoN mutant consistently emitted higher levels of luminescence. Data shown are from a representative experiment in which the average of values for three cultures of each strain are plotted. Error bars represent standard deviations.


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Colonization by the rpoN mutant.
 
Because colonization of E. scolopes requires flagella and {sigma}54 controls flagellar biogenesis, we determined whether colonization requires {sigma}54 by exposing juvenile squid to the rpoN mutant KV1513 or to its wild-type parent. Consistent with previous reports of nonmotile strains (5, 22), the rpoN mutant did not initiate symbiotic colonization, as measured by symbiotic bioluminescence and viable counts of squid-associated bacteria in a representative experiment (seven squid). In contrast to wild-type-exposed squid, which contained on average 3.46 x 105 CFU/squid, animals inoculated with KV1513 either were uncolonized (four of seven animals) or exhibited colonization levels of <100 CFU/squid (three of seven animals). The apparent low level of colonization in the latter squid group likely reflects bacteria aggregated on the surface of the light organ rather than bacteria present inside the light organ, as previously reported (22, 24).

Complementation with a functional copy of rpoN (pLD6) restored the ability to initiate symbiotic colonization (84 to 90% of animals inoculated with the rpoN mutant versus 90 to 96% of animals inoculated with the wild-type parent). This rate of initiation was greater than that reported (49%) for the complemented flrA mutant (22) and likely reflects differences in the ways the assays were performed. The colonization levels achieved by squid inoculated with the complemented rpoN mutant varied, but some animals achieved wild-type levels of colonization. Thus, while these data clearly demonstrate a role for rpoN in symbiotic initiation, a role for this regulator in subsequent stages cannot be assessed with the tools currently available; further investigation of its role in symbiotic colonization will require construction of strains with a tightly regulated promoter upstream of the rpoN gene.


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Summary.
 
This work shows that {sigma}54 of V. fischeri plays multiple roles. It controls flagellar biogenesis and, thus, motility. It contributes to biofilm development, nitrogen assimilation, and the regulation of bioluminescence. Finally, {sigma}54 plays an essential role in the establishment of symbiotic colonization, most likely due to its requirement for motility. Whether other {sigma}54-dependent traits contribute to symbiotic initiation or to later stages of colonization remains to be determined. This work provides a foundation for a deeper understanding of the contributions to symbiotic colonization of {sigma}54-dependent determinants such as motility, bioluminescence, and biofilm development.

Nucleotide sequence accession number. The 2.5-kb chromosomal insert from plasmid pES2-2 has been assigned GenBank accession number AY082659.


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ACKNOWLEDGMENTS
 
We thank Cindy R. DeLoney for examining the rpoN mutant by transmission electron microscopy, Erika Simel for her assistance in cloning and sequencing the rpoN gene, Therese M. Bartley for expert technical assistance, and members of our labs for critical reading of the manuscript.

This work was supported by NIH grant GM59690 awarded to K.L.V.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Immunology, Loyola University Chicago, 2160 S. First Ave., Bldg. 105, Rm. 3860A, Maywood, IL 60153. Phone: (708) 216-0869. Fax: (708) 216-9574. E-mail: kvisick{at}lumc.edu. Back


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Applied and Environmental Microbiology, April 2004, p. 2520-2524, Vol. 70, No. 4
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.4.2520-2524.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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