JOURNAL OF BACTERIOLOGY, Nov. 1997, p. 6851–6854
0021-9193/97/$04.0010
Copyright © 1997, American Society for Microbiology

Vol. 179, No. 21NOTES
Cloning of a Vibrio alginolyticus rpoN Gene That Is Requiredfor Polar Flagellar Formation
IKURO KAWAGISHI, MASANORI NAKADA, NORIKO NISHIOKA,
AND MICHIO HOMMA*Division of Biological Science, Graduate School of Science,
Nagoya University, Chikusa-Ku, Nagoya 464-01, Japan
Received 7 July 1997/Accepted 28 August 1997

A fragment of DNA was cloned which complemented a polar flagellum-defective (pof) mutation of Vibrio
alginolyticus. The fragment contained two complete and two partial open reading frames (ORFs) (ORF2 and -3
and ORF1 and -4, respectively). The presumed product of ORF2 has an amino acid sequence with a high degreeof similarity to that of RpoN, which is an alternative sigma factor (s54) for other microorganisms. The otherORFs are also homologous to the genes adjacent to other rpoN genes. Deletion analysis suggests that ORF2
complements the pof mutation. These results demonstrate that RpoN is involved in the expression of polar
flagellar genes.

Cloning of the pof gene. Most of the strains and plasmids
used in this work have been listed previously (28). Plasmid
pSU18 and chromosomal DNA from V. alginolyticus 138-2
were digested with EcoRI and BamHI and ligated. These DNA
libraries were transferred into YM14 cells by electroporation
as described previously (16), and the cells were inoculated onto
0.3% agar VC plates (0.3% agar in VC medium [0.5% Polypeptone, 0.5% yeast extract, 0.4% K2HPO4, 3% NaCl, 0.2%glucose]) supplemented with chloramphenicol. The only colony which spread on the plate was isolated, and the plasmid
DNA from the clone was recovered (Fig. 1A). It contained a2.4-kb EcoRI fragment (Fig. 1B). The plasmid was named
pVIK14 and was used for further analysis. When plasmid
pVIK14 was introduced into YM14 cells, the resulting cells
spread on 0.3% agar VC plates as fast as wild-type cells (datanot shown), and the formation of polar flagella was confirmedby high-intensity dark-field microscopy (12). Therefore, weconcluded that the EcoRI fragment contained the pof gene.
When the plasmid pVIK14 was introduced into other pof mutants, YM44 cells also acquired motility.
The 2.4-kb fragment was hybridized to chromosomal DNA
digested by EcoRI in high-stringency conditions (Fig. 2). Single
bands of 2.4 and 5 kb were detected in V. alginolyticus andV. parahaemolyticus, respectively. In the two pof mutants, the2.4-kb band was detected. No band was detected in Salmonella
typhimurium or Escherichia coli.
Nucleotide sequence of the pof gene. We then subcloned thevarious regions of the 2.4-kb fragment into the vector pSU21,whose multiple cloning site is different from that of pSU18(Fig. 1B). Only pMSA6 complemented the pof defect of
YM14. Next, we determined the entire nucleotide sequence ofthe 2.4-kb EcoRI fragment by the dideoxy chain termination
method with the Sequenase version 2.0 DNA sequencing kit(United States Biochemical Corp., Cleveland, Ohio). [a-32P]
dCTP (3,000 mCi/mmol; Amersham Japan, Tokyo) was usedfor radioactive labeling.
Two complete and two partial open reading frames (ORFs),
all in the same orientation, were identified in the fragment andMany bacteria move by means of rotating flagella, locatedeither peritrichously or at a pole on the cell body. Certain
marine Vibrio species are unique in that they have two types offlagella: a single polar flagellum suited for swimming in a liquid
environment and numerous lateral flagella suited for swarming
over the surfaces of animate or inanimate objects (2, 4, 20, 24).
In Vibrio parahaemolyticus and the closely related Vibrio alginolyticus, it has been shown that polar and lateral flagella arepowered by different ion-motive forces: the coupling ion of thepolar flagellar motor is sodium and that of the lateral flagellar
motor is a proton (5, 15).
The lateral flagella are synthesized under viscous conditions(2, 6, 24, 31). An increase in viscosity is a cue for induction oflateral flagellar expression (6). Surprisingly, a decrease in therotation of the polar flagellum is sensed and lateral flagellar
expression is induced (14, 23). Thus, the polar flagellum functions not only as a locomotive organelle but also as a mechanosensor which couples viscous drag to cell differentiation.
The polar flagella of V. alginolyticus rotate very fast, up to 1,700
rps, and the rotation is stable (22, 27). Furthermore, strictly
speaking, the synthesis of polar flagella may not be constitutive. It should be more or less coupled with the cell division
cycle, since most of the Vibrio cells have a single polar flagellum at one of the cell poles.
To investigate these unique features of the polar flagellum inV. alginolyticus, we wanted to clone the genes involved. From alateral flagellum-defective mutant, we have isolated many polar flagellum-defective mutants which are defective in bothpolar and lateral flagellar formation (12, 15, 28). In this study,
using one of those strains as a recipient for shotgun cloning byelectroporation (16), we cloned a pof gene involved in polar
flagellar formation. Nucleotide sequencing of the gene and theflanking region revealed that the pof gene encodes an alternative sigma factor, s54 (RpoN).

* Corresponding author. Phone: 81-52-789-2992. Fax: 81-52-7893001. E-mail: g44416a@nucc.cc.nagoya-u.ac.jp.
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FIG. 2. Southern hybridization with the rpoN coding region. Total chromosomal DNAs, which were prepared for strains ST1 (S. typhimurium), W3110(E. coli), and YM14, 138-2, YM44, and BB22 (V. parahaemolyticus) were digested with EcoRI and separated by agarose gel electrophoresis. Southern hybridization was performed by using a probe prepared from an EcoRI fragment ofpVIK14 according to a manual from the manufacturer (ECL direct nucleic acidlabeling and detection system; Amersham).

FIG. 1. Restriction map and complementation assay. (A) Swimming recoveryof YM14 cells carrying the plasmid pVIK14. Single colonies of the chloramphenicol-resistant transformants with pSU18 (vector) or pVIK14 and of the hoststrain (YM14) were inoculated on a 0.3% agar VC plate and incubated at 30°C.
(B) Restriction map of plasmid pVIK14 and its deletion derivatives. The plasmids carry the fragments indicated by solid lines. Their abilities to complement
the defect of strain YM14 are indicated on the right side of the map. The coding
regions, ORF1, -2, -3, and -4, were predicted from the DNA sequence.

named ORF1, ORF2, ORF3, and ORF4 in the order shown inFig. 1B. The noncoding regions between the ORFs are 54, 27,
and 5 bp. From the results of the complementation assay, weconcluded that the longest ORF (ORF2; 489 amino acids) isthe pof gene (Fig. 1B).
A search of the protein database revealed that the deduced
amino acid sequence of the pof gene product is very similar to
those of the rpoN gene products (s54) of a wide variety ofbacteria (Fig. 3). The identity of ORF2 with the various RpoN
sequences is about 60%. We found all the diagnostic featuresof RpoN (i.e., a glutamine-rich region, an acidic central region,a helix-turn-helix motif, and an RpoN box) in the deduced pof
product. We therefore concluded that this gene encodes s54
and named the gene rpoN. However, the mutants did not show
glutamine auxotrophy and they grew on ammonium ions as thesole nitrogen source.
ORF1, ORF3, and ORF4 were also similar to the rpoNlinked genes (Fig. 4). The order of the homologous genes is thesame as those in other species, such as E. coli (30). The ORF1
protein may be an ATP-binding protein and a member of theABC transporter family (11), and the ORF1 homolog wassuggested to be an essential gene in Rhizobium meliloti (1). Thefunction of the ORF3 protein (95 amino acids), whose gene isjust downstream of rpoN, is not known. The ORF4 protein ishomologous to an enzyme, IIA, of the phosphoenolpyruvatedependent phosphotransferase system (30).
Implications of regulation of polar flagellum gene expression. The extensive similarity of its gene product to s54 in manyother bacteria and the existence of the three rpoN-flanking
genes indicate that the pof gene encodes an alternative sigmafactor, s54 (RpoN). This is consistent with the previous findingthat a gene named motY, which encodes a component of thesodium-driven polar flagellar motor, has a sequence very similar to the consensus of the s54-dependent promoters (28). In
V. parahaemolyticus, a potential s54 consensus promoter sequence in addition to the s28 consensus promoter may direct
the genes for hook-associated proteins or flagellin (25). Fur-FIG. 3. Amino acid sequence comparison of ORF2 with s54 from variousbacteria. The sequences were derived from Klebsiella pneumoniae, S. typhimurium, P. putida, and Azotobacter vinelandii. Residues identical to those of
ORF2 are indicated by periods, and gaps are indicated by hyphens. The percent
identities of ORF2 with the various RpoN sequences are shown at the ends of thesequences.

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flagellum-related genes are transcribed from s54-dependent
promoters (3, 35). As suggested for P. aeruginosa (32) and C.
crescentus (9), it is possible that s54 and s28 in marine Vibrio
spp. may regulate the expression of polar flagellum genes atdifferent levels in the regulatory hierarchy. Interestingly, nos54-dependent promoter has been described in any gene for
peritrichous flagella. Considering that polar flagella seem to besynthesized once in a cell cycle and that rpoN expression is cell
cycle-dependent in C. crescentus (8–10), we speculate that theregulation of s54 activity may be responsible for the determination of polar flagellation.
Nucleotide sequence accession number. The sequence dataof the 2.4-kb fragment have been deposited with DDBJ underaccession no. AB006709.
We thank Linda McCarter for critically reading the manuscript andS. Kojima for assistance in the construction of a plasmid.
This work was supported in part by grants in aid for scientificresearch to I.K. from the Ishida Foundation and from the Ministry ofEducation, Science and Culture of Japan and to M.H. from the research and development program of NEDO of Japan.

FIG. 4. Amino acid sequence comparison of the rpoN-flanking gene productsof ORF1 (A), ORF3 (B), and ORF4 (C) of V. alginolyticus with those of various
bacteria. The sequences were derived from Klebsiella pneumoniae, S. typhimurium, P. aeruginosa, Azotobacter vinelandii, Thiobacillus ferrooxidans, Alcaligenes eutrophus, Rhizobium meliloti, and Bradyrhizobium japonicum. Residues
identical to those of ORF1, ORF3, or ORF4 are indicated by periods, and gaps
are indicated by hyphens.

thermore, the predicted product of flaK, which is located in acluster of polar flagellum genes of V. parahaemolyticus, is homologous to NtrC, a s54-interacting regulatory protein (33).
Therefore, we suggest that some, if not all, polar flagellumrelated genes, including those involved in flagellation, motility,and chemotaxis, are transcribed from s54-dependent promoters.
In E. coli and S. typhimurium, genes involved in flagellar
formation and function form contiguous clusters and are organized into a regulon (for reviews, see references 7 and 21).
Most of them are transcribed by RNA polymerase holoenzyme
containing s28, encoded by fliA, which itself is a member of theregulon (18, 19). In V. parahaemolyticus also, expression of atleast one lateral flagellum gene coding for the filament subunit
protein (flagellin) depends on s28, encoded by lafS (26). In
other bacteria, another alterative sigma factor, s54, is activatedby environmental changes, such as lack of nutrients, to express
the genes involved in responses to the environment (17). In
several bacteria with polar flagella, such as Pseudomonas
aeruginosa (34), Pseudomonas putida (13), Caulobacter crescentus (9), and Vibrio anguillarum (29), rpoN mutants do not
produce flagella. Furthermore, it has been shown that some

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