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Infection and Immunity, June 2005, p. 3749-3753, Vol. 73, No. 6
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.6.3749-3753.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Genetic Basis for Conversion of Rough-to-Smooth Colony Morphology in Actinobacillus actinomycetemcomitans
Ying Wang,
Amy Liu, and
Casey Chen*
University of Southern California School of Dentistry, Los Angeles, California
Received 18 January 2005/
Accepted 8 February 2005

ABSTRACT
The basis of the rough-to-smooth conversion of
Actinobacillus actinomycetemcomitans was examined. Smooth variants often contained
mutations at the
flp promoter region. Replacing the mutated
flp promoter with the wild-type promoter restored the rough
phenotype. The expression level of the
flp promoter was

100-fold
lower in smooth than in rough strains. Mutations of the
flp promoter are a cause of the rough-to-smooth conversion.

TEXT
Gram-negative, facultatively anaerobic
Actinobacillus actinomycetemcomitans is a major periodontal pathogen (
1,
20). Fresh oral isolates
of
A. actinomycetemcomitans are invariably fimbriated and form
small (

1-mm), rough-surface, translucent colonies with an internal
star-shaped structure (
2,
9,
15,
20). After repeated in vitro
passages, the rough-colony morphotype may yield nonfimbriated
smooth-colony variants that grow as large, round, opaque colonies
on agar (
2,
9,
15,
16). Occasionally, the rough-to-smooth transition
goes through an intermediate phase in which the colonies are
translucent but smooth surfaced (
9). Genes for the fimbria biogenesis
of
A. actinomycetemcomitans reside in a 12-kb
flp operon that
contains 14 genes,
flp-1-flp-2-tadV-rcpCAB-tadZABCDEFG (
6,
8-
12,
14). The transcription-initiation points of the operon were
located at 101 and 102 nucleotides upstream of
flp-1 (
5). Two
consensus elements, 10 (TATAAT) and 35 (TTGCAT),
separated by 16 nucleotides, of the canonical
70 promoter sequence
were identified upstream of the transcription-initiation points
(
5).
Many pathogenic bacteria are capable of phase variation and colonial morphology shift, which depends on the expression of surface proteins (3, 13, 18). While the rough-to-smooth conversion of A. actinomycetemcomitans occurs spontaneously, the reverse smooth-to-rough conversion has not been substantiated. We postulated that the rough-smooth conversion in A. actinomycetemcomitans is due not to a phase variation mechanism of the fimbria expression but to some mutational event of the flp operon. This study aimed to determine whether mutations at the promoter region of the flp operon might explain the rough-to-smooth conversion of A. actinomycetemcomitans.
Eighteen A. actinomycetemcomitans strains were examined (Table 1). The culture media and conditions for A. actinomycetemcomitans were as described previously (16). The sequences of the flp promoter of these strains were determined by direct sequencing of the PCR products amplified from this region. A 1-kb DNA fragment encompassing the flp-1-flp-2 genes and 300 bp upstream of flp-1 was amplified with the forward primer F-Ev24 (5'-TCGCGATATCTCTAAATCCACACA; an EcoRV site is underlined), which is located 300 bp upstream of flp-1 (see Fig. 1 for its location). Three different reverse primers were used. Most of the study strains were amplified with the reverse primer orfB-X, 5'-TATCTAGAACGGAATAATGGCGAATA (an XbaI site incorporated) located in orfB (Fig. 1). Two other reverse primers, orfB-B1, 5'-CAGGATCCAGCAGCGAGAGCGTTAT, and orfC-R, 5'-GCACTGAAATGATCAAGAGC, were used for strains that failed to be amplified with the primer orfB-X. PCR was carried out for 30 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min. PCR products were purified by the PCR purification columns (QIAGEN) and sequenced by the USC School of Medicine Microchemical Core Facility.
Figure
2 is a summary of the sequencing results of the 35
site, the spacer, and the 10 site. More detailed information
on the entire sequenced regions may be accessed through GenBank.
All four rough clinical isolates (D7S, D9, D28, and 29R) had
a promoter essentially identical to that identified by Haase
et al. (
5). Two published
flp sequences from strain CU1000 (GenBank
AY157714) (
12) and strain 310a (GenBank
D83053) (
10) also contain
the same promoter sequence. Therefore, this sequence was designated
as the consensus sequence. The rough-colony strain HK1651 exhibited
a one-base variation in a semiconserved base at the 10
site (Fig.
2). In broth culture, strain HK1651 formed less compact
aggregates than did other rough clinical strains. Presumably,
this base variation of strain HK1651 may slightly reduce the
flp promoter strength. The flanking regions of the promoter
exhibited a greater sequence variation among strains, but the
variations did not correlate with the rough-smooth conversion
(data not shown).
Five of the eight smooth- or intermediate-colony derivative
strains and three of the six smooth-colony reference strains
exhibited sequence variations in the conserved 10 site.
Six of these variations were due to a single-nucleotide substitution,
and the remaining two variations were due to a single-nucleotide
deletion. A frequent mutation was the transition of the most
conserved T residue of the 10 site. This base was often
called "invariable" T, as it was conserved in 97% of
Escherichia coli promoters among 112 promoter sequences compiled, and mutations
of this base were demonstrated to severely affect a number of
promoters' activities in
E. coli (
7,
19). Strain JP2 exhibited
a single-nucleotide deletion in the spacer region between the
conserved 35 and 10 sites. Strain ATCC 29524 showed
a deletion of the entire 35 and part of the spacer region.
The promoter sequences of the remaining three derivative strains
(strains D7S-ID, D28-IA, and 29-SS) and the smooth reference
strain ATCC 29523 were identical to the consensus sequence or
the sequence of the parental strain (i.e., strain 29R).
The bacterial morphology was examined by transmission electron microscopy (TEM) by a previously described protocol (17). As expected, the smooth-colony strain D7S-SA was nonfimbriated, in contrast to its parental rough-colony strain D7S (Fig. 3). We have examined 15 other smooth-derivative strains from clinical isolates and have not detected the presence of fimbriae (data not shown). However, TEM of strain ATCC 29523 showed a few thin fibrils that resembled fimbriae (Fig. 3). It is interesting that strain ATCC 29523 has a wild-type flp promoter. We also noted a tendency of strain 29523 to aggregate in broth cultures. The low expression of fimbria may explain the aggregation of ATCC 29523 in broth.
The mutated
flp promoters of several smooth-colony variants
were replaced with a wild-type promoter. A 1-kb PCR DNA fragment
encompassing
flp-1-flp-2 and the promoter was amplified from
rough strain D7S using primers F-Ev24 and orfB-X (Fig.
1 shows
primer locations). The DNA was then digested with EcoRV and
XbaI, ligated to pTc-USS at the same sites, and transformed
into smooth-colony strains D7S-SA and D7S-SB by a natural transformation
protocol described previously (
16). The plasmid pTc-USS is a
pBluescript II KS (Stratagene) derivative that does not replicate
in
A. actinomycetemcomitans (
17). Therefore, the Tc
r transformants
should have the plasmid containing the wild-type
flp promoter
integrated in the chromosome by a single crossover. If the point
mutation in the
flp promoter accounted for the rough-to-smooth
conversion in strains D7S-SA and D7S-SB, insertion of a promoter
(presumably any well-expressed promoter) upstream of the
flp operon should restore the rough-colony phenotype. As expected,
the transformants were predominantly of rough-colony type (an
example is shown in Fig.
4). The integration of the plasmid
in the chromosome was verified in selected rough colonies by
PCR using two pairs of primers, F-Ev24/Umer and orfC-R/Rmer
(data not shown). We also replaced the promoter region of the
smooth variant D7S-SA with the corresponding promoter region
from different
A. actinomycetemcomitans strains. Again, the
1-kb promoter-
flp-1-
flp-2 region was amplified from the rough
strains HK1651 and 29R and smooth strains ATCC 29523 and ATCC
33384. Each PCR amplicon DNA was ligated to pTc-USS and transformed
into strain D7S-SA. The results showed that no rough transformants
were obtained with the donor DNA from strain ATCC 33384, while
most transformants with donor DNA from the other three strains
were predominantly rough-colony type (e.g., 75% of the transformants
were rough-colony type with donor DNA from strain ATCC 29523).
The results indicated that random mutations in the promoter
of the
flp operon are a mechanism for the rough-to-smooth conversion
of
A. actinomycetemcomitans.
Conversely, we replaced the
flp promoter of the rough-colony
strain D7S with the promoter obtained from smooth-colony variants.
A 2.3-kb DNA fragment that included the
flp promoter region
was amplified by PCR from each of the smooth-colony strains
JP2, Y4, and ATCC 29524 with primers orfC-R and flp-UE (5'-CTGAATTCTCGCTCAGATACGGA,
which is located at 1.1 kb upstream of the promoter region)
and directly used as the donor DNA to transform strain D7S.
To enrich the nonfimbriated bacteria, the transformed bacteria
were briefly incubated in broth. Fimbriated D7S bacteria formed
aggregates that either settled to the bottom of the culture
tube or adhered to the tube, while the nonfimbriated smooth-colony
variants would grow as a single-cell suspension. The top portion
of the undisturbed broth culture of the transformed D7S was
collected and plated on agar. The results showed that the colonies
were predominantly smooth. The
flp promoters of four smooth-colony
variants (two were transformed with the
flp promoter from strain
JP2 and one each was transformed with the
flp promoter from
strains Y4 and ATCC 29524) were amplified by PCR and sequenced.
The results showed that each of the
flp promoters of these smooth-colony
variants carried the specific sequence variation of the
flp promoter found in the corresponding donor strain JP2, Y4, or
ATCC 29524. The smooth-colony transformant derived from transformation
with donor DNA of strain ATCC 29524 displayed an additional
insertion of two nucleotides downstream of the 10 site,
which may have arisen from PCR amplification or transformation.
However, the insertion occurred at the nonconserved area and
was not expected to be the cause of the reduced expression of
the
flp operon. The results further substantiated the role of
the
flp promoter mutations in rough-to-smooth conversion.
The expression levels of flp promoters in rough- and smooth-colony bacteria were measured by inserting a firefly luciferase gene, luc, as the reporter gene at the rcpB site of the flp operon (Fig. 5). Briefly, 3.2-kb DNA containing the Spe marker and the rcpB-flanking DNA was amplified from a previously constructed
rcpB::Spe mutant of strain D7S (unpublished data) with a primer at 1.1 kb upstream of rcpB and another primer at 1 kb downstream of rcpB. This 3.2-kb DNA was cloned in pBluescript II KS at the EcoRV site to produce pB-
rcpB. Separately, a luc-Spe cassette plasmid was constructed based on pBluescript II KS and pBRluc (4), and the plasmid was named pLuc-Spe2. A 3-kb luc-Spe reporter-marker was amplified from pLuc-Spe2 using the primer Luc-B1, 5'-AGGGATCCTAGGAAGCTTTCCATGGA (the luc start codon is underlined), and the primer Spe-USS, 5'-AAAGTGCGGTTTACACTTACTTTAGTTTT. This luc-Spe marker was then used to replace the 1.1-kb Spe marker in pB-
rcpB to create pLuc-
rcpB (Fig. 5), which was used to transform A. actinomycetemcomitans strains. Transformants were verified by PCR and tested for luciferase production. Briefly, bacteria were grown on serum trypticase soy broth agar overnight and resuspended in tryptic soy broth at an optical density (OD) at 600 nm of 0.5, and luciferase was assayed by mixing 20 µl of luciferin (1 mM in 0.1 M sodium citrate, pH 7.0) with 80 µl bacterial suspension (OD at 600 nm = 0.1 to 0.6) at room temperature for 5 min, and the light was counted twice with the BetaScout Liquid Scintillation Tester (Perkin-Elmer Life Sciences). Enzyme activity was defined as (photon counts in 10 s background counts)/0.001 OD unit of bacteria. The results (Table 2) showed that the luc expression in strain D7S-SA, with a T-to-C transition in the flp promoter, was 80-fold lower than that in the wild type and that expression in strain D7S-SC, with a T deletion, was 130-fold lower than that in the wild type. Interestingly, the luc activity in strain ATCC 29523 was fourfold lower than that in strain D7S. Perhaps the low-level expression of the flp promoter in ATCC 29523 resulted in the scant expression of fimbria seen under TEM.
Our findings suggest that in vitro spontaneous rough-to-smooth
conversion of
A. actinomycetemcomitans commonly occurs due to
mutations at the 35 site, the spacer region, or the 10
site of the
flp promoter. Such mutations are not likely to be
reversible at a significant rate and may explain the lack of
smooth-to-rough conversion among
A. actinomycetemcomitans strains.
However, several smooth variants had an apparently wild-type
flp promoter. Mutation of the
flp promoter is not the only mechanism
of the rough-to-smooth conversion of this bacterium.

ACKNOWLEDGMENTS
This research was supported by NIDCR grant R01 DE12212.
We thank S. Goodman and O. Kay for their help in developing the luciferase reporter.

FOOTNOTES
* Corresponding author. Mailing address: Division of Primary Oral Health Care, University of Southern California School of Dentistry, 925 W. 34th Street, Los Angeles, CA 90089-0641. Phone: (213) 740-1075. Fax: (213) 740-6778. E-mail:
ccchen{at}usc.edu.

Editor: V. J. DiRita

REFERENCES
1 - Asikainen, S., and C. Chen. 1999. Oral ecology and person-to-person transmission of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. Periodontol. 2000 20:65-81.
2 - Fine, D. H., D. Furgang, J. Kaplan, J. Charlesworth, and D. H. Figurski. 1999. Tenacious adhesion of Actinobacillus actinomycetemcomitans strain CU1000 to salivary-coated hydroxyapatite. Arch. Oral Biol. 44:1063-1076.[CrossRef][Medline]
3 - Gally, D. L., J. A. Bogan, B. I. Eisenstein, and I. C. Blomfield. 1993. Environmental regulation of the fim switch controlling type 1 fimbrial phase variation in Escherichia coli K-12: effects of temperature and media. J. Bacteriol. 175:6186-6193.[Abstract/Free Full Text]
4 - Goodman, S. D., and Q. Gao. 1999. Firefly luciferase as a reporter to study gene expression in Streptococcus mutans. Plasmid 42:154-157.[CrossRef][Medline]
5 - Haase, E. M., J. O. Stream, and F. A. Scannapieco. 2003. Transcriptional analysis of the 5' terminus of the flp fimbrial gene cluster from Actinobacillus actinomycetemcomitans. Microbiology 149:205-215.[Abstract/Free Full Text]
6 - Haase, E. M., J. L. Zmuda, and F. A. Scannapieco. 1999. Identification and molecular analysis of rough-colony-specific outer membrane proteins of Actinobacillus actinomycetemcomitans. Infect. Immun. 67:2901-2908.[Abstract/Free Full Text]
7 - Hawley, D. K., and W. R. McClure. 1983. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 11:2237-2255.[Abstract/Free Full Text]
8 - Inoue, T., I. Tanimoto, H. Ohta, K. Kato, Y. Murayama, and K. Fukui. 1998. Molecular characterization of low-molecular-weight component protein, Flp, in Actinobacillus actinomycetemcomitans fimbriae. Microbiol. Immunol. 42:253-258.[Medline]
9 - Inouye, T., H. Ohta, S. Kokeguchi, K. Fukui, and K. Kato. 1990. Colonial variation and fimbriation of Actinobacillus actinomycetemcomitans. FEMS Microbiol. Lett. 57:13-17.[CrossRef][Medline]
10 - Ishihara, K., K. Honma, T. Miura, T. Kato, and K. Okuda. 1997. Cloning and sequence analysis of the fimbriae associated protein (fap) gene from Actinobacillus actinomycetemcomitans. Microb. Pathog. 23:63-69.[CrossRef][Medline]
11 - Kachlany, S. C., P. J. Planet, M. K. Bhattacharjee, E. Kollia, R. DeSalle, D. H. Fine, and D. H. Figurski. 2000. Nonspecific adherence by Actinobacillus actinomycetemcomitans requires genes widespread in bacteria and archaea. J. Bacteriol. 182:6169-6176.[Abstract/Free Full Text]
12 - Kachlany, S. C., P. J. Planet, R. Desalle, D. H. Fine, D. H. Figurski, and J. B. Kaplan. 2001. flp-1, the first representative of a new pilin gene subfamily, is required for non-specific adherence of Actinobacillus actinomycetemcomitans. Mol. Microbiol. 40:542-554.[CrossRef][Medline]
13 - Neidhardt, F. C., R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (ed.). 1996. Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C.
14 - Planet, P. J., S. C. Kachlany, R. DeSalle, and D. H. Figurski. 2001. Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc. Natl. Acad. Sci. USA 98:2503-2508.[Abstract/Free Full Text]
15 - Rosan, B., J. Slots, R. J. Lamont, M. A. Listgarten, and G. M. Nelson. 1988. Actinobacillus actinomycetemcomitans fimbriae. Oral Microbiol. Immunol. 3:58-63.[Medline]
16 - Wang, Y., S. D. Goodman, R. J. Redfield, and C. Chen. 2002. Natural transformation and DNA uptake signal sequences in Actinobacillus actinomycetemcomitans. J. Bacteriol. 184:3442-3449.[Abstract/Free Full Text]
17 - Wang, Y., W. Shi, W. Chen, and C. Chen. 2003. Type IV pilus gene homologs pilABCD are required for natural transformation in Actinobacillus actinomycetemcomitans. Gene 312:249-255.[CrossRef][Medline]
18 - Weiser, J. N., S. T. Chong, D. Greenberg, and W. Fong. 1995. Identification and characterization of a cell envelope protein of Haemophilus influenzae contributing to phase variation in colony opacity and nasopharyngeal colonization. Mol. Microbiol. 17:555-564.[CrossRef][Medline]
19 - Youderian, P., S. Bouvier, and M. M. Susskind. 1982. Sequence determinants of promoter activity. Cell 30:843-853.[CrossRef][Medline]
20 - Zambon, J. J. 1985. Actinobacillus actinomycetemcomitans in human periodontal disease. J. Clin. Periodontol. 12:1-20.[CrossRef][Medline]
Infection and Immunity, June 2005, p. 3749-3753, Vol. 73, No. 6
0019-9567/05/$08.00+0 doi:10.1128/IAI.73.6.3749-3753.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
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