Previous Article | Next Article 
J Bacteriol, March 1998, p. 1592-1595, Vol. 180, No. 6
Institut für Mikrobiologie und Genetik,
Georg-August-Universität, D-37077 Göttingen, Germany
Received 30 September 1997/Accepted 16 January 1998
A gene (comC) essential for natural transformation was
identified in Acinetobacter sp. strain BD413. ComC has a
typical leader sequence and is similar to different type IV pilus
assembly factors. A comC mutant (T308) is not able to bind
or take up DNA but exhibits a piliation phenotype indistinguishable
from the transformation wild type as revealed by electron microscopy.
Although natural
transformation is a broadly distributed property among
gram-negative soil bacteria, hardly anything is known about the
components involved in DNA uptake and their assembly into the
presumptive complex structures involved in DNA binding and uptake
and the regulation of competence induction. Acinetobacter spp. are ubiquitous in terrestrial and aquatic environments and have
been the subject of many studies of the genetics of their broad
catabolic capabilities (3-5). At least two
Acinetobacter sp. strains are highly competent for
natural transformation (1, 11). We chose a miniencapsulated
mutant of the highly competent Acinetobacter sp.
strain BD4 (11), designated Acinetobacter sp.
strain BD413, as a model microorganism to study natural transformation in gram-negative soil bacteria. We recently reported on the generation by random kanamycin marker insertion of five Acinetobacter
mutants completely defective in natural transformation from the highly transformable Acinetobacter sp. strain ADP239, a
pobA (the p-hydroxybenzoate hydroxylase gene)
mutant of strain BD413. Complementation studies of one such
mutant, T205, led to the identification of the competence factor ComP (14). To identify additional components of
the natural transformation system in BD413 and to gain further insights
into the biogenesis and the structure of the transformation system, we
analyzed another transformation-defective mutant, T308.
Characterization of mutant T308.
The strains and plasmids used
in this study are shown in Table 1.
Bacteria were grown in Luria-Bertani medium or in mineral medium
(14). We previously reported a high proficiency of DNA repair in mutant T308 and therefore excluded the possibility that the
transformation defect of T308 was the result of a RecA dysfunction (14). To characterize the mutant phenotype in more detail,
we compared the DNA binding and uptake of ADP239 (the transformation wild type) and T308 essentially as described previously
(14). These studies revealed that T308 exhibited a
66%-reduced level of DNase-sensitive DNA. It is further evident from
Fig. 1 that ADP239 took up DNA at a
linear rate, reaching a maximum of 16,700 cpm after 120 min. In
contrast, mutant T308 was completely defective in DNA uptake.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Identification and Characterization of a Novel Competence Gene,
comC, Required for DNA Binding and Uptake in
Acinetobacter sp. Strain BD413
ABSTRACT
Top
Abstract
Text
References
TEXT
Top
Abstract
Text
References
TABLE 1.
Strains and plasmids used in this study
View larger version (14K):
[in a new window]
FIG. 1.
DNA uptake by Acinetobacter sp. strain ADP239
and the transformation-defective mutant strain T308. Competent cells
(10 ml each; 109 cells ml 
1) of strain ADP239
and mutant strain T308 were incubated at 30°C in mineral medium with
40 ng of 
-35S-dATP-radiolabeled DNA per ml. At the time
intervals indicated, 0.5-ml samples were taken, and the radioactivity
of the cells was determined as described previously (14).
Cloning of T308 mutant allele and regeneration of T308 wild-type allele. All molecular procedures were standard techniques (20). Transformations of strain ADP239 and spot matings of the transformation-deficient recipients with recombinant Escherichia coli S17-1 donor cells were performed as described recently (14). The mutated chromosomal locus from T308 was recovered on a 9.8-kb SstI-XbaI fragment (pRT308-2 [Fig. 2]). Transformation of the wild-type strain, ADP239, with this 9.8-kb SstI-XbaI fragment gave rise to mutants which had phenotypes indistinguishable from that of mutant T308. This result shows that pRT308-2 carries all of the sequence information required to generate the transformation-defective phenotype of the mutant T308.
|
, transformability not restored; +,
transformability restored.
4 to 4.1 × 104 transformants (based
on viable counts) in the presence of saturating DNA concentrations.
These frequencies are comparable to wild-type transformation
frequencies, which is evidence for a complete restoration of the
wild-type transformation phenotype by pRT308. All transconjugants still
exhibited the nptII-encoded Kmr phenotype, and
plasmid pRT308 could be recovered from all of the transconjugants
examined, which indicates that the transformation deficiency of T308
was complemented by providing the recombinant plasmid pRT308 in
trans. Furthermore, these results indicate that the
insertion of the nptII marker gene into the genome of mutant T308 does not cause any polar effects on the genes that may be located
downstream of the marker-affected mutant locus. A variety of
derivatives of the 8.5-kb SstI-XbaI fragment were
constructed and tested for their ability to complement the mutation in
T308. These studies identified the 4.6-kb
ClaI-XbaI fragment (pRK9) as the smallest
fragment able to restore the wild-type transformation phenotype (Fig.
2). To confirm that the fragment generated from the recovered mutant
locus is identical to wild-type DNA, the wild-type gene was cloned from
XbaI-digested chromosomal DNA of strain BD413 into pRK415.
Plasmid pCL20 contained a 20.1-kb XbaI fragment and was
found to restore the wild-type transformation phenotype of mutant T308
(Fig. 2). The results of restriction analysis, Southern hybridizations,
and DNA sequencing revealed that the insert in pRK9 is identical to the
DNA cloned from the wild type. These results show that the
comC disruption in mutant T308 by nptII marker
insertion was caused by an allelic replacement recombination of
contiguous DNA fragments flanking the nptII gene, which
resulted in a marker gene insertion without causing any deletion or
duplication events.
Identification and characterization of comC, a gene
complementing the transformation defect of mutant T308.
The 4.6-kb
ClaI-XbaI fragment of pRK9 was inserted into
pBluescriptII KS, yielding pSE9 (Table 1), which was subjected to unidirectional deletions. The complete nucleotide sequence of the
ClaI-XbaI fragment (4,584 bp) in pSE9 was
determined for both strands, and sequence analysis revealed one
complete open reading frame, designated comC, of 3,624 bp
extending from nucleotide positions 717 (ATG) to 4340, spanning the
BamHI site used to generate mutant T308 by marker insertion.
comC is preceded by a well-conserved and well-placed
Shine-Dalgarno sequence. The complementation of the
transformation-defective mutant T308 was found to be independent of the
insert orientation with respect to the lac promoter, as shown for complementation with pRK9 in Fig. 2. This indicates that
comC is expressed under the control of its native promoter. A conserved 
70 promoter sequence
(TTGCGAN20TATTAA) was found within a
region 141 to 172 bp upstream of comC. The 39.4% G+C
content of comC is within the characteristic range of 37 to
45% for coding regions in Acinetobacter species, and the
codon usage was much like that found in previously sequenced
Acinetobacter genes (12, 24).
Features of ComC and similarity to type IV pilus assembly and adhesion factors. ComC contains 1,208 amino acids (aa), with a calculated molecular mass of 132 kDa. The N terminus exhibits structural features characteristic of the three domains of signal peptides (23): (i) a positively charged N terminus (Lys 8 and Arg 10), (ii) a hydrophobic core (Ala 11 to Ala 18), and (iii) a C-terminal domain containing small neutral residues (Lys 21 to Thr 24). These findings suggest that ComC is located, and acting, at the cell surface.
Database searches and sequence alignments revealed that ComC exhibits similarities to proteins involved in the assembly of type IV pili in pathogenic bacteria, such as PilC (1,037 aa; 21% identity) (19), PilC1 (1,038 aa; 22% identity), and PilC2 (1,048 aa; 22% identity) of Neisseria meningitidis (15); PilC1 (1,044 aa; 22% identity) and PilC2 (1,050 aa; 21% identity) of Neisseria gonorrhoeae (10); and PilY1 (1,161 aa; 21% identity) of Pseudomonas aeruginosa (2). The similarities of ComC to the type IV pilus biogenesis factors are in the same range as the similarities found for PilC2 to PilY1 and are not restricted to a certain region, whereas the similarities displayed by PilC2 to PilY1 are restricted to the C terminus. ComC and its homologs are also of similar size and possess large regions of hydrophilic amino acids.ComC is not essential for the biogenesis of pilus fibers and twitching motility. PilC1 and PilC2 of N. gonorrhoeae and the recently identified PilC of N. meningitidis exhibit dual functions in type IV pilus biogenesis and in transformation (16, 19). To address the question of whether ComC also displays such a dual function, the ultrastructures of ADP239, mutant T308, and transconjugant cells restored to natural transformation were analyzed. Electron micrographs confirmed our recent finding that cells of strain ADP239 possess two types of fimbriae (14): thin ones with a diameter of 3.5 nm, which appear in bundles, and thicker isolated fimbriae with a diameter of about 6 nm. The piliation phenotype of the transformation-defective mutant T308 was indistinguishable from the piliation of ADP239 cells (Fig. 3), indicating that ComC is not essential for the biogenesis of pili in Acinetobacter sp. strain BD413. The thick pili of Acinetobacter sp. strains have been found to mediate a special kind of surface translocation, termed twitching motility (8). Since the presence of thick pili does not preclude a functional defect of the pili, such as a defect in twitching, T308 was analyzed for its ability to perform twitching motility, which was monitored by the appearance of spreading zones along the central streak of growth of the cells on agar plates as described previously (14). These studies revealed that T308 was not impaired in twitching (data not shown) and therefore provide substantial evidence that the transformation factor ComC is not essential for pilus biogenesis.
|
Conclusions. The PilC of N. gonorrhoeae and PilY1 of P. aeruginosa have been localized in both the outer membrane and the fimbrial fractions (2, 6), and PilC was found especially in the fimbrial tip of N. gonorrhoeae (18), which is also suggested for PilY1 of P. aeruginosa (2). The similarity of ComC to PilC and PilY1 and the DNA binding and uptake studies suggest that ComC is exported and acts in a cell surface protein complex required for DNA binding and uptake in Acinetobacter sp. strain BD413. Analogous to the function once predicted for the multifunctional gonococcal PilC2, ComC might represent a basement protein or a molecular usher, required for the correct subunit presentation of a growing oligomeric structure mediating DNA transfer through the outer membrane. Homologous sets of pil-like genes are found not only in DNA transfer systems but also in bacterial protein secretion systems (for a review, see reference 9). These homologies might reflect a common scheme of macromolecular transport.
Nucleotide sequence accession number. The sequence of the open reading frame designated comC has been deposited in the GenBank database under accession no. AF027189.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by grant AV 9/4-1 from the Deutsche Forschungsgemeinschaft.
We are indebted to G. Gottschalk, Göttingen, for stimulating discussions and generous support. We are grateful to M. Madkour and F. Mayer, Göttingen, for help with the electron microscopy studies. Thanks are due to V. Müller, Munich, for suggestions concerning the transport studies.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Institut für Mikrobiologie und Genetik, Georg-August-Universität, Grisebachstrasse 8, D-37077 Göttingen, Germany. Phone: 49-551-394041. Fax: 49-551-393793. E-mail: BAVERHO{at}gwdg.de.
| |
REFERENCES |
|---|
|
Top
Abstract
Text
References
|
|---|
| 1. | Ahlquist, E. F., C. A. Fewson, D. A. Ritchie, J. Podmore, and V. Rowell. 1980. Competence for genetic transformation in Acinetobacter calcoaceticus NCIB8250. FEMS Microbiol. Lett. 7:107-109. |
| 2. | Alm, R. A., J. P. Hallinan, A. A. Watson, and J. S. Mattick. 1996. Fimbrial biogenesis genes of Pseudomonas aeruginosa: pilW and pilX increase the similarity of type 4 fimbriae to the GSP protein-secretion systems and pilY1 encodes a gonococcal PilC homologue. Mol. Microbiol. 22:161-173[Medline]. |
| 3. |
Averhoff, B.,
L. Gregg-Jolly,
D. Elsemore, and L. N. Ornston.
1992.
Genetic analysis of supraoperonic clustering by use of natural transformation in Acinetobacter calcoaceticus.
J. Bacteriol.
174:200-204 |
| 4. | DiMarco, A. A., B. Averhoff, E. E. Kim, and L. N. Ornston. 1993. Evolutionary divergence of pobA, the structural gene encoding p-hydroxybenzoate hydroxylase in an Acinetobacter calcoaceticus strain well-suited for genetic analysis. Gene 125:25-33[Medline]. |
| 5. |
DiMarco, A. A.,
B. Averhoff, and L. N. Ornston.
1993.
Identification of the transcriptional activator pobR and characterization of its role in the expression of pobA, the structural gene for p-hydroxybenzoate hydroxylase in Acinetobacter calcoaceticus.
J. Bacteriol.
175:4499-4506 |
| 6. |
Fussenegger, M.,
T. Rudel,
R. Barten,
R. Ryll, and T. F. Meyer.
1997.
Transformation competence and type-4 pilus biogenesis in Neisseria gonorrhoeae a review.
Gene
192:125-134[Medline].
|
| 7. |
Hartnett, G. B.,
B. Averhoff, and L. N. Ornston.
1990.
Selection of Acinetobacter calcoaceticus mutants deficient in the p-hydroxybenzoate hydroxylase gene (pobA), a member of a supraoperonic cluster.
J. Bacteriol.
172:6160-6161 |
| 8. | Henrichsen, J., and J. Blom. 1975. Correlation between twitching motility and possession of polar fimbriae in Acinetobacter calcoaceticus. Acta Pathol. Microbiol. Scand. Sect. B 83:103-115[Medline]. |
| 9. | Hobbs, M., and J. S. Mattick. 1993. Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: a general system for the formation of surface-associated protein complexes. Mol. Microbiol. 10:233-243[Medline]. |
| 10. | Jonsson, A. B., M. Rahman, and S. Normark. 1995. Pilus biogenesis gene, pilC, of Neisseria gonorrhoeae: pilC1 and pilC2 are each part of a larger duplication of the gonococcal genome and share upstream and downstream homologous sequences with opa and pil loci. Microbiology 14:2367-2377. |
| 11. |
Juni, E., and A. Janik.
1969.
Transformation of Acinetobacter calcoaceticus (Bacterium anitratum).
J. Bacteriol.
98:281-288 |
| 12. | Kaplan, J. B., P. Goncharoff, A. M. Seibold, and B. P. Nichols. 1984. Nucleotide sequence of the Acinetobacter calcoaceticus trpGDC gene cluster. Mol. Biol. Evol. 1:456-472[Abstract]. |
| 13. | Keen, N. T., S. Tamaki, D. Kobayashi, and D. Trollinger. 1988. Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene 70:191-197[Medline]. |
| 14. | Porstendörfer, D., U. Drotschmann, and B. Averhoff. 1997. A novel competence gene, comP, is essential for natural transformation of Acinetobacter sp. strain BD413. Appl. Environ. Microbiol. 63:4150-4157[Abstract]. |
| 15. | Rahman, M., H. Kallstrom, S. Normark, and A. B. Jonsson. 1997. PilC of pathogenic Neisseria is associated with the bacterial cell surface. Mol. Microbiol. 25:11-25[Medline]. |
| 16. |
Rudel, T.,
D. Facius,
R. Barten,
I. Scheuerpflug,
E. Nonnenmacher, and T. F. Meyer.
1995.
Role of pili and the phase-variable PilC protein in natural competence for transformation of Neisseria gonorrhoeae.
Proc. Natl. Acad. Sci. USA
92:7986-7990 |
| 17. | Rudel, T., H.-J. Boxberger, and T. F. Meyer. 1995. Pilus biogenesis and epithelial cell adherence of Neisseria gonorrhoeae pilC double knock-out mutants. Mol. Microbiol. 17:1057-1071[Medline]. |
| 18. | Rudel, T., I. Scheuerpflug, and T. F. Meyer. 1995. Neisseria PilC protein identified as type-4 pilus tip-located adhesin. Nature 373:357-359[Medline]. |
| 19. | Ryll, R., T. Rudel, I. Scheuerpflug, R. Barten, and T. F. Meyer. 1997. PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci. Mol. Microbiol. 23:879-892[Medline]. |
| 20. | Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. . Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. |
| 21. | Simon, R., U. Priefer, and A. Pühler. 1983. A broad host-range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:784-791. |
| 22. | Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268[Medline]. |
| 23. | von Heijne, G. 1994. Signals for protein targeting into and across membranes, p. 1-19. In A. H. Maddy, and J. R. Harris (ed.), Subcellular biochemistry. Plenum Press, New York, N.Y. |
| 24. | White, P. J., I. S. Hunter, and C. A. Fewson. 1991. Codon usage in Acinetobacter structural genes, p. 251-258. In K. Towner, E. Bergogne-Berezin, and C. A. Fewson (ed.), The biology of Acinetobacter. FEMS Symposia Series. Plenum Press, New York, N.Y. |
J Bacteriol, March 1998, p. 1592-1595, Vol. 180, No. 6
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Perez, F., Hujer, A. M., Hujer, K. M., Decker, B. K., Rather, P. N., Bonomo, R. A.
(2007). Global Challenge of Multidrug-Resistant Acinetobacter baumannii. Antimicrob. Agents Chemother.
51: 3471-3484
[Full Text] -
Bacher, J. M., Metzgar, D., de Crecy-Lagard, V.
(2006). Rapid Evolution of Diminished Transformability in Acinetobacter baylyi. J. Bacteriol.
188: 8534-8542
[Abstract] [Full Text] -
Gohl, O., Friedrich, A., Hoppert, M., Averhoff, B.
(2006). The Thin Pili of Acinetobacter sp. Strain BD413 Mediate Adhesion to Biotic and Abiotic Surfaces. Appl. Environ. Microbiol.
72: 1394-1401
[Abstract] [Full Text] -
Barbe, V., Vallenet, D., Fonknechten, N., Kreimeyer, A., Oztas, S., Labarre, L., Cruveiller, S., Robert, C., Duprat, S., Wincker, P., Ornston, L. N., Weissenbach, J., Marliere, P., Cohen, G. N., Medigue, C.
(2004). Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res
32: 5766-5779
[Abstract] [Full Text] -
Friedrich, A., Rumszauer, J., Henne, A., Averhoff, B.
(2003). Pilin-Like Proteins in the Extremely Thermophilic Bacterium Thermus thermophilus HB27: Implication in Competence for Natural Transformation and Links to Type IV Pilus Biogenesis. Appl. Environ. Microbiol.
69: 3695-3700
[Abstract] [Full Text] -
Friedrich, A., Prust, C., Hartsch, T., Henne, A., Averhoff, B.
(2002). Molecular Analyses of the Natural Transformation Machinery and Identification of Pilus Structures in the Extremely Thermophilic Bacterium Thermus thermophilus Strain HB27. Appl. Environ. Microbiol.
68: 745-755
[Abstract] [Full Text] -
Friedrich, A., Hartsch, T., Averhoff, B.
(2001). Natural Transformation in Mesophilic and Thermophilic Bacteria: Identification and Characterization of Novel, Closely Related Competence Genes in Acinetobacter sp. Strain BD413 and Thermus thermophilus HB27. Appl. Environ. Microbiol.
67: 3140-3148
[Abstract] [Full Text] -
Porstendörfer, D., Gohl, O., Mayer, F., Averhoff, B.
(2000). ComP, a Pilin-Like Protein Essential for Natural Competence in Acinetobacter sp. Strain BD413: Regulation, Modification, and Cellular Localization. J. Bacteriol.
182: 3673-3680
[Abstract] [Full Text] -
Graupner, S., Frey, V., Hashemi, R., Lorenz, M. G., Brandes, G., Wackernagel, W.
(2000). Type IV Pilus Genes pilA and pilC of Pseudomonas stutzeri Are Required for Natural Genetic Transformation, and pilA Can Be Replaced by Corresponding Genes from Nontransformable Species. J. Bacteriol.
182: 2184-2190
[Abstract] [Full Text] -
Busch, S., Rosenplänter, C., Averhoff, B.
(1999). Identification and Characterization of ComE and ComF, Two Novel Pilin-Like Competence Factors Involved in Natural Transformation of Acinetobacter sp. Strain BD413. Appl. Environ. Microbiol.
65: 4568-4574
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»