Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Sep;192(17):4436-44.
doi: 10.1128/JB.00442-10. Epub 2010 Jul 2.

DNA uptake sequence-mediated enhancement of transformation in Neisseria gonorrhoeae is strain dependent

Paul M Duffin  1 , H Steven Seifert
Affiliations

DNA uptake sequence-mediated enhancement of transformation in Neisseria gonorrhoeae is strain dependent

Paul M Duffin et al. J Bacteriol. 2010 Sep.

Abstract

Natural transformation is the main means of horizontal genetic exchange in the obligate human pathogen Neisseria gonorrhoeae. Neisseria spp. have been shown to preferentially take up and transform their own DNA by recognizing the nonpalindromic 10- or 12-nucleotide sequence 5'-ATGCCGTCTGAA-3' (additional semiconserved nucleotides are underlined), termed the DNA uptake sequence (DUS10 or DUS12). Here we investigated the effects of the DUS on transformation and DNA uptake for several laboratory strains of N. gonorrhoeae. We found that all strains showed efficient transformation of DUS containing DNA (DUS10 and DUS12) but that the level of transformation with DNA lacking a DUS (DUS0) was variable in different strains. The DUS-enhanced transformation was 20-fold in two strains, FA1090 and FA19, but was approximately 150-fold in strains MS11 and 1291. All strains tested provide some level of DUS0 transformation, and DUS0 transformation was type IV pilus dependent. Competition with plasmid DNA revealed that transformation of MS11 was enhanced by the addition of excess plasmid DNA containing a DUS while FA1090 transformation was competitively inhibited. Although FA1090 was able to mediate much more efficient transformation of DNA lacking a DUS than was MS11, DNA uptake experiments showed similar levels of uptake of DNA containing and lacking a DUS in FA1090 and MS11. Finally, DNA uptake was competitively inhibited in both FA1090 and MS11. Taken together, our data indicate that the role of the DUS during DNA transformation is variable between strains of N. gonorrhoeae and may influence multiple steps during transformation.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Schematic scale cartoon of transforming constructs. (A) The gyrB1 allele contains a G1285A mutation which confers resistance to Nal and contains a DUS10. Three constructs were created containing gyrB1 (dotted lines); all were cloned with the same 5′ primer but differing 3′ primers. One construct lacks a DUS (3′ primer anneals immediately upstream of DUS), one construct contains the DUS10 (3′ primer anneals to DUS10), and one construct contains the DUS12 (3′ primer anneals to DUS10 and contains “AT” nucleotides on 5′end). (B) The ksgA1 allele contains a G305A mutation which confers resistance to Ksg and contains a DUS12. Three constructs were created containing ksgA1 (dotted lines); all were cloned with the same 3′ primer but differing 5′ primers. One construct lacks a DUS (5′ primer anneals immediately downstream of DUS12), one construct contains the DUS10 (5′ primer anneals to the DUS10), and one construct contains the DUS12 (5′ primer anneals to the DUS12). (C) Schematic cartoon of iga::cat DUS0 and DUS12 constructs (dashed boxes). The DUS0 iga::cat construct contains the indicated region of the iga locus with the cat insertion. The iga::cat DUS12 construct contains an additional 382-bp region of N. gonorrhoeae DNA which contains the DUS12 (diagonal line-filled box).
FIG. 2.
FIG. 2.
The magnitude of DUS enhancement of transformation is strain dependent (A), and DUS phenotypes are not due to pilin sequence or pilin antigenic variation (B). (A) Strains FA1090, FA19, MS11, and 1291 were quantitatively transformed with iga::cat DUS0 and DUS12 and gyrB1 DUS10 and DUS0 plasmid DNA. Transformation efficiencies are plotted as resistant CFU/total CFU. Gray bars indicate iga::cat DUS12, white bars indicate DUS0 iga::cat, horizontal dashed lines indicate gyrB1 DUS10, and diagonal lines indicate gyrB1 DUS0 DNA. Error bars are SEM. *, P < 0.05 by Student's t test. (B) Strains FA1090 1-81-S2nv and MS11 1-81-S2, which cannot undergo pilin antigenic variation and are isogenic for pilE, were quantitatively transformed with gyrB1 and ksgA1 DUS0, DUS10, and DUS12 plasmid DNA. Transformation efficiencies are plotted as resistant CFU/total CFU. White bars indicate gyrB1 DUS0, gray bars indicate gyrB1 DUS10, black bars indicate gyrB1 DUS12, diagonal stripes indicate ksgA1 DUS0, horizontal dashed lines indicate ksgA1 DUS10, and vertical dashed lines indicate ksgA1 DUS12 plasmid DNA. Error bars are SEM. †, 2 repeats below limit of detection. *, P < 0.05 by Student's t test.
FIG. 3.
FIG. 3.
Competition of DUS0 and DUS10 gyrB1 transformation (A and B) or DNA uptake (C and D) by pHSS6 and pUP1. FA1090 (A) or MS11 (B) was transformed with gyrB1 DUS10 (triangles) or DUS0 (squares) in the presence of zero or a 10-, 100-, or 1,000-fold molar excess of pHSS6 (solid lines and shapes) or pUP1 (dashed lines and open shapes). Transformation efficiency is plotted as resistant CFU/total CFU. DNA uptake of 33P-labeled gyrB1 DUS10 (triangles) or DUS0 (squares) was measured in FA1090 (C) or MS11 (D) in the presence of zero or 10-, 100-, or 1,000-fold molar excess of pHSS6 (solid lines and shapes) or pUP1 (dashed lines and open shapes). DNA uptake is plotted as the percentage of DNA added. Error bars are SEM. *, P < 0.05 compared to results with no competitor by Student's t test. †, P < 0.05 by Student's t test.
FIG. 4.
FIG. 4.
DNA uptake of DUS10/12 and DUS0 DNA correlates with transformation efficiencies in MS11 but not FA1090. (A) Binding and uptake of 32P-labeled pHSS6 (DUS0) and pUP1 (DUS12) DNA in strains FA1090 and MS11. Cell-associated (bound plus taken up) DNA is shown in gray, and DNaser DNA is shown in white; data are graphed as a percentage of DNA added. Error bars are SEM. *, P < 0.05 by Student's t test. (B) Binding and uptake of 33P-labeled gyrB1 DUS0 and DUS10 DNA in strains FA1090 and MS11. Total cellular DNA is shown in gray, and DNaser DNA is shown in white; data are graphed as a percentage of DNA added. Error bars are SEM. *, P < 0.05 by Student's t test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aas, F. E., C. Lovold, and M. Koomey. 2002. An inhibitor of DNA binding and uptake events dictates the proficiency of genetic transformation in Neisseria gonorrhoeae: mechanism of action and links to type IV pilus expression. Mol. Microbiol. 46:1441-1450. - PubMed
    1. Aas, F. E., M. Wolfgang, S. Frye, S. Dunham, C. Lovold, and M. Koomey. 2002. Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression. Mol. Microbiol. 46:749-760. - PubMed
    1. Ambur, O. H., S. A. Frye, and T. Tonjum. 2007. New functional identity for the DNA uptake sequence in transformation and its presence in transcriptional terminators. J. Bacteriol. 189:2077-2085. - PMC - PubMed
    1. Anonymous. 2007. Update to CDC's Sexually Transmitted Diseases Treatment Guidelines, 2006: fluoroquinolones no longer recommended for treatment of gonococcal infections. MMWR Morb. Mortal. Wkly. Rep. 56:332-336. - PubMed
    1. Apicella, M. A., J. F. Breen, and N. C. Gagliardi. 1978. Degradation of the polysaccharide component of gonococcal lipopolysaccharide by gonococcal and meningococcal sonic extracts. Infect. Immun. 20:228-234. - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources