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1.  Genetic transformation of Neisseria gonorrhoeae shows a strand preference 
FEMS microbiology letters  2012;334(1):44-48.
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 a non-palindromic 10 or 12 nucleotide DNA uptake sequence (DUS10 or DUS12). We investigated the ability of the DUS12 to enhance single-stranded DNA (ssDNA) transformation. Given the non-palindromic nature of the DUS12, we tested whether both strands of the DUS equally enhance transformation. Recombinant single-stranded M13 phage harboring transforming DNA with the Watson DUS12, the Crick DUS12, or no DUS (DUS0) were constructed and circular ssDNA was purified. Southern blots of the purified DNA probed with strand-specific oligonucleotide probes showed greater than 10,000:1 ratio of ssDNA to contaminating dsDNA. The Crick strand of the DUS12 enhanced ssDNA transformation 180–470 fold over DUS0 ssDNA whereas the Watson strand of the DUS only modestly enhanced ssDNA transformation in two strains of N. gonorrhoeae. These data confirm that ssDNA efficiently transforms N. gonorrhoeae but that there is a strand preference, and that part of this strand preference is a greater efficiency of the Crick strand of the DUS12 in enhancing transformation.
PMCID: PMC3466376  PMID: 22676068
genetic exchange; pathogen; recombination
2.  DNA Uptake Sequence-Mediated Enhancement of Transformation in Neisseria gonorrhoeae Is Strain Dependent ▿  
Journal of Bacteriology  2010;192(17):4436-4444.
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.
PMCID: PMC2937394  PMID: 20601472
3.  Mismatch Correction Modulates Mutation Frequency and Pilus Phase and Antigenic Variation in Neisseria gonorrhoeae ▿  
Journal of Bacteriology  2009;192(1):316-325.
The mismatch correction (MMC) system repairs DNA mismatches and single nucleotide insertions or deletions postreplication. To test the functions of MMC in the obligate human pathogen Neisseria gonorrhoeae, homologues of the core MMC genes mutS and mutL were inactivated in strain FA1090. No mutH homologue was found in the FA1090 genome, suggesting that gonococcal MMC is not methyl directed. MMC mutants were compared to a mutant in uvrD, the helicase that functions with MMC in Escherichia coli. Inactivation of MMC or uvrD increased spontaneous resistance to rifampin and nalidixic acid, and MMC/uvrD double mutants exhibited higher mutation frequencies than any single mutant. Loss of MMC marginally enhanced the transformation efficiency of DNA carrying a single nucleotide mismatch but not that of DNA with a 1-kb insertion. Unlike the exquisite UV sensitivity of the uvrD mutant, inactivating MMC did not affect survival after UV irradiation. MMC and uvrD mutants exhibited increased PilC-dependent pilus phase variation. mutS-deficient gonococci underwent an increased frequency of pilin antigenic variation, whereas uvrD had no effect. Recombination tracts in the mutS pilin variants were longer than in parental gonococci but utilized the same donor pilS loci. These results show that gonococcal MMC repairs mismatches and small insertion/deletions in DNA and also affects the recombination events underlying pilin antigenic variation. The differential effects of MMC and uvrD in gonococci unexpectedly reveal that MMC can function independently of uvrD in this human-specific pathogen.
PMCID: PMC2798252  PMID: 19854909
4.  ksgA mutations confer resistance to kasugamycin in Neisseria gonorrhoeae 
The aminoglycoside antibiotic kasugamycin (KSG) inhibits translation initiation and thus the growth of many bacteria. In this study, we tested the susceptibilities to KSG of 22 low-passage clinical isolates and 2 laboratory strains of Neisseria gonorrhoeae. Although the range of KSG minimum inhibitory concentrations (MICs) was narrow (seven-fold), clinical isolates and laboratory strains fell into three distinct classes of KSG sensitivity, susceptible, somewhat sensitive and resistant, with MICs of 30, 60–100 and 200 μg/mL, respectively. Two genes have previously been shown to be involved in bacterial KSG resistance: rpsI, which encodes the 30S ribosomal subunit S9 protein; and ksgA, which encodes a predicted dimethyltransferase. Although sequencing of rpsI and ksgA from clinical isolates revealed polymorphisms, none correlated with the MICs of KSG. Ten spontaneous KSG-resistant (KSGR) mutants were isolated from laboratory strain FA1090 at a frequency of <4.4 × 10−6 resistant colony-forming units (CFU)/total CFU. All isolated KSGR variants had mutations in ksgA, whilst no mutations were observed in rpsI. ksgA mutations conferring KSG resistance included four point mutations, two in-frame and one out-of-frame deletions, one in-frame duplication and two frame-shift insertions. These data show a narrow range of susceptibilities for the clinical isolates and laboratory strains examined; moreover, the differences in MICs do not correlate with nucleotide polymorphisms in rpsI or ksgA. Additionally, spontaneous KSGR mutants arise by a variety of ksgA mutations.
PMCID: PMC2723803  PMID: 19097863
Mutagenesis; Gonorrhoea; Antibiotic resistance

Results 1-4 (4)