We first combined the guanine count (ie, the number of guanines in the polyG tracts) for all of the clones of each gene from all of the slides (pooled data). If the exudative environment selected a dominant gene expression, the guanine count would be normally distributed around the number of guanines selected by the PMN intracellular environment, and the breadth of the distribution would indicate the strength of the selective pressure. If the pooled data were not normally distributed, we could conclude that the exudative environment exerted no selective pressure on expression of that gene. The distributions for each gene are in Figure .
Figure 2. Frequency of the number of guanines in the polyG tracts of lgtA (A), lgtC (B), and lgtD (C) in urethral exudates containing Neisseria gonorrhoeae. Black bars indicate polyG tracts that yield an out-of-frame gene and a nonfunctional glycosyl transferase (more ...)
The pooled guanine counts for lgtA and lgtC were normally distributed (Figure ). LgtA had a mode of 11 guanines and a span of 9–13 guanines, which is consistent with moderately strong selection of a gene with 11 guanines (Figure A). An lgtA with 11 guanines would be IF, as frame for lgtA is 11 ± 3n Gs, and the organisms in the exudates would have functioning LgtA β1 → 3 glucosaminyl transferases.
The guanine count mode for lgtC was 9, with a narrow span of 8–10 (Figure B). Frame for lgtC is 10 ± 3n Gs, so the guanine count showed strong selection for an OOF lgtC and bacteria that do not have functioning α1 → 4 galactosyl transferases at the time of potential transmission.
The lgtD guanine count spanned 4 frames, as determined from the upstream start codon, and were designated frames 1 (8–10 guanines), 2 (11–13 guanines), 3 (14–16 guanines), and 4 (17–19 guanines), with the first guanine count for each frame being the IF count (Figure C). This distribution is consistent with only modest selective pressure for any one guanine count; however, the counts were not distributed equally within each frame. The guanine counts were concentrated in frames 2 and 3, and the modes of those frames were 13 and 15 guanines, respectively, which both yield OOF genes and nonfunctional LgtDs. IF lgtD guanine counts were the least common.
To ensure that the pooled data were not biased by any one patient's bacteria, we analyzed the clones from each patient's slide individually (Table ). The guanine counts were averaged to produce means for that patient for each gene, and the individual means then were averaged to generate an overall guanine count mean and 95% confidence interval (CI). To evaluate the consistency of the results and ensure that it was not slippage during PCR that accounted for the variation, we amplified genes from the same patient's slide 2 or 3 times and averaged those results (Table ).
Guanine Counts for lgtA, lgtC, and lgtD in Clones Amplified From Gonococci in the Gram-Stained Urethral Exudates of Infected Men
The mean lgtA guanine count (± SD) for 6 patients was 10.9 ± 0.42 (95% CI, 10.51–11.29). Because there is a single whole integer, 11, within the CI, we can conclude that this is the number of lgtA guanines in almost all gonococci during transmission and that we sequenced lgtA polyG tracts from enough patients to determine that this gene is IF in almost all of the bacteria. Consequently, we are confident that the gonococci in urethral exudates express paraglobosyl nLc4 LOS α chains.
The lgtC overall mean (±SD) from 7 men was 9 ± 0.11 guanines (95% CI, 8.92–9.10). Not only is there a single whole integer, 9, within the CI, but the interval was very narrow. Thus we can conclude that essentially all of the gonococci in the urethral exudates of infected men have an OOF lgtC with 9 guanines.
Given the distribution of lgtD guanine counts in the pooled data, we generated means within frames 2 and 3 of lgtD for individual men. The overall mean ( ± SD) was 11.99 ± 0.88 guanines (95% CI, 11.31–12.67) for frame 2 of lgtD and 14.98 ± 0.54 guanines (95% CI, 14.56–15.40) for frame 3 of lgtD (Table ). Frame for lgtD is 11 ± 3n Gs, so the single whole integers within these CIs, 12 and 15, respectively, would yield OOF lgtDs. We conclude that there is strong selection for an OOF lgtD gene and that most, but not all, gonococci in urethral exudates have a nonfunctioning LgtD β1 → 3 galactosaminyl transferase.
The MS11mkC lgtA, lgtC
, and lgtD
polyG tracts were consistent with those of the exudative bacteria (Table ) and predictive of the structures reported for this strain [1
was IF, with 11 guanines, as it was in the exudative bacteria, and its functional LgtA would catalyze production of its polylactosaminyl nLc4–8
α chains. lgtC
is OOF in MS11mkC with 9 guanines, as it is in the exudative bacteria [7
was OOF with 13 guanines.