The obligate human pathogen, Neisseria gonorrhoeae, is polyploid

Abstract

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts.

Figure 1. Flow Cytometry of Hoechst-Stained, Fixed Bacterial Cells

For each histogram, the x-axis shows fluorescence levels, which indicate the amount of DNA content per particle counted. The y-axis shows counts, which indicate the number of fluorescing particles or cells. Culture optical densities (OD ₆₀₀) are listed to the left of each corresponding histogram. o.n., overnight culture. (A) E. coli (Column 1) and Gc (Column 2) growth curves under standard laboratory conditions. At mid-log phase, part of the culture was treated with rifampicin ( E. coli) or tetracycline (Gc). Genome equivalents were determined from the stationary phase and rif-treated E. coli and are shown on the x-axis. The dotted line represents the division line for sorting into higher and lower fluorescent populations. (B) E. coli and gonococcal cultures grown to mid-log phase at 37 °C or 30 °C. Genome equivalents were determined from stationary phase and rif-treated E. coli as in (A).

Figure 2. Microscopic Analysis of Gc DNA Content

(A) Fluorescence microscopy of Hoechst-stained cells. Phase contrast, fluorescence, and merged images are shown for examples of each cell type observed. (i) monococci, (ii) diplococci. (B) Histogram of fluorescence intensities for exponentially growing and Tet-treated Hoechst-stained cells as determined from fluorescent micrographs.

Figure 3. Marker Frequency Determined by Microarray Analysis

Log2 ratio of the median differential hybridization signal of labeled DNA from untreated and Tet-treated exponentially growing cultures of N. gonorrhoeae. Gene number indicates the order of genes on the FA1090 genomic sequence starting with dnaA. The location of dnaA, pilC1, and the pilC1 associated terminus are indicated by symbols along with the location of the genes surrounding the dif site. The trend line was produced using polynomial regression with an order of 4.

Figure 4. Diagram of the FA1090 Chromosome

Quantitative PCR target sequences are shown, as well as putative origin sites, genes associated with bacterial origins, and the dif site which is linked to the terminus.

Figure 5. Model for Multiple Genomes per Monococcal Cell

The proposed model has a monococcal cell undergoing DNA replication and cell division over time as indicated in min below each drawing. Segregation of chromosomes to promote homozygosity is demonstrated by the dotted chromosomal DNA.