G D Biswas

Opposing selective forces for expression of the gonococcal lactoferrin receptor

All isolates of Neisseria gonorrhoeae express receptors that bind human transferrin (Tf). Although lactoferrin (Lf) is abundant on mucosa and in purulent exudates, many gonococci do not express an Lf receptor. The naturally occurring Lf receptor deletion mutant FA1090 (LbpB–LbpA–) is infectious, but a Tf receptor mutant of FA1090 is unable to infect male volunteers [Cornelissen, C.N., Kelley, M., Hobbs, M.M., Anderson, J.E., Cannon, J.G., Cohen, M.S., and Sparling, P.F. (1998) Mol Microbiol 27: 611–616]. Here, we report that expression of an Lf receptor in the absence of the Tf receptor was sufficient for infection, and that expression of both Lf and Tf receptors resulted in a competitive advantage over a strain that made only the Tf receptor in mixed infection of male volunteers. We confirmed that nearly 50% of clinical isolates do not make an Lf receptor. Surprisingly, about half of geographically diverse Lf – isolates representing many different auxotypes and porin serovars carried an identical lbpB lbpA deletion. Among Lf+ strains, all produced the integral outer membrane protein LbpA, but 70% did not express the lipoprotein LbpB. Thus, there are apparently selective pressures for expression of the Lf receptor in the male urethra that are balanced by others against expression of the Lf receptor in niches other than the male urethra.

Cloning and functional characterization of Neisseria gonorrhoeae tonB, exbB and exbD genes

Neisseria gonorrhoeae is able to utilize iron (Fe) from a variety of sources including transferrin (TF) and lactoferrin (LF). To gain insight into the molecular mechanisms used by gonococci to scavenge Fe from TF and LF, we cloned a 3.5 kb segment of wild‐type DNA that repaired the defect in tlu mutants, which are unable to take up Fe from either TF or LF despite exhibiting apparently normal ligand binding to the receptor. Nucleotide sequence determination identified three open reading frames (ORFs), designated ORF1, ORF2, and ORF3, which were arranged in tandem. The deduced amino acid sequence of the 852 bp ORF1 encoded a 28 kDa protein that exhibited 26–32% identity with TonB proteins of nine other bacteria. The 663 bp ORF2 predicted a 24 kDa protein and the 435 bp long ORF3 predicted a 15 kDa protein. These predicted protein sequences exhibited 32–38% and 24–36% identity, respectively, with ExbB and ExbD proteins of three other bacteria. Thus, the sequence comparison identified the ORF1, ORF2 and ORF3 as gonococcal homologues of the E. coli tonB, exbB and exbD genes. An insertional mutation in the tonB homologue resulted in the failure of gonococci to grow with TF, LF or human haemoglobin (HB) as sole Fe sources and in the inability to take up 55Fe from TF and LF. The tonB mutation did not prevent the utilization of Fe from citrate (CT) or haemin (HM). Binding of TF, LF and HB to whole cells in a solid‐phase binding assay was largely unaffected by the tonB mutation. We conclude that the pathways for utilization of Fe bound to TF, LF and HB but not to HM or CT were dependent on the TonB system.

Gene transfer in Neisseria gonorrhoeae.

The ability of bacteria to exchange deoxyribonucleic acid (DNA) endows these organisms with greater genetic variability and increased capability to adapt to changing environments. Many bacteria have evolved transformation and conjugation systems to effect this exchange, whereas others achieve it through the action of bacteriophages. In Neisseria gonorrhoeae no transducing bacteriophages have been identified, but conjugation and transformation both occur (10). Gonococci are extremely autolytic and therefore release DNA to neighboring cells (19). Transformation with chromosomal markers has been demonstrated between strains in laboratory-grown mixed cultures (32); similar transformation probably occurs in nature. Quite recently, antigenic and phase variation of gonococcal pili has been shown to be due in large part to release of DNA from autolyzing cells, with subsequent transformation of other competent cells in the population by variant pil sequences (32a; C. Haas and T. Meyer, personal communication). Gonococcal transformation has been studied extensively and has proven useful in the construction of isogenic strains for mapping antibiotic resistance genes and biosynthetic auxotrophs, and in studies of molecules implicated in the virulence of this organism (10, 34). Conjugation in N. gonorrhoeae is important because it results in mobilization of antibiotic resistance plasmids, but chromosomal genes cannot be transferred by conjugation (3, 10, 27, 31, 37, 44J. Thus, in the laboratory and in nature, transformation is the primary means of transfer of chromosomal genes. In this brief review, mechanisms of gene exchange will be emphasized, and some speculative comments on possible future developments are included.

The Role of Hydroxyl Radical in Chromosomal and Plasmid Damage in Neisseria Gonorrhoeae in vivo

Viable Neisseria gonorrhoeae exposed to streptonigrin generate intracellular hydroxyl radical detected by spin-trapping with 5,5-dimethyl-l-pyrroline-N-oxide; gonococci exposed to paraquat generate primarily superoxide (J. Biol. Chem., 262: 13404-143048, 1987). The use of streptonigrin and paraquat provide a model with which to examine the action and site(s) of hydroxyl radical-mediated damage. N. gonorrhoeae exposed to streptonigrin, but not paraquat, developed extensive chromosomal, plasmid, and RNA damage. Addition of excess Fe+3 to the reaction mixture enhanced intracellular hydroxyl radical formation by paraquat, detectable as DNA damage. Desferal and dimethyl sulfoxide allowed approximately 25% of protection of plasmid DNA damage as judged by linear scanning densitometry. These results demonstrate DNA and RNA damage in viable organisms exposed to intracellular redox stress and confirm the critical role of hydroxyl radical in this process.

Plasmids of the Gonococcus

There are at least four naturally-occurring plasmids in the gonococcus (Table 1). This paper will review the structure, origins and functions of these plasmids, insofar as known or can be reasonably inferred. Certain hybrid plasmids which have been of particular interest in delineating early steps in entry of DNA into competent gonococci are also discussed.