Opa expression correlates with elevated transformation rates in Neisseria gonorrhoeae.

The members of the genus Neisseria are naturally competent for DNA transformation, and there is considerable evidence indicating that horizontal exchange of chromosomal and plasmid DNAs occurs in these bacteria in vivo (23). For most naturally transformable bacteria, an organism (e.g., Bacillus subtilis or Haemophilus influenzae) becomes competent in vitro through starvation and/or other manipulations of the culture conditions (14, 29). However, the neisseriae are a little different in that competence is constitutively expressed in vitro (5). Nonetheless, some limitations are imposed on the gonococcal transformation process, as only genus-specific DNA can be efficiently taken up into a DNase I-resistant state due to a strong preference for a specific neisserial DNA uptake sequence within the donor DNA (10, 15). Consequently, gonococci can be transformed in the presence of excess amounts of nonspecific DNA, whereas only small amounts of neisserial DNA are required to competitively inhibit the process (8, 16). Studies of the mechanism of gonococcal transformation have relied heavily on the isolation of organisms with specific mutations that block some step in the transformation process. Using this approach, several gonococcal gene products that assist in the movement of donor DNA across the cell envelope have been identified. Gonococci exhibit (i) a general requirement for the presence of pili on the cell surface and Tpc protein for genetic competence (5, 13), (ii) involvement of PilT and PilC proteins in sequestering donor DNA into a DNase I-resistant state (4, 26, 38), (iii) involvement of ComL in the movement of DNA across the peptidoglycan layer (12), and (iv) involvement of ComA in transporting donor DNA across the cytoplasmic membrane (11). Furthermore, it has been shown that once the donor DNA has translocated into the cytoplasm, the RecA protein facilitates its incorporation into the host chromosome (21). Recently, the RecBCD recombination pathway was also shown to be involved in the transformation process (24). However, unlike all other organisms, gonococcal recD mutants are not hyperrecombinogenic for DNA transformation (6). For most DNA transformation systems, uptake of donor DNA into a DNase I-resistant state is generally considered to be the first step in the process. However, prior to this uptake is the actual recruitment of donor DNA onto the bacterial cell surface, and very little is known about the bacterial surface component(s) that participates in this early step. However, when considered simplistically, it would seem that any surface component that could contribute a positive surface charge should be a candidate for an electrostatic attractant of donor DNA (a complex polyanion) to the bacterial cell surface. Gonococci may provide an ideal model system to test this hypothesis (as well as explore the more general hypothesis that changes in cell surface charge can influence the biological properties of an organism) because, under most conditions encountered in vivo, gonococci express a unique group of proteins on their cell surfaces (the opacity [Opa] proteins) (9, 19, 20, 34) that theoretically should endow the organism with a positive cell surface charge. Colonies of cells expressing Opa protein display increased opacity when viewed under a phase-contrast microscope (30, 31). Moreover, the Opa proteins belong to a complex multigene family (3) and are one of three gonococcal surface constituents that vary in vivo (20, 34, 35). However, the unique feature of Opa expression that is pertinent to this study is that secondary structural algorithms of their predicted primary polypeptide sequences indicate that their surface-exposed segments should contain an excess of positively charged amino acids (3, 32). Consequently, if Opa expression does endow the organism with a positive cell surface charge, then Opa protein may preferentially attract complex polyanions (such as DNA) to the cell surface. This apparently is the case, as immobilized Opa proteins are able to bind radiolabelled DNA in a blot format, as well as promote DNA accretion in situ on intact bacteria (32). Furthermore, the endowment of a positive cell surface charge through Opa expression is further indicated by the results of physical studies that examined the relative electrophoretic mobility of Opa-expressing gonococci (32). In this study, I examined whether Opa-DNA interactions actually translate into an enhanced transformation phenotype for the gonococcus. The data show that Opa expression invariably correlates with an increased transformation efficiency. Moreover, by growing gonococci under conditions in which binding of nonspecific polyanions to the cell surface is limited, then the DNA transformability of the organism is significantly enhanced, with transformation now being inhibitable by nonspecific DNA and/or the complex polyanion heparin. Overall, the data suggest that the elaboration of a specific cell surface charge may indeed influence a basic biological function of the gonococcus.