Identification and functional characterization of the Neisseria gonorrhoeae lbpB gene product

Abstract

We cloned lbpB, encoding a predicted 80-kDa lipoprotein, upstream of lbpA. A nonpolar mutant (LbpB- LbpA+) had normal lactoferrin (LF) binding and grew normally with LF as an iron source, whereas LbpB- LbpA- and LbpB+ LbpA- strains had reduced binding of LF and did not grow with LF as an iron source. LbpB bound LF directly in an affinity purification, suggesting that LbpB might play a still-uncharacterized role in the LF iron utilization.

FIG. 1

Physical map of the N. gonorrhoeae chromosome showing genetic organization of the lbpBA operon region. The top line represents the restriction map of the cloned FA19 chromosome fragment. The organization of lbpBA genes including the direction of transcription as indicated by arrows is shown in the next line. Plasmid pUNCH192SB is a 1.1-kb Sau3AI fragment. Plasmids pUNCH142 and pUNCH127 contain 2.3-kb RsaI and 2.5-kb SspI fragments, respectively. The solid inverted triangle denotes the position of the aphA3 insertion in strain FA6965(pUNCH142), and the open inverted triangle denotes the position of the Ω insertion in strain FA6839(pUNCH127). L58 and L62 refer to the oligonucleotides used as probes in isolating the clones pUNCH142 and pUNCH192SB, respectively.

FIG. 2

Lineup of negatively charged residues in region one and region two of the gonococcal (NG) and meningococcal (NM) LbpB proteins. The numbers indicate the position in the unprocessed protein of the first amino acid listed. Amino acids identical between the two proteins are denoted by vertical lines; colons indicate highly conservative substitutions, and dots indicate less conservative substitutions.

FIG. 3

Expression of LbpB and LbpA in N. gonorrhoeae. Shown are Western blots containing total membranes prepared from Fe-starved cells probed with affinity-purified LbpB polyclonal antibody (A) and monoclonal LbpA antibody (B) (3). Lanes: 1, parent strain FA6815 (LbpB⁺ LbpA⁺); 2, FA6839 (LbpB⁻ LbpA⁻); 3, FA6985 (LbpB⁺ LbpA⁻); 4, FA6986 (LbpB⁻ LbpA⁺).

FIG. 4

Isotherms for binding of LF to lbp mutants. The curves represent the amount of specifically bound LF as a function of LF concentration. Each point represents the mean of four individual experiments. Symbols: ⧫, FA19 (LbpB⁺ LbpA⁺); ■, FA6965 (LbpB⁻ LbpA⁺); ○, FA6775 (LbpB⁺ LbpA⁻); ▴, FA6839 (LbpB⁻ LbpA⁻).

FIG. 5

Western blot of LF-agarose affinity-purified LbpB and LbpA from total membranes of Fe-starved FA6815 (LbpB⁺ LbpA⁺) (lanes A and D), FA6985 (LbpB⁺ LbpA⁻) (lanes B and E), and FA6986 (LbpB⁻ LbpA⁺) (lanes C and F). The blot was probed with antibodies to LbpA and then LbpB. The anti-LbpB antibody used here was raised against an LbpB His-tagged fusion protein. The 66-kDa band present in lanes A and B was recognized by antiserum elicited against full-length recombinant LbpB but not against an LbpB-specific peptide (Fig. 3 and data not shown). This protein band probably represents a breakdown product of holo-LbpB. Lanes A to C, total membranes (TM); lanes D to F, LF-agarose affinity-purified proteins. MW, molecular weight in thousands.