Pathogenic neisseriae can use hemoglobin, transferrin, and lactoferrin independently of the tonB locus

Abstract

Redundant TonB systems which function in iron transport from TonB-dependent ligands have recently been identified in several gram-negative bacteria. We demonstrate here that in addition to the previously described tonB locus, an alternative system exists for the utilization of iron from hemoglobin, transferrin, or lactoferrin in Neisseria meningitidis and Neisseria gonorrhoeae. Following incubation on media containing hemoglobin, N. meningitidis IR3436 (tonB exbB exbD deletion mutant) and N. gonorrhoeae PD3401 (tonB insertional mutant) give rise to colonies which can grow with hemoglobin. Transfer of Hb(+) variants (PD3437 or PD3402) to media containing hemoglobin, transferrin, and/or lactoferrin as sole iron sources resulted in growth comparable to that observed for the wild-type strains. Transformation of N. meningitidis IR3436 or N. gonorrhoeae PD3401 with chromosomal DNA from the Hb(+) variants yielded transformants capable of growth with hemoglobin. When we inactivated the TonB-dependent outer membrane hemoglobin receptors (HmbR or HpuB) in the Neisseria Hb(+) variants, these strains could not grow with hemoglobin; however, growth was observed with transferrin and/or lactoferrin. These results demonstrate that accumulation of iron from hemoglobin, transferrin, and lactoferrin in the pathogenic neisseriae can occur via a system that is independent of the previously described tonB locus.

FIG. 1

(A) The tonB exbB exbD deletion in N. meningitidis strains IR3436, PD3437, and PD3439 was confirmed by PCR. The tonB gene was amplified by colony PCR using N. meningitidis primers specific for the tonB gene (Table 2). Lanes: M, molecular size standard; 1, IR1072; 2, IR3436; 3, PD3437; 4, PD3438. Molecular sizes are indicated on the left; those on the right correspond to the approximate sizes of the expected amplified products. (B) Growth of N. meningitidis IR3436, PD3437, and PD3438 with various iron sources. A 10-μl volume of human hemoglobin (Hb) (10 mg/ml) or an iron-saturated human transferrin (Tf) (50 mg/ml) or lactoferrin (Lf) (25 mg/ml) was used. These solutions were applied to paper disks and placed onto the GCB-desferal (60 μM) plates seeded with bacteria (10⁸ CFU). Strain IR1072 is the wild-type strain from which IR3436 and PD3437 were derived. Growth was recorded after 18 to 36 h; results are from one experiment and are representative of three separate experiments.

FIG. 2

(A) Insertional inactivation of the tonB gene in N. gonorrhoeae strains PD3401, PD3402, and PD3404, was confirmed by PCR as described in the legend to Fig. 1. Lanes: M, molecular size standard; 1, 340; 2, PD3401; 3, PD3402; 4, PD3403. Molecular sizes are indicated on the left. (B) Growth of N. gonorrhoeae PD3401, PD3402, and PD3403 with various iron sources. Strains were examined for their ability to utilize various iron sources in a plate assay as described in the legend to Fig. 1. N. gonorrhoeae strain 340 is the wild-type strain from which strains PD3401 and PD3402 were derived. Growth was recorded after 18 to 36 h; results are from one experiment and are representative of three separate experiments. Hb, hemoglobin; Tf, transferrin.