Aerobactin Utilization by Neisseria Gonorrhoeae and Cloning of a Genomic DNA Fragment That Complements Escherichia Coli Fhub Mutations SUSAN E

Home , Aerobactin, Neisseria gonorrhoeae

JOURNAL OF BACTERIOLOGY, Aug. 1987, p. 3414-3421 Vol. 169, No. 8 0021-9193/87/083414-08$02.00/0 Copyright © 1987, American Society for Microbiology Aerobactin Utilization by Neisseria gonorrhoeae and Cloning of a Genomic DNA Fragment That Complements Escherichia coli fhuB Mutations SUSAN E. H. WESTt* AND P. FREDERICK SPARLING Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514 Received 17 February 1987/Accepted 28 April 1987 Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (flwCDB) genes. Hybridization was detected with IhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the ph id vector XSE4, and a clone was isolated that complemented theihuB mutation in derivatives ofE. coli BU736 and BN3307. These results suggest thatfJhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.

Most microbial iron acquisition systems consist of two product is apparently a very hydrophobic membrane protein components: a siderophore which is a soluble low-molecu- of 70,337 Mr as calculated from the nucleotide sequence (23); lar-weight iron-chelating compound and a specific cell sur- its exact location in the membranes of E. coli is uncertain. face receptor for the iron-siderophore complex (33). Iron The mechanism of aerobactin-mediated iron acquisition acquisition by Neisseria gonorrhoeae is different in that by gonococci has not been characterized. We confirmed that gonococci do not appear to produce siderophores (34, 45, gonococci can utilize aerobactin as a sole iron source. We 46). However, gonococci, like Escherichia coli (33) and found homology between the gonococcal genome and the Salmonella typhimurium (26), can utilize siderophores pro- cloned E. coli fhuB gene, but not with the fhuC, JhuD, or duced by other microorganisms. Yancey and Finkelstein (48) aerobactin biosynthesis and receptor genes. We describe the demonstrated that aerobactin and the related dihydroxamate identification and molecular cloning of the gonococcal JfhuB siderophores arthrobactin and schizokinen stimulate the homolog and demonstrate that it complements fhub muta- growth of iron-starved gonococci. tions in E. coli. Aerobactin is produced by many strains of E. coli and several other species of gram-negative bacteria (Enterobac- MATERIALS AND METHODS ter [Aerobacter] aerogenes, Enterobacter cloacae, Shigella sonnei, Shigella boydii, Shigella flexneri, Salmonella ari- Bacterial strains and plasmids. N. gonorrhoeae FA19 and zona, Salmonella austin, and Salmonella memphis) (16, 25, F62 were from laboratory stocks. Additional strains of N. 29, 35). The aerobactin operon in E. coli consists of the gonorrhoeae were obtained from J. R. Black, University of genes which code for the 74,000-molecular-weight (74K) Indiana, Indianapolis, and W. McKenna, University of aerobactin receptor (iutA) and for four enzymes required for North Carolina, Chapel Hill. The E. coli strains, plasmids, aerobactin biosynthesis (iucABCD [aerDBCA]) (2, 8). The and bacteriophage used in this study and their characteristics proteins IucA, IucB, IucC, and IucD catalyze the synthesis are listed in Table 1. To construct SW300, E. coli BU736 was of aerobactin from citrate and lysine (17). The aerobactin transformed with pSE103, and a malT(Con) allele linked to biosynthesis and receptor genes are arranged in a cluster transposon TnJO from E. coli BAR301 (provided by P. J. flanked by ISI-like inverted repeat sequences (29) and may Bassford, Jr.) was moved into this strain by P1 transduction. be either chromosomally or plasmid coded (29). In addition, Tetracycline-resistant transductants were scored for sensi- several chromosomally located genes, fhuBCD, are required tivity to phage lambda. To construct SW400, E. coli BN3307 for uptake of iron from aerobactin and other hydroxamate was transformed with pSE103 and then pColV-K30 was siderophores such as ferrichrome, rhodotorulic acid, and introduced by mating with LG1315 overnight in a static broth coprogen (6, 7, 15, 23, 37). ThefluBCD genes and theflzuA culture, followed by selection on kanamycin-LB agar. Kmr gene, which codes for the ferrichrome receptor, constitute a transconjugants were screened for production of colicin and single operon located at 3.5 min on the E. coli chromosome; aerobactin. Plasmids were transformed into the appropriate the gene order is JhuACDB (15). The protein products of strains by the method of Mandel and Higa (27). fhuC and .1 uD are inner membrane proteins (15). The fhuB Media and growth conditions. All Neisseria strains were maintained at 37°C in a CO2 incubator on GC agar (Difco Laboratories, Detroit, Mich.) supplemented with Kellogg * Corresponding author. supplements I and II (21). To deprive gonococci of iron, we t Present address: Department of Microbiology and Immunology, added 50 ,uM Desferal (Ciba-Geigy Corp., Summit, N.J.) to University of Rochester School of Medicine and Dentistry, Roch- either GC broth (1.5% Proteose Peptone no. 3 [Difco], 0.5% ester, NY 14642. NaCl, 0.4% K2HPO4, 0.1% KH2PO4, 0.8% NaHCO3, and 3414 VOL. 169, 1987 AEROBACTIN UTILIZATION BY N. GONORRHOEAE 3415

Kellogg supplement I) or the defined medium (DM) of Morse cinate and 50 ,ug of thiamine, 20 ,ug of tryptophan, 20 ,ug of and Bartenstein (31) lacking Fe(NO3)3 and supplemented phenylalanine, and 20 ,ug of tyrosine per ml). Aerobactin was with 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2- purified as described by Bindereif (A. Bindereif, M.A. ethanesulfonic acid). For some experiments, we used a thesis, University of California, Berkeley, 1980). The aero- modification of the Morse and Bartenstein medium, which bactin concentration was determined spectrophotometri- we designated chelexed defined medium (CDM) (S. E. H. cally at pH 7.0 in the presence of excess FeCl3 (18). West, Ph.D. thesis, University of North Carolina, Chapel Aerobactin was ferrated by addition of an appropriate Hill, 1986). Modifications included supplementation with 21 amount of FeCl3 to the purified preparation. mM HEPES, an increase in the sodium bicarbonate concen- Aerobactin utilization by N. gonorrhoeae. N. gonorrhoeae tration to 20 mM, reduction of the phosphate content to 4 strains FA19 and F62 were grown in DM supplemented with mM, and treatment of the medium with Chelex 100 (Bio-Rad either 50 ,uM Desferal, 50 pzM Desferal and 2 1±M fer- Laboratories, Richmond, Calif.). Cultures were incubated in riaerobactin, or 50 ,uM Desferal and 75 ,iM Fe(NO3)3 at 37°C a shaking (200 rpm) CO2 incubator at 37°C. E. coli strains in a shaking (200 rpm) CO2 incubator. The inoculum was an were maintained on LB agar supplemented with the appro- iron-deprived culture which had been grown to late log phase priate antibiotics (28). in DM plus 50 ,uM Desferal. For other experiments, Aerobactin. Aerobactin was prepared from E. coli gonococci were grown in either DM alone, DM supple- LG1315. Cells were grown for 48 to 72 h in Tris-succinate mented with 4 ,uM aerobactin, DM supplemented with 4 ,uM medium (T medium of Simon and Tessman [39] lacking aerobactin and 20 pLM Fe(NO3)3, or DM supplemented with glucose and FeCl3 and supplemented with 1% sodium suc- 20 FM Desferal. E. coli AN1937 was grown under the same conditions. Bioassay for aerobactin production. Culture supernatants TABLE 1. Characteristics of E. coli strains, plasmids, from N. gonorrhoeae FA19 and F62 were tested for aero- and bacteriophages bactin with a bioassay in which the indicator strain E. coli LG1522 was plated on M9 dipyridyl plates as described by Strain, plasmid, Characteristics Source or reference or bacteriophage Carbonetti and Williams (8). E. coli AN1937 was included to eliminate the possibility of nonspecific stimulation of the Strains indicator strain. Gonococci were grown in CDM, CDM AN1937 ara entA lac leu mtl proC P. Williams (47) supplemented with 20 ,M Fe(NO3)3, GCB, and GCB sup- rpsL supE thiffhuA trpE plemented with 50 ,uM apo-transferrin (Sigma Chemical Co., xyl LG1315 As AN1937, but pColV-K30 P. Williams (44) St. Louis, Mo.). Bacterial cells were removed by centrifu- LG1419 As AN1937, but pColV-K30 P. Williams (47) gation at 12,000 x g for 10 min, and the supernatants were iut filter sterilized. When required, the culture supernatants LG1522 ara fepA lac leu mtl proC P. Williams (8) were concentrated by lyophilization. rpsL supE thiflhuA trpE Cloacin sensitivity. Cloacin was isolated from Enterobac- xyl, pColV-K30 iuc ter cloacae N4049 (obtained from N. Carbonetti) and par- BR686 rpsL aroB cir thi tsx malT, V. Braun (6) tially purified as described by de Graaf et al. (12). N. pColV-K229 gonorrhoeae FA19 and F62 were deprived of iron by growth BU736 As BR686, but fhuB V. Braun (6, 23) in GC broth containing 50 ,uM Desferal. Approximately 107 SW300 As BU736, but malT(Con), This study pSE103 gonococci were spread onto a GC agar plate, and 10- and 20-pd of the cloacin were BN3307 F- proC trp thi lac Y rpsL J. INeilands (37) samples partially purified spotted galK ara entA mtl xyl azi onto the lawns. After overnight incubation, the plates were tsx supE44 fhuB panB observed for zones of inhibition where the cloacin was recA placed. E. coli LG1315 and AN1937 were grown in M9 SW400 As BN3307, but pColV- Thiis study dipyridyl broth and assayed for cloacin sensitivity as de- K30, pSE103 scribed for N. gonorrhoeae. M5158 c1857 N7 N53 AHM kil+ G. Walker (14) DNA hybridizations. Genomic DNA from N. gonorrhoeae RR1 F- hsd2O ara-14 proA2 28 FA19 was as described Stern et al. Plasmid lacY) galK2 rpsL20 xyl-5 prepared by (42). mtl-i supE44 A- DNA was isolated by an alkaline lysis procedure, followed LE392 F- hsdRS14 supE44 supF58 28 by equilibrium centrifugation in a cesium chloride-ethidium lacY1 or A(lacIZY)6 bromide density gradient (28). Restriction endonucleases galK2 galT22 metBi and other enzymes were obtained from Bethesda Research trpR55 A- Laboratories (Gaithersburg, Md.) and New England Biolabs (Beverly, Mass.). Nitrocellulose (BA85; Schleicher and Plasmids Schuell, Inc., Keene, N.H.) or nylon (Hybond-N; Amer- pCPN1 Recombinant plasmid J. INeilands (37) sham Corp., Arlington Heights, Ill.) filters for Southern encoding JhuACDB, Ampr hybridizations were prepared as described (40). For colony pCPN12 Recombinant plasmid J. INeilands (37) encoding fluB, Ampr filter hybridizations, suspensions of gonococci in GCB broth pABN5 Recombinant plasmid N. Carbonetti (3, 8) were spotted onto nitrocellulose filters (HATF; Millipore encoding iucABCD iutA, Corp., Bedford, Mass.). The filters were placed on GCB agar Ampr plates, incubated overnight at 37°C in a CO2 incubator, and pSE103 c1857, Kmr G. Walker (14) then processed as described (28). Hybridization probes, pKK177-3 Vector for pCPN12, Ampr J. INeilands (37) either gel-purified fragments from pCPN1, pCPN12, or pABN5 or N. gonorrhoeae FA19 genomic DNA, were nick Bacteriophages translated with [a-32P]dCTP (38) to a specific activity of at XSE4 Phasmid vector, Spr G. Walker (14) least 108 cpm/,lg of DNA. Hybridizations were carried out in XSW28 Recombinant phage, Spr This study 5x SSPE (28)-20 mM sodium PP,-2x Denhardt solution 3416 WEST AND SPARLING J. BACTERIOL.

(28)-0.2% sodium dodecyl sulfate (SDS)-100 ,ug of sheared denatured salmon sperm DNA per ml of hybridization fluid. Hybridizations were performed at 65, 60, or 55°C for 24 to 48 h as indicated. The final washes were performed in lx 100- SSPE-5 mM sodium PPi (pH 7.5)0.1% SDS at the hybridization temperature. Hybridizations under very stringent conditions were performed at 68°C for 24 h; the final washes 50. were done in 0.5x SSPE-5 mM sodium PP1-0.1% SDS at 680C. Cloning of a gonococcal genomic fragment which complements E. colifliuB mutants. To prepare phasmid DNA, XSE4 (14), a lysogen in E. coli M5158, was induced at 420C for 30 min and immediately chilled on ice. Cells were harvested by centrifugation at 4,000 x g, and phasmid DNA was isolated 0 .0 10- by an alkaline lysis procedure (28). Genomic DNA of high c molecular weight from N. gonorrhoeae FA19 was isolated as described by Hull et al. (20). Fragments (15 to 20 kilobases [kb]) of partially Sau3A-digested genomic DNA (1 ,ug) were 5. ligated to the 35-kb BamHI fragment of XSE4 (1 ,ug); the a- ligated DNA was packaged into phage particles with the %._ lambda in vitro packaging system from Bethesda Research - Laboratories (Gaithersburg, Md.). E. coli RR1 was infected 0 with the in vitro-packaged phage particles. Recombinent 100' phage were harvested as described (28) and used to infect E. 4- coli LE392. Infection of LE392 directly with in vitro- .r packaged phage resulted in a significantly lower titer of recombinant phage. A possible reason is that genomic DNA 50 from N. gonorrhoeae is very highly methylated at cytosine residues (36); E. coli HB101 and the recombination-pro- ficient derivative RR1 can be transformed at a higher fre- quency by cytosine-methylated DNA than other E. coli strains (5). Recombinant phage were used to infect E. coli SW400, a ffuB mutant containing pColV-K30 and pSE103. Transductants were selected on LB agar containing 25 ,ug of 10- kanamycin and 25 jig of spectinomycin per ml at 30°C and replica plated onto M9 dipyridyl plates containing both antibiotics (8). Recombinant phage DNA was isolated and purified by the procedure of Helms et al. (19). Assays to demonstrate complementation of E. col fluB _ I I 1 I I mutants. E. coli flzuB mutants, which cannot synthesize 0 2 3 4 5 enterobactin or lack the enterobactin receptor, are unable to grow on medium containing a hydroxamate siderophore. Time (Hours) Therefore, to demonstrate complementation of the ihuB mutation, we used M9 medium [M9 salts (28), 0.5% FIG. 1. Growth of N. gonorrhoeae with aerobactin as an iron Casamino Acids, 40 pg of tryptophan per ml, 0.4% glucose, source. N. gonorrhoeae FA19 (A) and N. gonorrhoeae F62 (B) were 1 ,ug ofthiamine per ml, 1 ,ug ofcalcium pantothenate per ml, grown in DM with the following supplements: (A) 50 ,uM Desferal, 2 mM MgCl2, 25 ,uM Fe(NO3)3, and 1.5% Bacto agar (Difco)] (0) 50 ILM Desferal and 2 ,uM aerobactin (100%, iron saturated), or supplemented with either 200 ,uM 2,2'-dipyridyl or 750 ,uM (N) 50 puM Desferal and 75 ,uM Fe(NO3)3. Desferal. SW300 and SW400 grew well on this medium when they contained either pCPN1 or pCPN12; they were unable (which gonococci cannot use as an iron source) and aero- to grow on this medium when they contained the vector bactin. However, the rate of growth of gonococci with plasmid pKK177-3. aerobactin was slower and the cell density attained at To assay for enterobactin production, tests strains were stationary phase was lower than when gonococci were removed with a toothpick onto a lawn of E. coli LG1419 grown in DM containing 50 ,uM Desferal and 75 ,uM (approximately 107 cells) on M9 dipyridyl plates. Enterobac- Fe(NO3)3 (Fig. 1). In other experiments, we grew N. gonor- tin production was indicated by growth of LG1419 around rhoeae FA19 and F62 in DM with 4 ,uM aerobactin, which the test strains. bound any iron initially present in the medium. Again, gonococci grew with aerobactin as the iron mediator but at a RESULTS slower rate than cells provided with Fe(NO3)3 (data not shown). To demonstrate that aerobactin had indeed bound Growth of N. gonorrhoeae with aerobactin as the sole iron all iron present in the medium, we grew E. coli AN1937, an source. Yancey and Finkelstein (48) reported that the fer- entA mutant, in DM containing 4 ,uM aerobactin and in DM rated forms of aerobactin, arthrobactin, and schizokinen containing 4 ,uM aerobactin and 20 ,uM Fe(NO3)3. Growth of stimulate growth of gonococci on GCB plates made iron- AN1937 was inhibited in DM containing aerobactin alone but limiting by addition of conalbumin. We confirmed this ob- not in DM containing aerobactin and excess Fe(NO3)3 (data servation by growing gonococci in DM containing Desferal not shown). VOL. 169, 1987 AEROBACTIN UTILIZATION BY N. GONORRHOEAE 3417

kb A B C D E F JhuBCD genes. Because any gonococcal analogs of these E. coli systems may not be fully homologous and therefore would not be detected under stringent hybridization conditions, we varied the hybridization stringency by altering the temperature. With the least stringent conditions (55°C), sequences which are at least 70%o homologous should be 2 31.2.-1- detected (1). The E. coli aerobactin biosynthesis genes (iucABCD) and 9.4 - MI ;. a portion of the iutA gene, which codes for the 74K aerobactin receptor, from pColV-K30 are located on a 6.8-kb 6.6 - 4. HindIII-EcoRI fragment, which has been cloned into the S vector pPlac to form the recombinant plasmid pABN5 (3). 4.3 - When this HindIII-EcoRI fragment was used to probe restriction endonuclease-digested DNA from N. gonorrhoeae FA19, we did not detect any homology even under the least stringent hybridization conditions (55°C) (data not shown). Prody and Neilands (37) cloned an 8.4-kb HindlIl frag- 2.3 - ment which encodes fhuC, fhuD, fhuB, and fhuA, the gene 2.0- for the ferrichrome receptor, into the vector pPlac to form the recombinant plasmid pCPN1. We used this HindlIl S fragment to probe restriction endonuclease-digested DNA from N. gonorrhoeae FA19 and detected homology under

Frogment 55 60° 65° kb 0.5-

-21.2 FIG. 2. Hybridization of E. colijfhuACDB sequences to genomic DNA from N. gonorrhoeae FA19. The 8.4-kb HindIll insert from pCPN1 was hybridized to genomic DNA from FA19 in 5x SSPE at -4. 60°C for 48 h. Lanes: A, 100 pg of pCPN12 digested with PstI; B to - 3.5 F, genomic DNA from N. gonorrhoeae FA19 digested with BgII (B), A AvaIl (C), Hinfl (D), DdeI (E), or HincII (F). 8- - I.e9 C- 4* - 1.3

Lack of aerobactin production by gonococci. In previous - 1.0 reports (46, 48), bioassays for possible gonococcal sidero- E - - 0.6 F - phore production were done with Salmonella typhimurium -0.6 LT2 enb-7 and Arthrobacter flavescens JG-9, which are unable to utilize aerobactin as an iron source (9, 35). G - Therefore, to determine whether gonococci produce aerobactin, we assayed gonococcal culture supernatants in an aerobactin-specific bioassay with E. coli LG1522 as the HP 8 S B BP X P H indicator organism (8, 9). No aerobactin was detected in 11 1 I sII III unconcentrated and 10-fold-concentrated culture supernatants from N. gonorrhoeae FA19 and F62 grown in CDM, A CDM supplemented with 20 puM Fe(NO3)3, GCB, and GCB B supplemented with 50 ,uM apo-transferrin. LG1522 was C stimulated by E. coli LG1315, which produces aerobactin. E. coli AN1937, which cannot synthesize enterobactin, was not E stimulated by the gonococcal culture supernatants. F - Cloacin resistance of gonococci. The bacteriocin cloacin G B produced by Enterobacter cloacae binds to the E. coli 74K 0 2 3 4 5 6 7 8 9 kb aerobactin receptor (2). Growth of E. coli strains that express the aerobactin receptor is inhibited by cloacin. We FIG. 3. Hybridization of N. gonorrhoeae FA19 genomic DNA to found that quantities of partially purified cloacin which a Bg11-SmaI-Pstl digest ofthe 8.4-kb HindIII fragment from pCPN1. inhibited growth of sensitive E. coli strains (LG1315 and The hybridizations were performed at 65, 60, and 55°C in 5 x SSPE LG1522) did not affect the growth of either N. gonorrhoeae for 48 h. The restriction map of the 8.4-kb HindIII fragment from FA19 or F62. pCPN1 (bottom) shows the sites for HindIll (H), PstI (P), BglI (B), Probing of gonococcal genomic DNA with cloned aerobactin SmaI (S), and XhoI (X). The locations of fhuABCD and their gonococci approximate sizes are indicated as described by Prody and Neilands biosynthesis, receptor, and utilization genes. Since (37) and Coulton et al. (11). The lane on the left is the ethidium utilized aerobactin but did not appear to synthesize aerobac- bromide-stained agarose gel of the Bgll-SmaI-PstI digest of the tin, we used Southern hybridization analysis to determine 8.4-kb HindlIl fragment. The seven fragments which were obtained whether there was any DNA sequence homology between are labeled A to G, and their locations are diagrammed below the genomic DNA from N. gonorrhoeae FA19 and the cloned restriction map. The lane on the right contains lambda DNA E. coli aerobactin biosynthesis, aerobactin receptor, or digested with Hindlll and EcoRI. 3418 WEST AND SPARLING J. BACTERIOL. moderately stringent hybridization conditions (60°C) but not under more stringent conditions (Fig. 2). To determine whether the gonococcal genome had homology with the entire HindIII fragment of pCPN1 or only a N portion of it, we probed specific fragments obtained from a ..).. 9 restriction endonuclease digestion of the 8.4-kb HindIII ;.~. C fragment from pCPN1 with 32P-labeled genomic DNA from N. gonorrhoeae FA19 under various hybridization stringencies. In control experiments, we easily detected hybridization of nick-translated genomic DNA to the cloned 2- gonococcal proline biosynthesis genes (41) (data not shown). In Fig. 3, the seven restriction endonuclease fragments obtained from a BgII-PstI-SmaI digest of the 8.4-kb HindIII fragment from pCPN1 are labeled A through G according to FIG. 5. Hybridization offlzuB sequences to XSW28 and genomic the size of the fragment. Under moderately stringent condi- DNA from N. gonorrhoeae FA19 and E. coli BR686. The 32p_ tions (60°C), we detected strong hybridization only to frag- labeled 1.4-kb PstI insert from pCPN12 was hybridized to AvaIl ment C, which encodes most of the fhuB gene (23). A very digest of FA19 chromosomal DNA, 2.5 ,ug (lane A) and 5.0 ,ug (lane faint hybridization signal was detected to fragment B, which B); XSW28, 250 ng (lane C); BR686 genomic DNA, 1.0 Fg (lane D) contains sequences downstream from fragment C, including and 2.5 ,ug (lane E); 100 pg of pCPN1 digested with Hindlll (lane F); a portion of the fhuB gene as determined by Koster and and 100 pg of pCPN12 digested with PstI (lane G). Hybridizations Braun (23). Under the least stringent conditions (55°C), we were performned in 5 x SSPE at 60°C for 48 h. did not detect hybridization of gonococcal genomic DNA to JhuACD or sequences upstream of the flu operon (11). Colony filter hybridization was used to determine whether Cloning a fragment of N. gonorrhoeae FA19 DNA that three other laboratory strains and 18 strains of N. gonor- complements E. coli fhuB mutations. To confirm that rhoeae recently isolated from both urogenital and dissemi- gonococci possess a protein with a function analogous to nated infections had any nucleotide sequence homology with that of the E. coli JhuB gene, we cloned a fragment of the cloned E. coli aerobactin biosynthesis and utilization gonococcal DNA that complemented E. colifhuB mutations. genes. A variety of auxotypes, including the arginine- We screened a genomic library of N. gonorrhoeae FA19 hypoxanthine-uracil type, and various protein I serotypes DNA in the phasmid vector XSE4 for complementation of (43) were included among these strains. The colony filters the E. colifhuB mutant BN3307 (37), which is also deficient were probed with the 8.4-kb HindIII insert from pCPN1 in enterobactin production. This strain cannot grow on (data not shown) and the 1.4-kb PstI fragment from pCPN12 iron-limiting medium unless either the phenolate siderophore (Fig. 4, probe A) at moderate hybridization stringencies enterobactin is supplied or the JhuB mutation is comple- (60'C). Hybridization was detected with both probes, indi- mented and a hydroxamate siderophore is supplied. cating that all strains tested had homology with the E. coli The recombinant XSE4 library was grown in E. coli LE392 fhuB gene. When we probed the same strains with the and then introduced into E. coli SW400. Of approximately SalI-EcoRI insert from pABN5, which contains the E. coli 2,000 spectinomycin and kanamycin-resistant colonies, 4 aerobactin biosynthesis and receptor genes, we did not grew when replica plated onto M9 dipyridyl plates contain- detect any homology at moderate (60°C) hybridization strin- ing kanamycin and spectinomycin. Plasmid DNA was pre- gency (Fig. 4, probe B). pared from three of these recombinants and transformed into LE392, and phage were purified from single plaques. The purified recombinant phage were used to infect SW300 and 4 5 2 3 4 SW400. All tested colonies that grew on LB plates with both 2 3 antibiotics at 30°C also grew when replica plated onto M9 A * - plates containing both antibiotics plus Desferal. To eliminate any possibility that these recombinant clones were rever- B 0 * * * e tants that could synthesize enterobactin, the Spr colonies were assayed for enterobactin production with E. coli LG1419 as the indicator strain. All Spr colonies of either D SW300 or SW400 were unable to stimulate growth of LG1419. However, E. coli LE392, which does produce E S* * enterobactin, was stimulatory. One recombinant clone containing an insert of about 15 kb of gonococcal DNA was chosen and designated XSW28. In the complenientation assays, this clone enabled SW300 and SW400 to grow on M9 Probe A Probe B dipyridyl plates as rapidly and luxuriantly as these strains FIG. 4. Hybridization of aerobactin biosynthesis and receptor containing the cloned E. colifhuB gene on either pCPN1 or genes and fluACDB genes to 22 strains of N. gonorrhoeae. Dupli- pCPN12. cate filters were prepared and hybridized in 5 x SSPE at 60°C for 48 To confirm that XSW28 had nucleotide sequence homol- h. Probe A is the 32P-labeled 1.4-kb PstI insert from pCPN1; probe ogy with the E. colifhub gene, we probed both XSW28 and B is the 32P-labeled SalI-EcoRI fragment from pABN5. N. gonor- DNA with the 1.4-kb PstI from rhoeae strains: A2, PT1759; A3, PT2435; Bi, PT1969; B2, WM28; FA19 genomic fragment B3, WM27; B4, WM26; B5, DC83-48; Cl, FA759; C2, FA854; C3, pCPN12, which is composed entirely ofjhuB structural gene FA1131; C4, FA1128Cx; C5, FA1127; Dl, FA1126; D2, JB118; D3, sequences except for 75 bp upstream offhuB (23). Hybrid- JB135; D4, JB118; D5, JB103; El, JB101; E2, FA1090; E3, MS11; ization was detected to a 4.3-kb AvaIl fragment present in E4, FA19; E5, F62. E. coli strains: F2, LG1315; F3, AN1937. both FA19 genomic DNA and DNA from XSW28 (Fig. 5, VOL. 169, 1987 AEROBACTIN UTILIZATION BY N. GONORRHOEAE 3419 lanes A to C) and to a 2.3-kb AvaIl fragment of E. coli BR686 Under conditions of reduced hybridization stringency, we (Fig. 5, lanes D and E). Under high-stringency conditions detected multiple fragments of the gonococcal genome that (680C), we probed FA19 genomic DNA, XSW28, XSE4, and hybridized weakly to E. coli fiuB sequences (Fig. 2). The E. coli BR686 genomic DNA with 32P-labeled XSW28. Hy- explanation is unclear, but this result could be due to bridization was detected to a 4.3-kb AvaIl fragment in FA19 incomplete restriction endonuclease digestion of genomic DNA and weakly to a 2.3-kb AvaIl fragment in E. coli BR686 DNA or hybridization to closely related genes, regulatory (data not shown).' Thus, the 4.3-kb AvaIl fragment from sequences, or small repeated sequences present in the )SW28 was of gonococcal origin and had nucleotide se- gonococcal genome (10). Koomey and Falkow (22) observed quence homology with the cloned E. coli fhuB gene. similar extraneous hybridization patterns under conditions Hybridization of E. colifJhuB to genomic DNA from several of reduced stringency when the gonococcal IgA protease species of bacteria. We probed EcoRI restriction endonucle- gene was used as a probe of chromosomal DNA from a ase digestions of genomic DNA from one strain each of pathogenic Neisseria species. Bacteroides fragilis, Bacillus subtilis, Haemophilus influ- Since gonococcal DNA was able to complement two enzae, Haemophilus parainfluenzae, Legionella pneumo- independent E. coli fhuB mutations, one of which is proba- phila, and Neisseria meningitidis and two Bordetella pertus- bly a deletion mutation (strain SW400) (37), gonococci sis strains with the 1.4-kb PstI fragment from pCPN12. The contain a functional equivalent tofhuB. The role offluB in fhuB probe hybridized to DNA from Bordetella pertussis aerobactin transport by E. coli is unknown; therefore, it is and N. meningitidis under conditions of moderate hybridiza- hazardous to speculate about the function of the gonococcal tion stringency (60°C) and to Bacteroides fragilis, H. influ- JhuB homolog. Nevertheless, fhuB could be involved in enzae, H. parainfluenzae, and Bacillus subtilis under condi- other iron transport systems in gonococci rather than limited tions of low hybridization stringency (55°C) (data not to aerobactin-mediated iron transport. Construction of mu- shown). Under conditions of low hybridization stringency, tations of the gonococcalj7huB homolog would allow testing theffhuB probe did not hybridize to L. pneumophila (data not to determine whether the function of huB is limited to the shown). The vector pKK177-3 either did not hybridize under transport of hydroxamate siderophores or whether fhuB is these conditions or hybridized to different restriction frag- involved in the transport of iron by other mechanisms such ments (data not shown). as the nonsiderophore-mediated removal of iron from transferrin and lactoferrin used by gonococci (45). DISCUSSION Gonococci cause-infections of mucosal surfaces, including Although gonococci can use aerobactin as a sole source of the pharynx, rectum, and cervix. Each of these sites has a iron, they apparently do not produce aerobactin. Aerobactin complex microbial flora, and at least one (the recetal site) was not detected in culture supernatants from N. gonor- contains some aerobactin-secreting organisms. It is conceiv- rhoeae FA19 and F62 in the aerobactin-specific LG1522 able that gonococci "borrow" aerobactin produced by other bioassay. Using hybridization conditions which will detect bacteria at such sites; if this were so, it might help explain sequences which are at least 70% homologous, we detected how those gonococci that are unable to utilize lactoferrin as nucleotide sequence homology between the gonococcal an iron source (30) are able to scavenge essential iron at genome and the E. coli ffhuB gene but not vwith other certain mucosal surfaces. components of the E. coli aerobactin system (iucABCD, Several bacterial species exhibited partial homology with iutA, andfluCD). Lack of detectable homology with probes afhuB-specific probe. This diverse group of microorganisms other thanfhuB indicates that if other" proteins are involved included both gram-negative and gram-positive organisms, in gonococcal aerobactin utilization, their genes are suffi- an obligate anaerobe, and several aerobic species. This ciently dissimilar from those in E. coli that they were not observation suggests thatfhuB may be a conserved gene. detected under these conditions. Finally, we note that there is a discrepancy in the litera- The mechanism by which gonococci utilize aerobactin- ture about whether plasmids such as pCPN12 will comple- bound iron is unclear. They were not susceptible to cloacin, ment E. colifhuB mutations. Prody and Neilands (37) found which binds to the E. coli aerobactin receptor, and they did that the 1.4-kb insert in pCPN12 was sufficient to comple- not contain detectable homology with the E. coli aerobactin ment two independent-huB mutations. Koster and Braun receptQr (iutA) gene. Gonococci might nonetheless possess a (23), however, estimated that plasmids with inserts as small structurally different protein which can bind the iron- as that in pCPN12 would not complementJhuB; 2 kb ofDNA aerobactin complex. It is also conceivable that aerobactin, a was necessary to provide complementation of their jhuB hydrophilic compound of molecular weight 620, can enter mutations. Using derivatives offhuB mutants from both the through a porin, bypassing a requirement for an aerobactin- Braun and Neilands collections and an assay for complemen- specific receptor. The porins of gonococci, in contrast to tation in which aerobactin was produced by the test strains those of E. coli, allow passage of saccharides with molecular but could not be utilized for growth unless the]huB mutation weights considerably higher than 620 (13). was complemented, we found that pCPN12 did complement The exact degree of homology between the E. coli fhuB both JhuB alleles. This is surprising, since fhuB appears to sequences and the gonococcal JhuB homolog can be deter- encode a 70K protein as determined by DNA sequence mined only'by comparison of nucleotide sequence data; analysis, showing an open reading frame of 1,977 base pairs however, the hybridization conditions used in this study (23). We have not yet attempted to determine the minimum indicate that the gonococcal sequences are at least 75% size of the gonococcal insert in XSW28 needed to provide homologous with the E. coli ffhuB gene. This degree of complementation or the size of the gonococcal FhuB protein sequence divergence has been observed in other families of homolog. bacterial genes, such as the tryptophan operon in enteric bacteria (4), E. coli heat-labile enterotoxin and cholera toxin ACKNOWLEDGMENTS (32), immunoglobulin A (IgA) protease genes of Neisseria This work was supported by Public Health Service grants and Haemophilus spp. (22), and DNA gyrase in Bacillus AI-15036 and AI-07001 from the National Institute of Allergy and subtilis and E. coli (24). Infectious Diseases. 3420 WEST AND SPARLING J. BACTERIOL.

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