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Home , Neisseria gonorrhoeae

JOURNAL OF BACTERIOLOGY, July 1996, p. 4224–4232 Vol. 178, No. 14 0021-9193/96/$04.00ϩ0 Copyright ᭧ 1996, American Society for Microbiology

Isolation and Analysis of a fur Mutant of gonorrhoeae

1 1,2 CHRISTOPHER E. THOMAS * AND P. FREDERICK SPARLING Departments of Microbiology and Immunology1 and Medicine,2 School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599

Received 28 November 1995/Accepted 9 May 1996

The pathogenic Neisseria spp. produce a number of iron-regulated gene products that are thought to be important in virulence. Iron-responsive regulation of these gene products has been attributed to the presence in Neisseria spp. of the Fur (ferric uptake regulation) protein. Evidence for the role of Fur in neisserial iron regulation has been indirect because of the inability to make fur null mutations. To circumvent this problem, we used manganese selection to isolate missense mutations of Neisseria gonorrhoeae fur. We show that a mutation in gonococcal fur resulted in reduced modulation of expression of four well-studied iron-repressed genes and affected the iron regulation of a broad range of other genes as judged by two-dimensional poly- acrylamide gel electrophoresis (PAGE). All 15 of the iron-repressed spots observed by two-dimensional PAGE were at least partially derepressed in the fur mutant, and 17 of the 45 iron-induced spots were affected by the fur mutation. Thus, Fur plays a central role in regulation of iron-repressed gonococcal genes and appears to be involved in regulation of many iron-induced genes. The size and complexity of the iron regulons in N. gonorrhoeae are much greater than previously recognized.

Although iron is the fourth most abundant element on earth, binds to specific sequences in the promoters of iron-repressible it can be a limiting nutrient in many bacterial systems (14). genes, blocking transcription. Most biochemical analyses have This is due in part to the relative insolubility of most common utilized Fur purified from E. coli (72). Alpha helices near the iron salts at neutral pH in an aerobic environment. In addition, amino terminus of the protein have been implicated in DNA in host- systems, the host frequently limits the avail- binding (55, 56), while a series of histidine residues near the ability of iron by producing iron-binding proteins as part of a middle and carboxyl terminus are thought to be involved in first line of defense against invading (16, 74). Many binding ferrous iron (54, 56). Many genes repressed by Fur are have overcome this limitation by evolving high-affinity involved in iron uptake, but iron starvation is also used by siderophore-receptor systems that allow the bacteria to solu- many pathogens as a signal that they are in a host environment bilize and accumulate the iron necessary for growth (49). A with resultant expression of certain virulence factors. These number of pathogens that live entirely within a single host virulence factors include a number of hemolysins (which could species, including the pathogenic Neisseria spp., have evolved be considered part of the pathogen iron-acquisition system) means of utilizing host iron-binding proteins directly as a and toxins (42). source of iron (44). N. gonorrhoeae produces a number of iron-regulated pro- Iron-containing enzymes play a central role in electron teins. Most of the well-studied iron-regulated proteins are in- transport and other processes required for aerobic life. Along volved in iron acquisition, including the transferrin-binding with iron’s enormous redox flexibility comes the potential to proteins, Tbp1 (22) and Tbp2 (2); the ferric binding protein, participate in Haber-Weiss-Fenton chemistry, which creates Fbp (48); the lactoferrin-binding protein, Lbp (11); and prob- highly reactive and toxic hydroxyl radicals (27). Because of the ably FrpB, whose function is not fully understood (7). The metabolic cost of these iron-uptake systems and the potential expression of all of these proteins is thought to be under the toxicity of excess intracellular iron, bacteria tightly regulate control of Fur. This conclusion is based partly on the presence their iron-uptake machinery. Many gram-negative bacteria of sequences in the promoters of the respective structural regulate intracellular iron levels by means of the fur (ferric genes which are similar to sequences found in the promoters of uptake regulator) gene product. The fur locus was first de- Fur-regulated genes in other species. The dependence on Fur scribed for Salmonella typhimurium (26) but has been best for iron regulation of these proteins has been difficult to test characterized for (42, 59). Recently, fur genes directly because of the inability to make a gonococcal fur null from Neisseria gonorrhoeae (6), (66), mutant. (40), Vibrio vulnificus (41), Vibrio anguillarum Many of the available fur mutants of different species have (67), (53), Pseudomonas putida (71), been isolated on the basis of constitutive expression of sid- pertussis (5, 15), ducreyi (8), Yersinia erophores or other iron-regulated products. Null mutations pestis (62), (35), have been successfully isolated for E. coli (3, 30) V. cholerae (78), and (73) have been cloned and (43), V. vulnificus (41), and Shigella flexneri (60). Unsuccessful sequenced. attempts to isolate insertional null mutations have been re- Fur is a 15- to 17-kDa protein that, in the presence of iron, ported for P. aeruginosa (53), P. putida (71), V. anguillarum (67), H. ducreyi (8), N. meningitidis (66), and N. gonorrhoeae (6). For P. aeruginosa (53) and V. anguillarum (67), as well as * Corresponding author. Mailing address: Division of Infectious for a number of other species, point mutations have been Diseases, University of North Carolina at Chapel Hill, 521 Burnett- successfully isolated by manganese selection (15, 31, 39, 63). Womack Clinical Sciences Bldg., Campus Box 7030, Chapel Hill, NC We used a variation of this manganese selection system to 27599. Phone: (919) 966-3661. Fax: (919) 966-6714. Electronic mail isolate a point mutation in N. gonorrhoeae fur and have begun address: [email protected]. to define the extent of the Fur regulon. We found that fur

4224 VOL. 178, 1996 NEISSERIA GONORRHOEAE fur MUTANT 4225

TABLE 1. Strains and used in this study

Strain or Description Reference(s) or source N. gonorrhoeae FA6256 F62 (pro-1 met-1 spc-3) 9, 36, 58 FA6710 FA6256 transformed to Cmr by pUNCH662 This work FA6829 FA6710 transformed to Smr by pUNCH644 This work FA6835 FA6829 subjected to Mn selection and screened for PhoA regulation defect This work FA6836 FA6710 transformed to Smr by FA6835-1 (fur-1) chromosomal DNA This work FA6846 FA6710 transformed to Emr by pUNCH650 (fur wild type) This work FA6847 FA6710 transformed to Emr by pUNCH665 (fur-1) This work FA6848 FA6836 (fur-1) transformed to Emr by pUNCH650 (fur wild type) This work

E. coli DH5␣MCR FϪ mcrA mcrB mrr ␾80dlacZ⌬M15 ⌬(argF-lac)U169 recA1 endA1 hsdR hsdM supE44 Bethesda Research Laboratories ␭Ϯ thi-1 gyrA96 relA1 H1780 araD139 ⌬(argF-lac)U169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR fur 31 fiu␭::placMu53

Plasmids pACYC184 ori p15a Cmr Tcr 20 pBluescript- ori pMB1 Apr Stratagene II SKϪ pNC62 Source for por-1 with downstream mTn3(Cm) insertion 18 pHP45⍀ Source for ⍀ interposon fragment (Smr)52 pUP1 pHSS6 containing synthetic gonococcal DNA uptake sequence (Kmr)25 pUCErm Source for the ermC gene (Emr)70 pUNCH601 Source for the amino-terminal meningococcal fur probe 66 pUNCH604 Source for the full-length and carboxyl-terminal meningococal fur probe 66 pUNCH605 Source for the fbpP-phoA transcriptional fusion 66 pUNCH634A pBluescript-II SKϪ containing the upstream HincII-to-EcoRI fragment of FA6256 fur This work pUNCH635B pBluescript-II SKϪ containing the downstream EcoRI-to-HincII fragment of FA6256 fur This work pUNCH641 pACYC184 containing the reconstructed FA6256 fur gene This work pUNCH644 pUNCH641 with SmaI-digested ⍀ interposon ligated into the filled-in downstream XhoI site This work pUNCH648 pUNCH641 with the closest upstream MluI site destroyed This work pUNCH650 pUNCH648 with the filled-in HindIII-SmaI ermC fragment ligated into the filled-in downstream XhoI site This work pUNCH655 pUNCH650 with the AflII-to-BspHI internal fur fragment replaced by a PCR product derived from FA6836 (fur-1) This work pUNCH660 pUP1 containing por-1 with downstream mTn3(Cm) insertion This work pUNCH661 pBluescript-II SKϪ containing fbpP-phoA transcriptional fusion cassette This work pUNCH662 pUNCH660 with the fbpP-phoA cassette ligated into the mTn3(Cm) filled-in AccI site This work

controls expression of all the tested gonococcal iron-repressed frozen at Ϫ20ЊC until needed. CDM was converted to solid medium by supple- proteins. mentation with 0.5% (wt/vol) potato starch and 1.5% (wt/vol) washed agarose (19). Gonococci were labeled with 14C-amino acids by growth in CDM with the MATERIALS AND METHODS following modifications. The concentrations of all of the amino acids in CDM were reduced by one-half to yield reduced amino acid CDM (RACDM), effec- Bacterial strains, plasmids, and growth conditions. Bacterial strains and plas- tively doubling the specific activity of 14C-amino acids added during labeling. A mids used in this study are listed with their relevant properties in Table 1. N. total of 18.75 ␮Ci of 14C-amino acid mixture (catalog no. CFB.104; Amersham gonorrhoeae strains were routinely maintained on GC-medium base (Difco Lab- Corp., Arlington Heights, Ill.) per ml of CDM was added prior to the 3-h growth oratories, Detroit, Mich.) supplemented with Kellogg’s supplement I (36) and period. These modifications yielded gonococcal cultures that were labeled to 12.5 ␮M FeNO (GCB) at 36ЊC in an atmosphere of 5% CO . Strain DH5␣MCR 3 2 approximately 50,000 dpm/␮g of protein. (Bethesda Research Laboratories) was used as the host for all plasmid manip- Alkaline phosphatase assays. Alkaline phosphatase activity of toluene-treated ulations in E. coli and was maintained on Luria-Bertani medium (57) supple- bacterial cultures was measured by the method of Kreuzer et al. (37), except that mented with as necessary. Antibiotics were included in media at the bacteria were harvested by centrifugation and resuspended in assay buffer concentrations as follows: ampicillin, 100 ␮g/ml; streptomycin, 100 ␮g/ml; chlor- amphenicol, 30 ␮g/ml for E. coli and 0.65 ␮g/ml for N. gonorrhoeae; erythromy- prior to toluene treatment. Activity was expressed in Miller (47) equivalent units cin, 300 ␮g/ml for E. coli and 2 ␮g/ml for N. gonorrhoeae. (MEUs). One MEU was defined as the amount of alkaline phosphatase in a In experiments comparing iron-limited and iron-replete gonococcal cultures, culture with an original OD600 of 1.0 that produced a change in the OD405 of the bacteria were grown in chemically defined medium (CDM) treated with Chelex- assay mixture of 0.001 in 1 min. The chromogenic substrate 5-bromo-4-chloro- 100 (Bio-Rad Laboratories, Hercules, Calif.) to remove any residual iron (77). 3-indolylphosphate (BCIP) was incorporated into GCB plates at 25 ␮g/ml (GCB- All manipulations of CDM were done with either disposable plastic or acid- BCIP) to screen alkaline phosphatase expression as indicated. The PhoA regu- washed glassware to avoid incidental iron contamination. Iron-replete CDM latory phenotype was evaluated on solid media by placing a 0.25-in. (ca. 0.64 cm) cultures were supplemented with ferric chloride to 100 ␮M. CDM cultures were paper disk saturated with 500 nmol of an iron chelator (Desferal [deferoxamine inoculated to an optical density at 600 nm (OD600) of 0.14 with the growth from mesylate]; Ciba-Geigy, Basel, Switzerland) near the center of a GCB-BCIP plate. nonconfluent GCB plates incubated for 16 h. Cultures were then grown for 1 h This effectively created a gradient of chelator, with available iron increasing with at 36ЊCina5%CO2shaking incubator, back diluted to an OD600 of 0.05, and distance from the chelator disk on a plate, allowing the qualitative assessment of grown for an additional 3 h. Cultures were then harvested by centrifugation at PhoA expression. room temperature, resuspended in 10 mM HEPES (N-2-hydroxyethylpiperazine- Mn selection. Mn selection was performed by a modification of the method of NЈ-2-ethanesulfonic acid) (pH 7.4) at 1/40 of the original culture volume, and Hantke (31). Selection of Mn-resistant mutants was done on GCB plates sup- 4226 THOMAS AND SPARLING J. BACTERIOL.

was blunted by filling in with Klenow fragment) (Fig. 1A). The Cmr gene on pUNCH662 remained intact after this construction. pUNCH662 was now ready to be transformed into N. gonorrhoeae. Cloning and modification of gonococcal fur. Isolation of a genomic clone of the fur region was necessary both to confirm the linkage of a mutation with the fur locus and to determine the exact nature of the mutation. Our approach to cloning the fur locus relied heavily on our experience with meningococcal fur (66) and the work of Berish et al. (6). We cloned the ϳ1.4-kb HincII fur fragment in two pieces, separated at the EcoRI site (Fig. 1B). The 1.3- to 2.0-kb fragments of a FA6256 HincII chromosomal digest were isolated and digested with EcoRI. This DNA was then ligated into pBluescript II SKϪ digested with EcoRI and either HincII or SmaI. These partial libraries were transformed into DH5␣MCR and screened by hybridization to the insert from pUNCH604 (1.5-kb meningo- coccal HincII fur-containing fragment). Hybridizing clones were then screened in duplicate by hybridization to either the 600-bp MluI-to-EcoRI fragment from pUNCH601 (encoding the amino terminus of meningococcal Fur) or the 500-bp EcoRI fragment of pUNCH604 (encoding the carboxyl terminus of meningo- coccal Fur). The amino terminus-encoding HincII-to-EcoRI fragment of gono- coccal fur in similarly digested pBluescript II SKϪ was designated pUNCH634A. The carboxyl terminus-encoding EcoRI-to-HincII fragment of gonococcal fur in EcoRI-SmaI-digested pBluescript was designated pUNCH635B. The identity of these clones was confirmed by ethidium bromide-stained gel and Southern blot hybridization. Gonococcal fur was reassembled by ligating the SalI-to-EcoRI fragment of pUNCH634A and the EcoRI-to-BamHI fragment of pUNCH635B into SalI- BamHI-digested pACYC184 (Fig. 1B). This clone was designated pUNCH641 and was capable of complementing the E. coli fur mutant, H1780 (31), as judged by regulation of its Fur-regulated reporter fusion. The ⍀ interposon (52) was then inserted into the XhoI site downstream of fur (Fig. 1B). The SmaI-digested ⍀ fragment derived from pHP45⍀ (52) was cloned into the Klenow fragment- treated XhoI site of pUNCH641 to yield pUNCH644. When pUNCH644 was transformed back into FA6710 to yield FA6829, it made movement of the resident fur allele into new genetic backgrounds simple by virtue of the selectable marker now linked to fur. FA6829 was used as the parent strain for the Mn selection discussed below. FIG. 1. Restriction map showing the construction of the gonococcal reporter The cloned gonococcal fur locus was subjected to two additional modifications construct (A) and fur clones (B). Shown are linear restriction maps of plasmids to permit the reintroduction of defined fur mutations into the chromosome of N. described in Table 1 (inserts only). For simplicity, only the pertinent restriction gonorrhoeae (Fig. 1B). The first of these modifications was disruption of one of sites have been shown. Relevant genes are indicated by boxes with their orien- the two MluI sites upstream of fur in pUNCH641. This was accomplished by tation indicated by the arrows. The hairpins in panel A indicate the position of filling in a partial MluI digest of pUNCH641 with Klenow fragment and religat- inverted repeats recognized as the gonococcal uptake sequence and frequently as ing. Clones were screened for loss of the MluI site proximal to fur, and one clone transcriptional terminators (29). The MluI site inside a slashed circle in panel B lacking this site was designated pUNCH648. The second modification was the shows the position of the MluI site that was lost during the construction of insertion of a selectable marker downstream of fur. The ermC gene of pUCErm pUNCH648. The X in the middle of fur shows the position of the fur-1 mutation (70) was isolated as a 1.2-kb SmaI-HindIII fragment and was cloned into the present in pUNCH655. XhoI site downstream of fur (both insert and vector were made blunt ended with Klenow fragment). The resulting construct was designated pUNCH650. The loss of the upstream MluI site allowed the screening of transformants for an upstream crossover event. The insertion of ermC downstream allowed selection of recom- plemented with 200 ␮M MnCl2 (GCB-Mn) or on CDM plates containing 2.5 ␮M binants. 30% Fe-saturated human transferrin (44) (Sigma Chemical Co., St. Louis, Mo.) PCR cloning and sequencing of the fur allele. The mutant fur gene of FA6836 and 10 ␮M MnCl2 (CDM-Tf-Mn). Mn plates were always used fresh, and as was PCR amplified with Taq DNA polymerase. The amplification mixture con- observed by Hantke (31), selection was Mn concentration sensitive. Less than a sisted of 50 pmol of each primer, 25 nmol of each deoxynucleoside triphosphate, twofold variation of Mn concentration in either direction interfered with efficient 1/10 volume of 10ϫ Taq buffer, 160 ng of chromosomal DNA, and5UofTaq selection of Mn-resistant isolates. Gonococci from 18-h GCB plates were heavily DNA polymerase in a volume of 100 ␮l. The primers used were oligonucleotide streaked onto Mn-selective media and allowed to incubate for 48 h at 37ЊCin5% 601-5 (5Ј-CGA-TAA-TCA-TAC-GCT-TAA-GC-3Ј; bp 136 to 155) and oligonu- CO2. Healthy colonies were restreaked onto the same media to confirm a resis- cleotide 604-6 (5Ј-GCA-TAG-GCA-GGA-TCG-GG-3Ј; bp 838 to 854). The tant phenotype and then screened for alteration of PhoA expression phenotype primers and their numerical positions were based on the published meningococ- on GCB-BCIP media. cal fur sequence (66). This mixture was subjected to 35 cycles of 94ЊC for 1 min, Cloning and DNA manipulations. Routine cloning techniques were performed 45ЊC for 1 min, and 72ЊC for 1 min. The PCR products from several reactions as described by Sambrook et al. (57). Plasmids were isolated from E. coli by the were pooled, gel isolated, digested with AflII and BspHI, and gel isolated again. Qiagen DNA purification system (Qiagen, Inc., Chatsworth, Calif.). Chromo- This fragment was then cloned into AflII-BspHI-digested pUNCH650 to yield somal DNA from N. gonorrhoeae was purified by a modification of the method of pUNCH655. To avoid misinterpreting Taq polymerase errors as changes in Stern et al. (64) using polyoxyethylene sorbitan monolaurate (Tween 20; Sigma FA6836 fur, multiple isolates of pUNCH655 were pooled and sequenced in Chemical Co.) instead of Triton X-100 in the lysis step. In general, DNA- parallel with the parent plasmid (pUNCH650). An individual isolate of modifying enzymes were purchased from New England Biolabs, Inc. (Beverly, pUNCH655 was also sequenced to confirm that it had no PCR errors, and this Mass.) and used according to the manufacturer’s specifications. DNA fragments isolate was used for all further manipulations. were isolated as necessary by the Gene Clean system (Bio 101, Inc., La Jolla, Protein sample preparation and Western blot (immunoblot) analysis. Whole- Calif.) or the Magic DNA clean-up kit (Promega Corp., Madison, Wis.). cell lysates for Western blots were prepared by bringing samples frozen in 10 mM Construction of an iron-responsive reporter strain. An iron-responsive enzy- HEPES (pH 7.4) to 2% (wt/vol) sodium dodecyl sulfate (SDS). Samples were matic reporter strain of N. gonorrhoeae was constructed to aid in the isolation of mixed and boiled for 2 min. Samples were then assayed for protein content by the a mutation in the gonococcal fur gene. A transcriptional fusion between the bicinchoninic acid assay (Pierce, Rockford, Ill.) and balanced to 1 ␮g of protein promoter of fbp and a promoterless phoA gene (66) was cloned into the region per ␮lin1ϫsample loading buffer (Hoefer Scientific Instruments, San Francisco, downstream of por-1, the gene of N. gonorrhoeae. The plasmid pNC62 has Calif.). Proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS- a mTn3(Cm) insertion downstream of the por (18). The NotI PAGE) (38) and transferred to Nitroplus membranes (Micron Separations, Inc., insert from pNC62 was subcloned into pUP1, which had been partially digested Westborough, Mass.) or BA85 (Schleicher & Schuell, Inc., Keene, N.H.) by the with NotI, to yield pUNCH660 (Fig. 1A). pUP1 has a synthetic gonococcal DNA method of Towbin et al. (69). The percentage of acrylamide in the polyacrylamide uptake sequence to aid its transforming efficiency (25). The fbp-phoA fusion from gel was adjusted as necessary for differently sized proteins. Blots that were to be pUNCH605 (66) was subcloned into KpnI-HincII-digested pBluescript II SKϪ on probed with antibodies were blocked with 2% (wt/vol) bovine serum albumin in a KpnI-to-NdeI fragment (the NdeI site was blunted by filling in with Klenow phosphate-buffered saline with azide (137 mM NaCl, 2.7 mM KCl, 10 mM fragment) to yield pUNCH661 (not shown). The fbp-phoA fusion was then Na2HPO4, 1.8 mM KH2PO4, 0.1% NaN3 [pH 7.4]) for a minimum of 1 h. isolated from pUNCH661 as an EcoRV fragment and cloned into the unique Antibodies were diluted in blocking buffer and used at concentrations as follows AccI site within mTn3(Cm) of pUNCH660 to yield pUNCH662 (the AccI site for a minimum of 1 h: polyclonal anti-Tbp1 at 1:300 (22), polyclonal anti-FrpB VOL. 178, 1996 NEISSERIA GONORRHOEAE fur MUTANT 4227 at 1:2,000 (12), anti-Fbp monoclonal antibody RT26 at 1:1,000 (45), and anti-PIA which to carry the Fur-regulated reporter fusion into the chro- monoclonal antibody 1EA at 1:5,000 (24). Blots were then washed for 10 min mosome (fig. 1 and Materials and Methods). The construction three times in TBST (20 mM Tris [pH 7.5], 500 mM NaCl, 0.05% Tween 20). Polyclonal antibodies were detected with a 1:2,000 dilution of protein A-alkaline of pUNCH662 afforded an efficient means to move an iron- phosphatase conjugate (Sigma). Monoclonal antibodies were detected with anti- responsive reporter fusion into the chromosome of N. gonor- mouse immunoglobulin G–alkaline phosphatase conjugate (Bio-Rad) at a dilu- rhoeae. Chloramphenicol-resistant transformants of FA6256 tion of 1:2,500. Blots were developed with 337 ␮g of nitroblue tetrazolium per ml (furϩ) were screened for iron-regulated alkaline phosphatase and 175 ␮g of BCIP per ml in TMS (100 mM Tris [pH 9.5], 50 mM MgCl2, 100 mM NaCl). Tbp2 was detected by binding of human transferrin essentially as expression by streaking them onto GCB-BCIP plates (which described by Cornelissen et al. (22) with the exception that blots were developed contain abundant iron) and placing a paper disk containing 500 with enhanced chemiluminescent substrate (Amersham Corp.) and exposed to nmol of Desferal (an iron chelator) near the center. One iso- X-AR5 film (Eastman Kodak Co., Rochester, N.Y.). The blots, film, and Coo- late which showed a blue colony phenotype only in the area massie blue-stained gel shown in Fig. 2 were scanned on a ReliSys 2412 flatbed scanner (ReliSys, Milpitas, Calif.) and annotated and assembled in Adobe Pho- just outside the zone of no growth around the Desferal disk (a toshop (Adobe Systems, Inc., Mountain View, Calif.). region of iron-stressed growth) was chosen and designated Two-dimensional PAGE (2-D PAGE). Samples for 2-D PAGE were prepared FA6710. This strain produced approximately 60 MEUs of al- 14 by collecting bacteria labeled with C-amino acids by centrifugation at room kaline phosphatase under CDM iron-replete conditions and temperature. Bacteria were then rinsed twice in a decreasing volume of Hanks buffered salt solution plus supplements (HBSSϩS) (137 mM NaCl, 5.4 mM KCl, 400 MEUs under CDM iron-limiting conditions, an induction 337 ␮MNa2HPO4, 441 ␮MKH2PO4, 171 ␮M CaCl2, 105 ␮M MgCl2), collected ratio of about sixfold. This indicated that we had successfully by centrifugation, and resuspended in 100ЊC osmotic lysis buffer (10 mM Tris constructed an iron-responsive reporter strain of N. gonor- [pH 7.4], 0.3% SDS) to approximately 10 ␮g/␮l. The lysate was cooled and rhoeae. As a by-product of this transformation, FA6710 was treated for 5 min on ice with a 1/10 volume of a mixture of bovine pancreatic converted from the porin serotype PIB (FA6256) to the porin nucleases to reduce viscosity. The nuclease mixture contained 50 mM MgCl2, 100 mM Tris (pH 7.0), 500 ␮g of RNase A type IIIA (Sigma) per ml, and 1,000 ␮g serotype PIA (pUNCH662). This transition was judged by the of DNase I type II (Sigma) per ml. Following nuclease treatment, samples were reaction of FA6710 with monoclonal antibody 1EA, which diluted 1:1 in SDS boiling buffer (5% SDS, 5% ␤-mercaptoethanol, 10% glycerol, recognizes the amino terminus of PIA and confirmed that the 60 mM Tris [pH 6.8]) and frozen at Ϫ70ЊC until needed. Each sample was run in triplicate on 2-D gels to control for gel-to-gel vari- reporter construct had recombined into the porin locus. This ability and to allow for the application of statistical tests. Approximately 1 ␮Ci of alteration had no observed effect on the regulation of several labeled protein was loaded per gel. 2-D electrophoresis was performed according iron-repressed genes including tbpA, frpB, and fbp (data not to the method of O’Farrell (51) by Kendrick Laboratories, Inc. (Madison, Wis.), shown). as follows. Isoelectric focusing was carried out in glass tubes of 2.0 mm in inner diameter, with 2.0% Resolytes pH 4 to 8 ampholines (BDH from Hoefer Sci- Isolation of a fur mutant. Using the reporter strain FA6710 entific Instruments) for 9,600 V ⅐ h. The final tube gel pH gradient for this set of (and its derivatives), we were able to screen large numbers of ampholines was measured by a surface pH electrode. After equilibration for 10 isolates for alterations in their ability to repress the fbp-phoA min in SDS sample buffer (10% glycerol, 50 mM dithiothreitol, 2.3% SDS, and fusion. Initial attempts to generate a fur mutant involved ran- 0.0625 M Tris [pH 6.8]), the tube gel was sealed to the top of a stacking gel, which was on top of a 10% acrylamide slab gel (0.75 mm thick). SDS-slab gel electro- dom mutagenesis with ethyl methylsulfonate (10) and screen- phoresis was carried out for about4hat12.5 mA per gel. The following ing of colonies on GCB-BCIP (iron-rich complex medium with 14C-molecular weight markers were added to a well in the agarose that sealed the chromogenic PhoA substrate) plates for PhoA-derepressed tube gel to the slab gel: myosin, 200,000; phosphorylase b, 97,400; bovine serum isolates. Although these experiments yielded isolates which albumin, 69,000; ovalbumin, 46,000; carbonic anhydrase, 30,000; and lysozyme, 14,300. After electrophoresis, the gels were dried onto paper. were affected in their ability to repress expression of the alka- Dried gels were imaged directly on a Molecular Dynamics Phosphoimager SF line phosphatase fusion (i.e., blue on high-iron GCB-BCIP (Molecular Dynamics, Sunnyvale, Calif.). Images were exported to 8-bit tagged plates), none of them proved to be linked to fur (data not image file format, cropped, inverted, and imported into the BioImage Analysis shown) or the reporter construct. One potential explanation Software system (BioImage Products, Ann Arbor, Mich.). By using the portion of this software package designed for analysis of 2-D PAGE, approximately 1,000 for this class of mutations was a defect in inorganic iron uptake spots were detected on each of the 12 images. Of these, 800 of the most which reduced intracellular iron levels and therefore dere- prominent and reproducible spots were quantitated and matched to a single pressed the normally iron-repressed reporter fusion. These reference image. Approximately two-thirds of the spots were automatically mutants were not studied further. matched by the software. The remaining one-third of the spots were matched manually. Automated matches were proofread during the manual matching Manganese selection (31) has been used in several other process. When necessary, spot boundaries were drawn manually to resolve con- species to isolate fur mutants (15, 31, 39, 53, 63, 67). Although flicts and discrepancies. Tables of spot quantitation and matching data were it was possible to isolate Mn-resistant isolates under conditions exported from the BioImage Analysis Software system and manipulated in Mi- of excess inorganic iron on GCB plates (complex medium), crosoft Excel. The integrated intensity of each of the spots was normalized both internally within each triplicate set and externally between samples. Internal none of the isolates screened showed any defect in iron regu- normalization using all the spots was referenced to one of the three gels of each lation as measured by their phenotype on GCB-BCIP medium sample. This normalization minimized systematic gel-to-gel variability. External (data not shown). However, when Mn selection was applied to normalization between samples was referenced to the wild-type, iron-replete FA6829 on CDM plates (defined medium) on which the only sample. This normalization used only spots that were less than twofold iron regulated in the wild-type strain and controlled for sample-to-sample variation. source of iron was human transferrin (CDM-Tf-Mn), approx- Spots were classified as iron repressed (Frp) or iron induced (Fip) and Fur imately 50% of the Mn-resistant colonies were altered in their dependent (Furd) or Fur independent (Furi). Spots that exhibited greater than ability to repress the fbp-phoA reporter construct. These twofold variation depending on iron availability with a greater than 95% confi- PhoA-derepressed isolates were designated FA6835 isolates. dence level in Student’s t test were considered to be iron regulated. Final judgments were made by visual comparisons by both authors. The difference in the ability of Mn to affect the regulation phenotype of the reporter fusion under these two selection conditions probably stems from the poorly understood way in RESULTS which Mn semispecifically selects for fur mutations (see Dis- Creation of an iron-responsive reporter strain. The first step cussion). in seeking a neisserial fur mutation was the development of an DNA from each FA6835 isolate was transformed back into efficient means to screen for alterations in iron regulation. To FA6710 selecting for the ⍀ interposon. Approximately one- this end, we transformed a transcriptional fusion between the third of the FA6835 Mn-resistant isolates tested (14 of 47) had fbp promoter and a promoterless phoA gene (previously used a phenotype which cotransformed at high frequency with the to assess neisserial fur regulation in E. coli [66]) into the neis- fur-linked ⍀ interposon. This transformation also helped to serial chromosome. This was accomplished by cloning the re- ensure that the strains were isogenic except for the fur region. porter fusion into a nesserial gene to provide homology with A representative group of transformants chosen on the basis of 4228 THOMAS AND SPARLING J. BACTERIOL. their phenotype on GCB-BCIP plates was characterized fur- ther by growth under iron-limiting and iron-replete conditions. Samples from these cultures were quantified for PhoA activity. The ability of isolates to regulate PhoA activity in quantitative assay paralleled their phenotype on GCB-BCIP plates. Isolates varied from near-wild-type regulation to almost no regulation (data not shown). Since a goal of this study was to investigate the breadth of the Fur regulon, the strain showing the most profound defect, a transformant obtained with DNA from FA6835 isolate 1, was chosen for further study. This transfor- mant was designated FA6836, and its fur allele was designated fur-1. Preliminary studies by SDS-PAGE and Western blot of FA6836 indicated that it was derepressed for each of three tested Frps (Fbp, FrpB, and Tbp1). To determine the exact nature of the fur-1 mutation in FA6836, we PCR amplified, cloned, and sequenced the fur open reading frame. The mutation was a missense mutation FIG. 2. Western blot analysis of a fur mutant. Lanes 1 and 2 contain whole- changing tyrosine 82 to a cysteine (DNA codon, tat to tgt). The cell lysates of FA6846 (furϩ) grown under iron-replete and iron-limiting condi- cloned version of the fur-1 mutation (pUNCH655) as well as tions, respectively. Lanes 3 and 4 contain whole-cell lysates of FA6847 (fur-1) grown under iron-replete and iron-limiting conditions, respectively. (A) Coo- the wild-type fur allele (pUNCH650) was also transformed massie blue-stained 10% polyacrylamide gel with 10 ␮g of protein loaded per back into FA6710 to ensure that further characterization was lane. Molecular mass markers are indicated along the left edge of panel A. (B done on a completely defined isogenic pair of strains desig- and C) Western-type blots of 7.5% polyacrylamide gels loaded with 30 ␮gof nated FA6846 (furϩ) and FA6847 (fur-1). FA6847 had a phoA protein per lane probed for Tbp1 and Tbp2, respectively. (D) Western blot of a 7.5% polyacrylamide gel loaded with 10 ␮g of protein per lane probed for FrpB. phenotype indistinguishable from that of the other gonococcal (E) Western blot of a 10% polyacrylamide gel loaded with 10 ␮g of protein per strains containing fur-1 (data not shown). FA6836 was trans- lane probed for Fbp. (F) Western blot of a 10% polyacrylamide gel loaded with formed to a wild-type phoA phenotype by using pUNCH650 200 ng of protein per lane probed for Por. The approximate positions of the (FA6848), demonstrating that the phenotype was due solely to proteins detected in panels B through F are indicated in panel A by the con- ϩ necting lines. The digital images for this figure were generated with a ReliSys the fur allele (data not shown). FA6846 (fur ) and FA6847 2412 flatbed scanner and annotated with Adobe Photoshop. (fur-1) were passaged to isolate nonpiliated, transparent colony phenotypes to minimize the possibility of misidentifying a change in expression of or Opa proteins as an effect of the mutation. The opacity phenotype (OpaϪ) was confirmed by rates of these strains were statistically indistinguishable under Western blot with monoclonal antibody 4B12 (1). iron-limiting conditions: 88 Ϯ 7 min for FA6846 (furϩ) and Effect of a fur mutation on known iron-regulated genes. 82 Ϯ 5 min for FA6847 (fur-1)(Pvalue ϭ 0.47). Strains FA6846 (furϩ) and FA6847 (fur-1) produced five iron- Analysis of 2-D PAGE. We investigated additional changes regulated proteins to which we had probes. These proteins in the protein expression profile by 2-D PAGE. Representative were FrpB, Tbp1, Tbp2, Fbp, and PhoA (from the fbp-phoA 2-D gels of the isogenic pair of strains, FA6846 (furϩ) and fusion). Lbp was not produced by these strains since they were FA6847 (fur-1), grown under iron-replete and iron-limiting derivatives of F62, a natural lactoferrin-receptor mutant (13). conditions, are shown in Fig. 3. Each of the four samples was Alkaline phosphatase expression was used as the screen for the run and analyzed in triplicate to enable statistical analysis. This fur-1 mutation in this strain and was monitored during all was necessary because of the incomplete nature of the muta- strain manipulations. Quantitatively, modulation of PhoA ex- tion being analyzed (sixfold to twofold reduction in reporter pression was reduced in CDM from more than sixfold in regulation). Spots were classified as constitutive, Fe induced FA6846 (furϩ)(63UFeϩ,384UFeϪ) to less than twofold in (Fip) or Fe repressed (Frp). Regulated spots were then clas- FA6847 (fur-1)(251UFeϩ,444UFeϪ). Analysis of the sified as either Fur independent (Furi) or Fur dependent expression levels of the remaining four iron-regulated proteins (Furd) on the basis of whether they remained regulated in the is shown in Fig. 2. Duplicate gels were transferred and probed mutant. Of the 800 spots analyzed, 106 spots were iron regu- in Western blots. A Coomassie brilliant blue-stained gel and a lated at least twofold (P Յ 0.05), but only 60 of these spots Western blot probed with anti-Por monoclonal antibody 1EA could be visually confirmed. Of these 60 spots, 15 were iron were included as controls to show that samples were balanced repressed (Frps) and 45 were iron induced (Fips). Three of the for total protein. Although these Western blots were not en- four Frps which were assayed by Western blot (Tbp1, FrpB, tirely quantitative, there was an obvious effect of the fur mu- and Fbp) were not expected to be visible on these 2-D gels tation on the regulation of all four tested iron-repressed pro- because of their extremely basic predicted pIs. The fourth teins. (Tbp2) may be visible as one or both of the two Furd Frp spots Effect of a fur mutation on bacterial growth. Mutations in fur in the upper right region of the gels (Tbp2 is 85 kDa, with a have been observed to have pleiotropic effects in a number of predicted pI of 7.5). All 15 Frps were at least partially depen- species (28, 43, 61, 68). FA6847 (fur-1) exhibited a significant dent on Fur for their regulation (Furd). The iron-induced spots reduction in colony size on solid media, but this growth defect consisted of 28 that were Furi and 17 that were Furd. In many was less pronounced in liquid cultures. To test whether this cases, the differentiation between Furi and Furd Fips was sub- growth defect was significant under the conditions to be used tle, and the majority of them (25 of 45) were modulated less to 14C label these strains for 2-D analysis, FA6846 (furϩ) and than threefold by iron. A schematic representation of the po- FA6847 (fur-1) were grown on three separate days in sition of the regulated spots is presented in Fig. 4. The size of

RACDM Ϯ 100 ␮M FeCl3. The exponential doubling time of the symbols is not proportional to the size of the spot or to the FA6846 (furϩ) (59 Ϯ 4 min) was significantly extended to 75 Ϯ magnitude of its regulation. In general, the Furd Frp class 5 min in FA6847 (fur-1)(Pvalue ϭ 0.0007) but only when contained the most visually convincing examples although grown under iron-rich conditions (100 ␮M FeCl3). The growth some of each of the two classes of Fips also were visually VOL. 178, 1996 NEISSERIA GONORRHOEAE fur MUTANT 4229

FIG. 3. 2-D polyacrylamide gels of 14C-labeled gonococcal whole-cell lysates. The upper panels show gels loaded with 1 ␮Ci of labeled FA6846 (furϩ) grown under iron-replete (left) and iron-limiting (right) conditions. The lower panels show gels loaded with 1 ␮Ci of labeled FA6847 (fur-1) grown under iron-replete (left) and iron-limiting (right) conditions. Phosphoimages of the gels are oriented with the isoelectric focusing dimension horizontal and the SDS-PAGE dimension vertical. The approximate surface pH of isoelectric focusing tube gels is indicated along the horizontal axis. The approximate positions of SDS-PAGE molecular mass standards are indicated in kilodaltons along the vertical axis. The digital images for this figure were generated with a Molecular Dynamics Phosphoimager SF and annotated with Adobe Photoshop. convincing. Examples of each of the three observed classes of iron-regulated proteins are presented in Fig. 5.

DISCUSSION

Previous evidence of the role of Fur in iron regulation of gonococcal genes consisted of the observation that all se- quenced iron-repressed gonococcal (and meningococcal) genes contained sequences in their proposed promoters similar to those found in the promoters of Fur-regulated genes of other species (Fur-binding sequences). In several cases, it was also shown that E. coli Fur was capable of interacting with the neisserial promoters by either gel shift (6), fur-dependent reg- ulation in E. coli (7, 66), or Fur titration assay (65). Obtaining direct evidence that the expression of these iron-repressed genes is controlled by Fur has been hampered by the inability to make fur null mutants. Attempts to create insertional null mutations were unsuccessful (references 6 and 66 and unpub- lished data) for poorly understood reasons. Some gene prod- ucts that are required for viability may be produced only in the presence of Fur. Alternatively, constitutive expression of all of the Fur-repressed genes may be detrimental to the growth of FIG. 4. Symbolic representation of 2-D gels. The relative positions of the the bacteria because of the resulting intracellular iron over- detected iron-regulated spots are indicated by the symbols; each type of symbol represents a different class. Filled symbols indicate iron-repressed spots, and load. This type of explanation is supported by the work of d Touati et al., who observed that fur mutants of E. coli produced open symbols indicate iron-induced spots. Circles are Fur spots and squares are Furi spots. The approximate pI and molecular mass (in kilodaltons) of the spots iron overload, leading to oxidative stress and DNA damage are indicated on the horizontal and vertical axes, respectively. The amounts of (68). The inability to isolate N. gonorrhoeae fur null mutations protein in each spot and the degree of regulation are not shown. 4230 THOMAS AND SPARLING J. BACTERIOL.

TABLE 2. Comparison of iron-regulated spot class distributions

Frpa Fipb Sp. (source or reference) Furi Furd Furi Furd Salmonella typhimurium (28) 6 25 1 9 Vibrio cholerae (43) 17 22 4 3 (61) 9 14 2 4 Neisseria gonorrhoeae (this study) 0 15 28 17

a Iron-repressed proteins. b Iron-induced proteins.

were only indirect symptoms of alterations in growth rate; however, the analyzed differences should have also included FIG. 5. Examples of the different classes of 2-D gel spots. Each column of most if not all of the proteins that were truly iron and/or Fur panels comes from a different set of duplicate gels. The first two columns of panels are from gels loaded with whole-cell lysate of FA6846 (furϩ) grown under regulated and contained within the limits of resolution and iron-replete and iron-limiting conditions, respectively. The second two columns detection of the 2-D gels. Controlling for differences in protein of panels are from gels loaded with whole-cell lysate of FA6847 (fur-1) grown expression caused by alterations in growth rate rather than the under iron-replete and iron-limiting conditions, respectively. The spot of interest stimulus being tested has been a problem inherent in many in each case is the spot closest to the center of the panel. Each row represents a different class of spot. Class A spots were iron repressed and Furd. A total of 15 analyses and would be difficult if not impossible to control for class A spots were detected. Class B spots were iron induced and Furi. A total of in the type of experiments presented here. 28 class B spots were detected. Class C spots were iron induced and Furd. A total Further analysis of other mutations isolated after Mn selec- of 17 class C spots were detected. The digital images for this figure were gen- tion could aid in functional analysis of Fur protein structure. erated with a Molecular Dynamics Phosphoimager SF and annotated with Adobe Photoshop. The analyzed mutation was a missense mutation, converting Tyr-82 to Cys. Position 82 is conserved in the sequenced fur homologs as either a tyrosine or, more commonly, a phenylal- anine. This mutation is only 6 residues upstream of one of the may be due to differences in the response of gonococci and E. proposed metal binding motifs, (H)-H-H-X2-C-X2-C (34) (the coli to oxidative stress (32) and/or DNA damage (17). first histidine is not conserved in Neisseria spp.). It is possible A key tool in isolating a mutation in N. gonorrhoeae fur was that this mutation affects the metal binding domain’s ability to the construction of the iron-regulated fbp-phoA reporter bind metals or its specificity in binding those metals. Alterna- strain, FA6710. The observed sixfold iron regulation of alka- tively, the mutation might affect the ability of this domain to line phosphatase activity in FA6710 was sufficient to allow transmit information regarding metal binding to the more ami- screening of large numbers of isolates for the inability to reg- no-terminal proposed DNA binding domain (23, 54–56). This ulate the reporter construct, although the level of fbp-phoA mutation also falls on the boundary of one of the alpha helix regulation was minimal compared with the iron regulation of regions proposed by Wertheimer et al. (75) and could alter this Fbp expression in wild-type gonococci. This discrepancy could region’s conformation as well. However, this mutation’s effect be due to other levels of regulation imposed on native Fbp that on intracellular levels of Fur (i.e., effects on stability and/or are not imposed on the truncated, out-of-context, fbp promoter expression level) will need to be studied before any of these sequences included in the reporter construct (66). mechanisms can be further explored. Mn selection is a relatively specific method for isolating fur The effects of the mutation were broad, influencing all of the mutations in many species of bacteria (15, 31, 39, 53, 63, 67). iron-repressed proteins to which probes were available. Anal- Although the mechanism of this selection is poorly understood ysis by 2-D PAGE showed that all 15 of the observed iron- (31), we based our modification of this technique on the repressed spots were at least partially derepressed under high- premise that the selection works through the ability of Mn to iron conditions in the fur-1 mutant (i.e., all were Furd). We substitute for ferrous iron, causing Fur to bind its cognate observed a surprisingly large number of iron-induced proteins, binding sequence (4). If the sole source of iron supplied to the some of which were Furd and others of which were Furi. These bacteria requires derepression of a specific uptake system, the observations were in contrast to the results of similar experi- bacterium will starve for iron unless it acquires a mutation ments with S. typhimurium (28), V. cholerae (43), and Y. pestis which at least partially derepresses the iron-uptake system. (61), in which there are multiple Furi as well as Furd Frps and The ability of Mn to affect the fidelity of DNA polymerases relatively fewer Fips (both Furd and Furi). A complete com- (46) may also influence the efficiency of this selection. parison of the number of spots of each class observed in each The mutants isolated by Mn selection varied in the degree of of these species is shown in Table 2. their inability to repress fbp-phoA in the presence of iron. Since We quantitated spots in triplicate and tested for the signif- a goal of our study was definition of the Fur regulon, we icance of regulation by standard Student’s t test. This may have focused on only the strongest mutation. Even this mutation increased our ability to detect both small spots and spots sub- had a notable effect on the growth phenotype on iron-rich solid ject to subtle regulation (two- to threefold), perhaps explaining medium (small colonies) and to a lesser extent an effect on the relatively large number of spots that we noted in each class growth rate in Feϩ RACDM. There was no difference in (especially Fips). The sensitivity of the present method is illus- growth rate in FeϪ RACDM as was expected if it is assumed trated by the finding that almost one-half of spots meeting the that Fur is active only in the presence of iron. The absolute twofold-or-greater, P Յ 0.05 criteria were eliminated from alteration of growth rate was greater for iron deprivation of further analysis because they could not be verified visually. wild type than the alteration of growth rate caused by the fur We attempted to keep the analysis of the Fur regulon as mutation under Feϩ RACDM conditions. It is possible that broad as possible, but it does have certain limitations. Identi- some of the spots indicated as being affected by iron and/or fur fication of proteins was limited to those of appropriate molec- VOL. 178, 1996 NEISSERIA GONORRHOEAE fur MUTANT 4231 ular mass and pI and of sufficient abundance to be detected 6. Berish, S. A., S. Subbarao, C. Y. Chen, D. L. Trees, and S. A. Morse. 1993. and resolved in this 2-D PAGE system. We did not differen- Identification and cloning of a fur homolog from Neisseria gonorrhoeae. Infect. Immun. 61:4599–4606. tiate independent proteins from isoforms of the same proteins, 7. Beucher, M. D., and P. F. Sparling. 1995. Cloning, sequencing, and charac- which may have inflated the number of spots detected. The terization of the gene encoding FrpB, a major iron-regulated, outer mem- strength of the statistical analysis was limited by the small brane protein of Neisseria gonorrhoeae. J. Bacteriol. 177:2041–2049. number of replicates (n ϭ 3). Some spots which are actually 8. Biegle, S., C. E. Thomas, and C. Elkins. Cloning and sequencing of a fur homolog. Gene, in press. iron regulated may have been missed because of inconsistent 9. Biswas, G. D., S. A. Lacks, and P. F. Sparling. 1989. Transformation-defi- quantitation, and a few regulated spots may be artifactual. It is cient mutants of piliated Neisseria gonorrhoeae. J. Bacteriol. 171:657–664. possible that part of the response to excess iron (especially Furi 10. Biswas, G. D., T. Sox, E. Blackman, and P. F. Sparling. 1977. Factors Fips) could have been a response to oxidative stress caused by affecting genetic transformation of Neisseria gonorrhoeae. J. Bacteriol. 129: 983–992. iron overload. Foster and Hall documented significant overlap 11. Biswas, G. D., and P. F. Sparling. 1995. Characterization of lbpA, the struc- between the iron and several other stress-regulated modulons tural gene for a lactoferrin receptor in Neisseria gonorrhoeae. Infect. Immun. in S. typhimurium (28). Another limitation of these experi- 63:2958–2967. ments was that the bulk of the analysis was done on a single 12. Black, J. R., D. W. Dyer, M. K. Thompson, and P. F. Sparling. 1986. Human immune response to iron-repressible outer membrane proteins of Neisseria mutation. However, an analysis of this complexity would be meningitidis. Infect. Immun. 54:710–713. impractical on multiple mutant strains. 13. Blanton, K. J., G. D. Biswas, J. Tsai, J. Adams, D. W. Dyer, S. M. Davis, Further work will be necessary to determine the function G. G. Koch, P. K. Sen, and P. F. Sparling. 1990. Genetic evidence that and identity of all of the regulated proteins observed in the 2-D Neisseria gonorrhoeae produces specific receptors for transferrin and lacto- ferrin. J. Bacteriol. 172:5225–5235. PAGE analysis. Although the effects of iron on gonococcal 14. Briat, J.-F. 1992. Iron assimilation and storage in prokaryotes. J. Gen. Mi- protein expression have been investigated for more than 17 crobiol. 138:2475–2483. years (50), the functions of relatively few of these proteins are 15. Brickman, T. J., and S. K. Armstrong. 1995. fur gene understood (2, 11, 21, 22). Most of the iron-repressed proteins restores iron repressibility of siderophore and protein expression to dereg- ulated Bordetella bronchiseptica mutants. J. Bacteriol. 177:268–270. are thought to be involved in iron acquisition. West and 16. Bullen, J. J., H. J. Rogers, and E. Griffiths. 1978. Role of iron in bacterial Sparling previously noted that some gonococcal proteins were infection. Curr. Top. Microbiol. Immunol. 80:1–35. expressed only under iron-sufficient conditions (Fips) (76), but 17. Campbell, L. A., and R. E. Yasbin. 1979. Deoxyribonucleic acid repair these proteins remain little studied. Along with the obvious capacities of Neisseria gonorrhoeae: absence of photoreactivation. J. Bacte- riol. 140:1109–1111. roles of these proteins in iron acquisition and storage, gono- 18. Carbonetti, N., V. Simnad, C. Elkins, and P. F. Sparling. 1990. Construction cocci may also use Fe availability to detect changes in the in of isogenic gonococci with variable porin structure: effects on susceptibility vivo environment, thereby affecting other properties such as to human serum and antibiotics. Mol. Microbiol. 4:1009–1018. attachment or invasion of host cells (33). 19. Catlin, B. W. 1973. Nutritional profiles of Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica in chemically defined media and the use It may be possible to isolate more profound or conditional of growth requirements for gonococcal typing. J. Infect. Dis. 128:178–194. null mutations in fur with nonstandard growth conditions. Nev- 20. Chang, A. C. Y., and S. N. Cohen. 1978. Construction and characterization of ertheless, the experiments presented here clearly indicate that amplifiable multicopy DNA cloning vehicles derived from the p15A cryptic the Fur regulon extends well beyond the known gonococcal miniplasmid. J. Bacteriol. 134:1141–1156. 21. Chen, C.-Y., S. A. Berish, S. A. Morse, and T. A. Meitzner. 1993. The ferric iron-regulated proteins and that Fur is the central regulator for iron-binding protein of pathogenic Neisseria spp. functions as a periplasmic gonococcal iron repression. Other mutations in gonococcal fur transport protein in iron acquisition from human transferrin. Mol. Micro- may be of value in further defining the Fur regulon and/or biol. 10:311–318. dissecting the functional domains of Fur. The location and 22. Cornelissen, C. N., G. D. Biswas, J. Tsai, D. K. Paruchuri, S. A. Thompson, and P. F. Sparling. 1992. Gonococcal transferrin-binding protein 1 is re- function of the many Fur- and/or iron-regulated genes will quired for transferrin utilization and is homologous to TonB-dependent almost certainly be the subject of future studies. outer membrane receptors. J. Bacteriol. 174:5788–5797. 23. Coy, M., and J. B. Neilands. 1991. Structural dynamics and functional do- mains of the Fur protein. Biochemistry 30:8201–8210. 24. Elkins, C., N. H. Carbonetti, V. A. Varela, D. Stirewalt, D. G. Klapper, and ACKNOWLEDGMENTS P. F. Sparling. 1992. Antibodies to N-terminal peptides of gonococcal porin are bactericidal when gonococcal is not sialylated. Mol. We thank Hillel Koren, Robert Devlin, and especially Bill Reed for Microbiol. 6:2617–2628. help with and the use of the BioImage system. We thank Sally Berish 25. Elkins, C., C. E. Thomas, H. S. Seifert, and P. F. Sparling. 1991. 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