©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Production and Oxidation of Indole by Haemophilus influenzae(*)

(Received for publication, May 9, 1994; and in revised form, November 4, 1994)

Terrence L. Stull (1)(§)(¶) Lisa Hyun (1) Christine Sharetzsky (1) James Wooten (1) John P. McCauley Jr. (2) Amos B. Smith III(§) (2)

From the  (1)Departments of Pediatrics and Microbiology/Immunology, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129 and the (2)Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

During growth in high concentrations of iron nitrate, H. influenzae produces compounds reactive in biochemical assays for hydroxamates. Mixing experiments established that nitrate was responsible for inducing these compounds. Analysis by ^1H and C NMR and high resolution mass spectrometry identified the active species as 2,2-bis(3`-indolyl)indoxyl. Bacterial production of the latter compound has been previously observed only in Pseudomonas aureofaciens. A mutant defective in the production of 2,2-bis(3`-indolyl)indoxyl was constructed by marker insertion. The formation of indole and 2,2-bis(3`-indolyl)indoxyl was quantitated by reverse-phase high pressure liquid chromatography during growth in high concentrations of nitrate. The mutant produced high concentrations of indole, but only minimal amounts of 2,2-bis(3`-indolyl)indoxyl, and also proved to be defective in nitrate reduction. These data suggest that indole may function as an electron donor for nitrate reductase in H. influenzae.


INTRODUCTION

Many microbes produce hydroxamates for use as extracellular high affinity iron binding agents (i.e. siderophores). For example, aerobactin is an iron-repressible dihydroxamic acid citrate siderophore produced by Aerobacter (Enterobacter) aerogenes(1) , Shigella species(2) , Klebsiella(3) , and Escherichia coli(4) . The outer membrane protein receptor for the ferrihydroxamate complex is co-regulated with the enzymes which synthesize the hydroxamates. In such systems, hydroxamate production is maximal under low iron conditions. In E. coli the iron level is in turn regulated by the interaction of elemental iron with the Fur repressor protein (5) .

Recently, we observed that the growth of H. influenzae in high concentrations of iron nitrate gave a positive result in biochemical assays specifically designed to detect hydroxamates(6) . The paradox of putative hydroxamate formation in the presence of high concentrations of iron nitrate suggested that the hydroxamates might bind iron for an alternative function. Alternatively, nitrate itself might induce the production of compounds active in the hydroxamate screening assays. The goals of this investigation were A) to characterize the production and determine the structures of the compounds reactive in the hydroxamate screening assays, and B) to explore their biological functions by preparing mutant strains incapable of forming the reactive species.


MATERIALS AND METHODS

Bacteria and Media

The bacterial strains and plasmids used are listed in Table 1. For routine culture, H. influenzae was grown in brain-heart-infusion broth supplemented with 10 µg/ml beta-NAD and 10 µg/ml heme. Modified Haemophilus defined medium (mHDM) (^1)was employed for growth under defined conditions(6) .



Biochemical Assays

Biochemical assays designed to detect hydroxamates were performed by the methods of Csaky (7) and Atkin and Neilands(8) . Nitrate reductase was detected via the method of Conn (9) .

Screening for the Production of 2,2-Bis(3`-indolyl)indoxyl (BII)

An acid-plate technique was developed to screen H. influenzae strains for production of BII. Cells were suspended at a concentration of 10^9 colony-forming units/ml in pH 7.4 phosphate-buffered saline, and 10 µl of the suspension was spotted onto a fresh supplemented brain-heart infusion agar plate containing 10M iron nitrate. Four strains were tested on each plate, along with E1a and Rd as positive and negative controls, respectively. Following overnight incubation, 50 µl of 10 N HCl was placed on each colony. Observation of a purple or pink halo after 15 min similar to the positive control was interpreted as positive.

Production and Purification of Indoles

Filtered supernatants from overnight growth of strain E1a in mHDM containing 0.1 mM nitrate were acidified to pH 2 by addition of concentrated HCl. The resultant solution was extracted with chloroform and concentrated under a stream of N(2), and the residue was suspended in methanol. The chloroform-extracted cell pellet and uninoculated media served as controls. Samples were analyzed by thin layer chromatography (TLC) on 250-µm silica gel plates (Uniplate, Analtech Inc, Newark, DE) using chloroform/methanol (135:1.5) for development. Compounds were visualized with FeCl(3) in 0.1 N HCl. After preparative TLC on 500-µm silica gel plates, band 4 was isolated and further purified by flash chromatography on silica gel, with chloroform/methanol (97:3) as eluant.

Mass Spectrometry

Samples were ionized using chemical ionization with ammonia (CI, NH(3)), and analyzed using a VG ZAB-E high resolution spectrometer.

Nuclear Magnetic Resonance Analysis

Carbon and proton NMR were recorded on a Bruker AM-500 spectrometer. Chemical shifts were measured relative to internal Me(4)Si (^1H and C, 0.00) or chloroform (^1H, 7.24; C, 77.0). Two-dimensional homonuclear and heteronuclear experiments were also performed. Short and long range couplings were determined via the latter methods. ^1H and C NMR assignments (Table 2) were made via homonuclear decoupling of the proton spectrum to assign the connectivity, heteronuclear multiple quantum correlation to correlate the protons with the carbons, and heteronuclear multiple bond correlation to determine the positions of the protons with respect to the fully substituted carbon atoms.



Synthesis of BII

BII was synthesized as described previously, using peracetic acid for the oxidation of indole(10, 11) (see (28) for a more recent synthesis than that described in (10) ).

Insertion Mutagenesis and Transformation of H. influenzae

Chromosomal DNA was isolated from a stationary phase culture of H. influenzae by repeated phenol-chloroform extractions of an SDS-proteinase XIV-treated cell lysate(12) . Insertion mutagenesis was performed as described previously(13) . Restriction fragments resulting from primary, partial digestion of chromosomal DNA with Sau3A were ligated at a concentration (1 µg/300 µl) that favors intramolecular ligation. The resultant cyclic molecules were then subjected to secondary digestion with the same concentration of Sau3A used initially, providing complementary ends for ligation to the BamHI-digested TSTE mutagenesis element, a neomycin cassette constructed from Tn5. This ligation was carried out at a higher concentration to promote intermolecular ligation (1 µg/20 µl). Approximately 10^9 competent H. influenzae were transformed with 0.5-1 µg of the recombinant molecules. Transformations were performed with M-II medium(14) . Organisms containing TSTE insertions were detected by growth on supplemented brain-heart infusion agar containing 15 µg/ml Rb.

To prepare for mutagenesis of the gene(s) encoding production of BII, chromosomal DNA from the BII-producing strain E1a was transformed into the nonproducing strain Rd. The latter transforms at a very high frequency and therefore is an excellent host strain for construction of insertion mutants. Replicas of plates were screened for production of BII by the acid-plate technique. One Rd transformant, HI1662, produced indole and BII and was selected for further investigations. Using homologous DNA, strain HI1662 was subjected to mutagenesis using the TSTE element(13) . Approximately 2000 Rb-resistant transformants were screened for the production of BII by the acid-plate method. One mutant, HI1663, produced no detectable BII, and a Southern blot analysis of HI1663 DNA probed with P-labeled TSTE revealed a single insertion (data not shown). To confirm that the TSTE element interrupted the gene responsible for the production of BII, the parent strain HI1662 was transformed with chromosomal DNA from strain HI1663; BII production was not detected in any of the resultant Rb-resistant transformants. Finally, strain E1a was transformed with DNA from strain HI1663. One Rb-resistant BII-nonproducing transformant, strain HI1664, was used for further investigations.

Quantitation by HPLC

Purified indoles were dissolved in methanol and subjected to reverse-phase HPLC on a C-18 column with methanol/water (62:38) as eluant. The absorbance of the effluent was monitored at 278 nm. Peaks were identified and quantitated relative to standard solutions of indole and synthetic BII.


RESULTS AND DISCUSSION

Induction of Reactive Compounds

Pidcock et al.(6) reported that H. influenzae grown in high concentrations of iron nitrate produced compounds reactive in the hydroxamate biochemical assays described by Csaky (7) and by Atkins and Neilands(8) . To identify the substance responsible for the induction of the reactive compounds, 10M or 10M concentrations of the following iron and nitrate sources were added to the media: iron nitrate, iron chloride, iron pyrophosphate, ferrous ammonium sulfate, and sodium nitrate. Only the supernatants of organisms grown in the presence of 10M nitrate produced reactive compounds.

Characterization of Production by TLC Analysis

Supernatants of H. influenzae grown in a high concentration of nitrate were extracted with chloroform:methanol. TLC analysis revealed six separate, colored bands with R(F) values of 0.0, 0.07, 0.12, 0.2, 0.45, and 0.6 (Fig. 1). These bands were not detected in extracts of the cell pellet or in the uninoculated media controls. Band 4 proved to be by far the major component. Hence, this band was selected for detailed structural analysis.


Figure 1: Thin layer chromatograph of compounds isolated from supernatants of H. influenzae E1a grown in mHDM containing 0.1 mM nitrate. LaneA, composite extract; laneB, purified band 2; laneC, purified band 3; laneD, purified band 4; laneE, purified band 5; laneF, extract of the cell pellet of E1a after growth in 50 ml of mHDM. The faint bands 1 and 6 could not be further purified. The compounds were visualized by spraying with FeCl(3) in 0.1 N HCl.



Structural Analysis

Low resolution mass spectroscopy of band 4 yielded a (M + 1) ion at 364 atomic mass units. A high resolution exact mass measurement (CI, NH(3)) gave m/z 364.1370 (calculated for CH(18)N(3)O, 364.1449). These results suggested a molecular formula of CHN(3)O.

The ^1H, C, and two-dimensional NMR analyses then led to the proposal of the BII structure (Fig. 2). To confirm this assignment, an authentic sample of BII was synthesized as described earlier (10, 11) (see also (28) ). The synthetic and natural materials proved to be indistinguishable by TLC, NMR, infrared, and mass spectroscopy analyses (Fig. 3). Bacterial production of the compound has been previously observed only in Pseudomonas aureofaciens(15) .


Figure 2: Structure of 2,2-bis(3`-indolyl)indoxyl (CHN(3)O).




Figure 3: NMR and mass spectrometric comparison of naturally produced (i.e. isolated) BII (top panel) with authentic, synthetic BII (bottom panel). Left, 500 MHz ^1H NMR (d(6)-Me(2)SO), intensity versus chemical shift. Right, chemical ionization mass spectroscopy (NH(3)), relative abundance versusm/z. See Table 2for NMR chemical shift assignments.



Reaction of BII in Biochemical Assays for Hydroxamates

BII was reactive at a concentration of 10 µg/ml in the two principal assays used for detection of hydroxamate production(7, 8) . Thus, the testing of extracts of broth supernatants in screening assays for hydroxamates led to the isolation of an indole derivative that gave a false positive assay for hydroxamates. Whereas false positive reactions with unspecified compounds have been reported previously for one of these assays(8) , our data indicate that certain indoles produce false positive results in both assays. These assays therefore should be used with caution, especially with indole-producing organisms. However, hydroxylamine is a direct metabolite of nitrites(16) , and our data do not exclude the possibility that hydroxylamines are also produced by H. influenzae.

Construction of an E1a Mutant Incapable of BII Production

Indole is produced by bacteria either as a precursor to tryptophan or as a degradation product thereof(17) . Previously described byproducts include skatole, indoxyl derivatives, indican, indigo, and indirubin. Hamill et al. reported the formation of BII by P. aureofaciens upon addition of indole (500 µg/ml) to the media(15) . Aerobic oxidation of indole results in the nonenzymatic generation of BII(18) , but the possibility of nonenzymatic oxidation of indole to BII was not investigated in the Hamill studies. To determine whether the production of BII by H. influenzae is enzymatically mediated, a mutant (HI1664) were constructed via insertion of the TSTE mutagenesis element (vide supra). The selected mutant had a single insertion, as determined by Southern blot analysis, and no BII could be detected using the acid-plate technique.

HPLC Analysis of Indole and BII Production

To confirm the lack of BII production by HI1664 and to further characterize its formation by E1a, these isogenic strains were grown in media containing 10M nitrate. Timed aliquots were removed and the indoles isolated from the cell-free supernatants. For the wild-type strain E1a, concentrations of indole and BII in the supernatant attained maximum values of 7.1 and 13.0 µg/ml after 8 and 12 h of growth, respectively. In contrast, the peak concentration of indole in the HI1664 mutant supernatants was 35.0 µg/ml at 12 h, and the concentration of BII remained <1.0 µg/ml at all times. The structure of BII and the accumulation of indole during the growth of strain HI1664 suggest that indole serves as a precursor of BII.

Nitrate Reductase Activity of Isogenic Strains

A role for nitrate reductase was suggested by the production of BII, which may arise via oxidation of indole, during growth in high concentrations of nitrate. Metabolism of nitrate can be initiated by its reduction to nitrite by nitrate reductase; this process occurs in certain bacteria, algae, fungi, and plants but not in animals(19) . In bacterial nitrate metabolism, separate nitrate reductases are expressed for the assimilatory and dissimilatory (respiratory) pathways(20, 21, 22, 23) . Structural genes, genes responsible for detecting environmental stimuli, and regulatory genes are all required for the expression of nitrate reductase(20, 24) . Nitrate reduction is characteristic of the genus Haemophilus, although the molecular genetics of this process have not been characterized.

To determine whether nitrate reductase may mediate the production of BII, the method of Conn was employed to screen the isogenic strains for the ability to reduce nitrate(9) . The parent strains E1a and HI1662, which produced BII, also reduced nitrate. In contrast, the mutants HI1663 and HI1664 which were defective in BII production were also defective in nitrate reductase activity. Although pyridine nucleotide usually serves as the electron donor for nitrate reduction, other compounds, such as formate, also can provide electrons(25) . Our data suggest that indole may provide electrons for the reduction of nitrates in H. influenzae. Less likely alternatives are that insertion of the mutagenesis element may have interrupted expression of both nitrate reductase and another redox enzyme, or that the insertion may have interrupted expression of a gene regulating both BII production and nitrate reduction. Further analysis of these mutants will be useful for defining the molecular genetics of nitrate reduction by H. influenzae.


FOOTNOTES

*
This work was supported in part by Grant AI29611 from NIAID, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Direct correspondence on chemistry to A. B. S.

Current address: Dept. of Pediatrics, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190-3030. Tel.: 405-271-4401; Fax: 405-271-8710.

(^1)
The abbreviations used are: mHDM, modified Haemophilus defined medium; BII, 2,2-bis(3`indolyl) indoxyl; HPLC, high pressure liquid chromatography; Rb, ribostamycin.


ACKNOWLEDGEMENTS

We acknowledge the technical contributions of Dr. George Furst (NMR) and Mr. John Dykins (mass spectrometry).


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