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
-NAD and 10 µg/ml heme. Modified Haemophilus defined medium (mHDM) (
)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
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 10
M 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
, 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
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
), 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
Si (
H and
C,
0.00) or chloroform (
H,
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.
H 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
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, 10
M or 10
M 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 10
M 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
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
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
) gave m/z 364.1370 (calculated for
C
H
N
O, 364.1449). These results
suggested a molecular formula of
C
H
N
O.The
H,
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
(C
H
N
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
H NMR (d
-Me
SO),
intensity versus chemical shift. Right, chemical
ionization mass spectroscopy (NH
), 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 10
M 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.