From the Howard Hughes Medical Institute, Departments of Medicine and Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
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Hemoglobin homologs are being identified in an expanding number of unicellular prokaryotic and eukaryotic organisms. Many of these hemoglobins are twodomain proteins that possess a flavin-containing reductase in their C terminus. Determination of a function for these flavohemoglobins has been elusive. A Salmonella typhimurium strain harboring a deletion in the flavohemoglobin gene shows no difference in growth under oxidative stress conditions but displays an increased sensitivity to acidified nitrite and S-nitrosothiols, both of which produce nitric oxide. The effect is seen aerobically or anaerobically, indicating that oxygen is not required for flavohemoglobin function. These results suggest a role for the bacterial flavohemoglobins that is independent of oxygen metabolism and provide evidence for a bacterial route of protection from nitric oxide that is distinct from oxidative stress responses.
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INTRODUCTION |
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Hemoglobins are a superfamily of ancient, oxygen-binding hemoproteins with remarkable three-dimensional similarity despite the drifting of primary amino acid sequence during evolution (1). Classic biochemical, structural, and genetic studies have established the intravascular hemoglobin tetramer and the intramuscular hemoglobin monomer (myoglobin) as the primary facilitators of mammalian oxygen delivery (2). Hemoglobins have also been isolated from a diverse array of invertebrate animals, plants, protozoa, fungi, and bacteria (3-5). The role of globins in nonmammalian organisms is largely speculative, often because of their intractability to genetic manipulation. The discovery of hemoglobins in microorganisms has broadened the experimental approaches available to include globin gene expression and deletion studies.
Logically, the initial assessments of hemoglobins found in microorganisms have emphasized the diffusion and metabolism of oxygen. Transcription of the hemoglobin from the aerobic bacterium Vitreoscilla is elevated under oxygen-limiting conditions (6, 7). Heterologous overexpression of the Vitreoscilla globin allows growth to higher cell densities in Escherichia coli and nearly doubles the growth rate of transgenic tobacco, suggesting an ability to increase the availability of intracellular oxygen (8, 9). Overexpression of the Vitreoscilla hemoglobin can also rescue E. coli terminal oxidase mutants, indicating that this protein can not only deliver molecular oxygen but also participate in productive electron transport (10). Members of an emerging family of two-domain hemoglobins (flavohemoglobins) from other microorganisms share substantial sequence identity to the single-domain Vitreoscilla globin in their N termini and exhibit homology to flavoprotein oxidoreductases in their C termini. Spectroscopic studies on the purified flavohemoglobin native to E. coli (designated hmp1) indicate that the redox state of the flavin moiety in the reductase domain is influenced by oxygen availability in the heme pocket. Therefore, hmp has been postulated to be a sensor, with oxygen-regulated modulation of reductase activity toward undefined substrate(s) in vivo (11).
Gene expression of many flavohemoglobins is regulated by changes in oxygen concentration, being induced by microaerobic or anaerobic conditions in the bacteria Alcaligenes eutrophus and Bacillus subtilis (12, 13) but enhanced by hyperoxic conditions in the yeast Saccharomyces cerevisiae (14, 15). A sensitivity to certain oxidative stress conditions has been reported for a S. cerevisiae flavohemoglobin deletion strain lacking functional mitochondria (15). The superoxide generator paraquat (methyl viologen) stimulates E. coli hmp promoter activity, but sensitivity of a flavohemoglobin deletion strain to superoxide was not reported (16).
Clues to a role in the metabolism of nitrogen oxides have come from
other flavohemoglobin genetic studies. A strain with a disrupted
flavohemoglobin gene in the strictly respiring bacterium A. eutrophus does not accumulate nitrous oxide as a transient intermediate during denitrification. Surprisingly, growth under denitrification conditions is normal, and generation of the pathway end
product, dinitrogen, is not diminished (17). In the presence of
nitrogen oxides, both the B. subtilis and E. coli
flavohemoglobin promoters fused to a lacZ reporter show
induced -galactosidase activity (13, 18). Correlates exist between
transcriptional activation by oxidative stresses and nitric oxide. The
SoxRS and OxyR transcription factors, known to respond to superoxide
and hydrogen peroxide, are also activated by NO in E. coli,
with soxRS and oxyR mutants displaying an
increased sensitivity to NO from macrophages and to the NO-donating
S-nitrosothiols, respectively (19, 20). The microbiostatic
action of NO and NO-producing derivatives may result from NO
modification of protein thiols, analogous to the oxidation of thiols
caused by oxygen-mediated damage (20). Factors involved in the nitrogen
oxide induction of the hmp gene in E. coli are
unknown but are independent of the SoxRS system (18). We now report a
role for the Salmonella hmp in specific protection from
nitric oxide that is independent of oxygen and its metabolites.
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EXPERIMENTAL PROCEDURES |
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Cloning and Deletion of the Salmonella typhimurium hmp-- All PCR reactions used Klentaq/LA (W. Barnes, Washington University, St. Louis, MO). Using the RN1 primer (5'-GATCGAATTCATGCTTGACGCACAAACCAT-3') from the published upstream S. typhimurium glyA sequence (21) and a degenerate primer RN4 (5'-GATCGGATCCNAGNAC(C/T)TT(A/G)TGNGGNCC(A/G)AA(A/G)CA(C/T)TC(A/G)TA-3') based on the C terminus of the E. coli hmp (22), the S. typhimurium hmp gene was amplified by PCR from ATCC S. typhimurium strain 14028s genomic DNA. The product was cloned into the TA cloning vector (Invitrogen) and found to contain an open reading frame homologous to other hmp genes. For creation of an hmp insertion/deletion construct, PCR products were generated using SKO1/SKO2 primers for hmp upstream/5' sequence and SKO3/SK04 primers to hmp 3' sequence. The sequences of the knockout primers are as follows: SKO1, 5'-GATCGAATTCTTTTACGCAAACGATTACCTA-3'; SKO2, 5'-GATCCCGGGTAGAAGTGGGCGGTCAG-3'; SKO3, 5'-GATCGGATCCTTACAGGACCTTATGCGGACC-3'; SKO4, 5'-GATCCCGGGCGAAAGAACAGCATACCGC-3'.
The two PCR primers internal to the gene (SKO2/SKO4) were created with SmaI sites to facilitate the ligation of the 1.4-kilobase pair kanamycin resistance cassette from pUC-KIXX (Amersham Pharmacia Biotech), creating the final knockout construct (pSKO). This construct was transduced byMaterials-- All chemicals were purchased from Sigma. S-Nitrosothiols were made as described previously (27). Briefly, the S-nitroso-derivatives of glutathione (GSNO) or N-acetylated cysteine (SNAC) were prepared by making 1 M solutions of the thiol in 1 N HCl and adding an equal volume of 1 M sodium nitrite. M9 minimal salts medium (pH 7.0): 7 g of Na2HPO4, 3 g of KH2PO4, 1 g of NH4Cl, 0.5 g of NaCl, 2 g of glucose, 120 mg of MgSO4, 15 mg of CaCl2, and 40 mg of thiamine/liter. For plates, agar was added to 1.5%. When needed, ampicillin (100 µg/ml) or kanamycin (50 µg/ml) was added to the medium.
Growth Conditions-- For nitrite growth curves, 1 × 104 cells of overnight cultures grown in LB-ampicillin were diluted into 250-ml culture flasks with 100 ml of fresh LB-ampicillin medium containing various concentrations of sodium nitrite. Cells were shaken at 225 rpm at 37 °C, and growth curves were measured by a Klett colorimeter with a red filter. Minimal inhibitory concentrations for oxidative stress compounds and NO derivatives were obtained by taking overnight cultures grown in LB and diluting in phosphate-buffered saline. 1 × 104 bacteria were then added to a test tube with 1 ml of M9 medium containing the inhibitory compound, which varied in concentration from tube to tube by 2-fold. The tubes were incubated with shaking at 37 °C. The minimal inhibitory concentration is the concentration of the compound where no visible growth occurred after 24 h. For plate cultures, 106 cells of overnight cultures grown in LB-ampicillin medium were washed in phosphate-buffered saline and streaked onto M9-ampicillin plates containing 10 mM GSNO. For liquid growth curves, 104 cells of overnight cultures grown in LB-ampicillin were diluted into 250-ml culture flasks with 100 ml of LB-ampicillin medium containing 500 µM GSNO. Cells were shaken at 225 rpm at 37 °C, and growth curves were measured. For anaerobic growth, 104 cells of LB-grown overnight cultures were diluted in phosphate-buffered saline and inoculated into 250-ml culture flasks containing 100 ml of LB-ampicillin medium. The flasks were then sealed with a two-holed stopper and ultrapure argon (Cee-Kay, St. Louis, MO) was vigorously bubbled into the medium for 30 min. The flasks were then incubated without shaking at 37 °C. Anaerobic indicator strips were used (Becton Dickinson Microbiology Systems) to confirm a proper seal.
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RESULTS |
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To determine if flavohemoglobins are involved in protection from oxidative and/or nitric oxide-mediated stress, we cloned and deleted the hmp gene of S. typhimurium. The coding region of the S. typhimurium hmp predicts a two-domain hemoglobin/reductase protein with 94% amino acid identity to the E. coli hmp (22). As in E. coli, the gene upstream of the S. typhimurium hmp is the divergently transcribed glyA, encoding serine hydroxymethyltransferase (21). Sequence similarity to the E. coli hmp locus, however, abruptly halts after the hmp stop codon. Instead of glnB (22), the cadC gene, a regulator of lysine decarboxylase in E. coli (28), is directly downstream of the S. typhimurium hmp (Fig. 1A). The hmp gene does not appear to be part of an operon with cadC. The 189-bp intergenic region contains a 12-bp inverted repeat downstream of the hmp thought to terminate the hmp transcript and standard basal promoter elements immediately upstream of cadC (not shown). For phenotypic characterization, we created a strain wherein most of the hmp coding region was replaced with a KanR cassette. This was confirmed by Southern blot analysis (Fig. 1B).
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No detectable growth differences between the deletion
(hmp) and wild-type strains were observed
under standard in vitro growth conditions in 100% oxygen,
air, hypoxic, or anaerobic conditions (not shown). With 3 mM nitrite at pH 7, no significant difference in growth
between strains was detected (Fig.
2A). Increasing the nitrite
concentration to 30 mM, however, resulted in a substantial increase in the doubling time of the hmp
strain (Fig. 2A). Nitrite will protonate to form
HNO2, which quickly dismutates to produce several species
of nitrogen oxides, including NO (29, 30). When the pH of the solution
was lowered to 6 to promote nitrite protonation, a delay in growth of
the hmp
strain was seen with only 3 mM nitrite (Fig. 2B). These results suggest that
it is not nitrite but nitrogen oxides created by the protonation and
subsequent dismutation of nitrite, that produce the difference in
growth between the wild-type and hmp
strains.
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Given the association of both hemoglobin and nitric oxide-mediated
stress responses to oxygen metabolites, we were interested in
determining whether the flavohemoglobin protects against both O2 and NO-mediated insult. Cultures were assayed for the
minimal inhibitory concentrations of oxidative and nitric
oxide-producing stressors. Both the superoxide generator paraquat and
hydrogen peroxide inhibited growth of the wild-type and mutant strains equally (Table I). Profound disparities
between wild-type and hmp strains were seen
with the NO-donating S-nitrosothiols. These compounds
possess a broad spectrum antimicrobial activity, presumably by donating
nitrosonium (NO+) to critical intracellular thiol targets,
disruption of iron-sulfur clusters, and by damage to DNA (31-33). The
NO derivatives of both GSNO and SNAC inhibited the growth of the
hmp
strain by approximately an order of
magnitude more than wild-type (Table I). We also wanted to ascertain
whether the flavohemoglobin is involved in protection from
peroxynitrite (OONO
), which is formed by the reaction of
O2
with NO· and is a major effector in the
NO-dependent bacteriocidal activity of macrophages (34).
The minimal inhibitory concentrations of the peroxynitrite generator
3-morpholinosydnonimine hydrochloride (35) were identical between the
two strains (Table I), indicating that hmp does not protect from
peroxynitrite.
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To further characterize the involvement of hmp in the NO-meditated
stress response, both wild-type and hmp
strains were cultured in medium containing GSNO. Whether on plates (Fig. 3A) or in liquid culture
(Fig. 3B), the parental organism was able to grow, whereas
the growth of the hmp
strain was greatly slowed. Growth
of the hmp
strain could be restored by reintroduction of
the hmp gene on a low copy number plasmid. A slight toxicity
of the hmp rescue plasmid was seen in liquid culture, even
in the wild-type strain (Fig. 3B). A similar restoration
from the nitrite-mediated growth inhibition was observed using the
hmp-expressing plasmid (not shown). To determine if the
flavohemoglobin requires oxygen to exert its protective effect against
NO donors, liquid cultures were subjected to anaerobiosis. The
hmp
cultures grown anaerobically continued to
be hypersensitive to GSNO (Fig. 3C), suggesting that the
flavohemoglobin is important in nitric oxide-mediated stress protection
even in the absence of oxygen.
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DISCUSSION |
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The flavohemoglobin gene is hypothesized to have been present in
organisms before the divergence of prokaryotes and eukaryotes (36) and,
like cytochrome oxidase, may predate the existence of significant
levels of atmospheric oxygen (37). Interaction with NO may be a
universal feature of hemoglobins throughout phylogeny. Even the
vertebrate hemoglobins, well understood as facilitators of oxygen
delivery, display physiologically important interactions with nitric
oxide. Human hemoglobin binds NO tightly in the heme pocket and may
assist in NO sequestration (38). S-Nitrosohemoglobin, formed
by a NO bound to the thiol group of Cys-93 in the -chain, may be a
major intermediate in transduction of the NO vasodilating signal in
humans (39, 40). Formation of a physiologically relevant
S-nitrosoflavohemoglobin in Salmonella or another
microorganism is unlikely. No cysteine residues are conserved in the
heme binding domain of these molecules, and indeed there are no
cysteines at all in heme binding domain of the Salmonella
hmp. Instead the NO moiety likely interacts with hmp inside the heme
pocket. This type of interaction is supported by experimental evidence
showing that E. coli hmp can form a nitrosylated complex
with a reduced heme iron (41). NO production can occur endogenously in
E. coli using nitrite as an electron acceptor. Ferrous iron,
nitrate reductase, and low pH can compete with the preferred enzymatic
reduction of nitrite to NH3 and instead create nitric oxide
(42-44). NO compounds also threaten bacteria from other sources,
including the S-nitrosothiols produced in the vertebrate
host (45, 46).
The flavohemoglobin may function by simply sequestering NO in its heme pocket, allowing other enzymes to detoxify the moiety. The homocysteine molecule, which forms a more stable S-nitrosylated form than GSNO or SNAC, may also serve as a NO sink in Salmonella. Mutants deficient in homocysteine biosynthesis are 2-3-fold more sensitive to S-nitrosothiols by zone of inhibition assays (47). Alternatively, hmp may directly detoxify NO by an oxidoreductase reaction, leading to a less toxic nitrogen oxide product. It should be noted, however, that no NO reductase activity could be demonstrated using the A. eutrophus flavohemoglobin (17). Another intriguing possibility is that hmp, upon binding NO in its heme domain, might be able to transduce a NO signal through its reductase domain, leading to the positive regulation of protective genes. A hemoprotein-containing, two-component system that senses oxygen levels exists in Rhizobium, where the FixL protein regulates its N-terminal kinase activity via oxygen bound to its C-terminal heme binding domain (48). Whichever mechanism applies, it is apparent that this bacterial hemoglobin can function independently of oxygen to protect the organism from stress effected by nitric oxide.
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ACKNOWLEDGEMENTS |
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We thank E. Groisman and F. Solomon for provision of S. typhimurium strains and for helpful suggestions and J. Gordon, M. Caparon, and V. Miller for critical reading of this manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF020388.
To whom correspondence should be addressed: Howard Hughes Medical
Institute, Depts of Medicine and Molecular Microbiology, Washington
University School of Medicine, P. O. Box 8230, 660 S. Euclid Ave., St.
Louis, MO 63110. Tel.: 314-362-1514; Fax: 314-362-1232; E-mail:
goldberg{at}borcim.wustl.edu.
1 The abbreviations used are: hmp, flavohemoglobin; GSNO, S-nitrosylated glutathione; SNAC, S-nitrosylated N-acetylcysteine; PCR, polymerase chain reaction; bp, base pair(s); WT, wild type.
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REFERENCES |
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