©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Saccharomyces cerevisiae Flavohemoglobin Gene Expression (*)

(Received for publication, November 1, 1994; and in revised form, December 27, 1994)

Michael J. Crawford David R. Sherman (§) Daniel E. Goldberg (¶)

From the Howard Hughes Medical Institute, Departments of Medicine and Molecular Microbiology, Washington University Medical School and the Jewish Hospital of St. Louis, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Saccharomyces cerevisiae hemoglobin is a flavoprotein of unknown function. It shares extensive sequence homology with the globin of Candida as well as those of several bacterial species. We have studied its gene regulation in order to better understand its purpose in the cell. Transcriptional analyses indicate that, in sharp contrast to the bacterial globins of Vitreoscilla and Alcaligenes eutrophus, the S. cerevisiae globin message is induced during logarithmic growth and under oxygen-replete conditions. Transcription of the S. cerevisiae hemoglobin gene is positively regulated by the transcription factors heme-activated protein (HAP) 1 and HAP2/3/4, which respond to intracellular heme levels. Anaerobically, there is a low level, HAP-independent induction of hemoglobin mRNA. Unlike other systems influenced by the HAP2/3/4 transcription factor complex, no activation of hemoglobin expression by growth in non-fermentable carbon sources is observed. Flavohemoglobin gene disruption does not alter cell viability or growth in a variety of oxygen conditions and carbon sources. Physical and genetic mapping of the S. cerevisiae flavohemoglobin gene places it on chromosome seven near the formyltetrahydrofolate synthase (ADE3) locus. These data indicate that, despite the high degree of homology, the S. cerevisiae globin may have a function distinct from those proposed for bacterial globins.


INTRODUCTION

Hemoglobins have been detected spectrally and their genes cloned from organisms representing all kingdoms of life, indicating a primordial origin and a continuing widespread requirement for this protein(1, 2, 3) . Although the role of vertebrate hemoglobins as facilitators of oxygen diffusion is well established, the function of globins in invertebrate animals, as well as in plants, protozoa, fungi, and bacteria, is generally unclear. Clues to the function of the microorganismal globins have come through heterologous expression of hemoglobin from the aerobic bacteria Vitreoscilla. Overproduction of the Vitreoscilla globin (VtHb) (^1)allows increased cell densities and rescues terminal oxidase mutants in Escherichia coli, indicating that VtHb is capable not only of delivering molecular oxygen but also of facilitating its reduction in vivo(4, 5) . Hemoglobins recently sequenced from the bacteria E. coli and Alcaligenes eutrophus and the yeasts Saccharomyces cerevisiae and Candida norvegensis are two-domain proteins with nearly 40% sequence identity(6, 7, 8, 9) . Their N-terminal regions share substantial sequence homology with the single-domain VtHb, whereas the C terminus contains a reductase domain with potential binding sites for flavin (FAD) and NADPH. Because of the additional domain, this class of globins has also been called flavohemoglobins. The native VtHb interacts with a separate reductase, making it a two-polypeptide analog of the flavohemoglobins(10) .

Gene regulation studies of the bacterial globins suggest a correlation in the functions of these proteins. Hemoglobin levels increase over 50-fold in Vitreoscilla as cell density increases and oxygen tension drops(11) . There is also an increase in reductase levels under the same conditions, suggesting coordinate expression of the hemoglobin and reductase activities in Vitreoscilla(10) . Remarkably, the oxygen regulation of Vitreoscilla hemoglobin can also be seen using the native VtHb promoter expressed in E. coli(12, 13) . Transcriptional fusions to the hemoglobin promoter show a 5-7-fold increase in reporter gene expression when cells are shifted to microaerophilic conditions or when cells reach the end of log-phase growth. Limited oxygen supply also causes a 20-fold increase in A. eutrophus globin levels(14) . On the basis of promoter sequence homology, the FNR and NARL transcription factors, which control anaerobic gene expression in E. coli, have been proposed to affect expression of the A. eutrophus globin(9) . A putative binding site for FNR is also found in the promoter of the Vitreoscilla gene, and E. coli mutants lacking the FNR gene product do not show microaerophilic activation of heterologous VtHb transcription(15) .

In S. cerevisiae, transcription factors involved in oxygen regulation of gene expression have been well characterized(16, 17, 18) . Heme, the biosynthesis of which requires molecular oxygen in eukaryotes (19) , is an effector molecule involved in the sensing of intracellular oxygen levels. For aerobically induced genes, HAP1 and the HAP2/3/4 complex activate transcription in the presence of heme(16) . Heme can also inhibit the transcription of hypoxic or anaerobically expressed genes through the ROX1 and ORD1 transcription factors(20, 21) . To investigate possible functions for the S. cerevisiae flavohemoglobin, we have initiated gene regulation and disruption studies. Our results indicate that S. cerevisiae hemoglobin expression is regulated by cell density and oxygen in a manner different from that of the bacterial globins, suggesting a separate and as yet undefined role for this protein in aerobic yeast metabolism.


MATERIALS AND METHODS

Strains and Plasmids Used

The S. cerevisiae strains used in this study are listed in Table 1. To facilitate deletion of the yeast hemoglobin gene, 3` portions of the S. cerevisiae flavohemoglobin (YHG) coding region were fused upstream of 5` portions (22) by polymerase chain reaction (PCR). A unique BamHI restriction site, necessary for the subsequent transformation in yeast, was incorporated into the joint between gene fragments. The fused sequence was subcloned into plasmid pRS305 (Leu selection), and the S. cerevisiae hemoglobin-deletion strain YD7 was constructed by -recombination into YM4134(23) .



The yeast hemoglobin reporter gene construct (pYH10) was generated by subcloning a SpeI-NheI restriction fragment containing 1.6 kb of YHG promoter into the SpeI site of the lacZ expression plasmid yIP357R(23) . Standard techniques in the manipulation of these shuttle vectors and propagation of E. coli were used(24) . Yeast transformations were performed using the LiAc method as described (25) and plated onto selective media(26) .

Media and Growth Conditions

For RNA analyses of growth phase, oxygen curve, and heme/transcription factor-related expression, cultures were grown in YPGal media (1% yeast extract, 2% Bactopeptone, 2% galactose) at 30 °C. Media components were from Difco or Sigma. The hem1 strain was supplemented with 10 µg/ml ergosterol with 0.1% Tween 80 and/or 100 µg/ml -aminolevulinic acid. For analysis of carbon source regulation, cultures were isolated in midlog phase grown in YP media with 2% glucose, galactose, or raffinose. Cultures grown in glycerol, lactate, and ethanol were supplemented with 4% carbon source and 0.1% galactose. For growth at various oxygen tensions, a small inoculum of YM4134 overnight culture was placed in a 250-ml side-arm flask containing 100 ml of YPGal supplemented with 10 µg/ml ergosterol and 0.1% Tween 80. Cultures were immediately placed in a closed system wherein defined oxygen concentrations (from 100 to 0%, balanced with nitrogen) were bubbled from tanks (Genex) through the media. The oxygen tensions below 0.03% were monitored by using an anaerobic indicator strip (BBL Microbiology Systems). Cultures were grown for at least 12 h at 30 °C with shaking. Growth was monitored by a Klett meter until midlog phase was reached. Then the flasks, still subjected to the flow of gas, were placed on ice for 20 min. 5-ml aliquots were subsequently removed for RNA extraction.

Extraction and Analysis of RNA

RNA was isolated from cells using the glass bead method in the presence of phenol/chloroform(27) . 10 µg of total RNA was loaded onto 1.2% agarose gels containing 3% formaldehyde. After electrophoresis, gels were ethidium-stained to monitor RNA integrity and then transferred to a Magnagraph nylon membrane (Micron Separations, Inc.). To ensure equal loading of mRNA, the membrane was hybridized to a probe made from the S. cerevisiae actin (ACT1) gene. All probe templates were created by polymerase chain reaction using the following primers based on the published sequences of YHG, ACT1, TIF51B, and COX5(8, 28, 29, 30) : YHG 5`, 5`-ATGCTAGCCGAAAAAACCC-3`; YHG 3`, 5`-CTAAACTTGCACGGTTGAC-3`; ACT1 5`, 5`-GGTTGCTGCTTTGGTTATTG-3`; ACT1 3`, 5`-TTAGAAACACTTGTGGTGAAC-3`; TIF51 5`, 5-ATGTCTGACGAAGAACACAC3`; TIF51 3`, 5`-CTAATCAGATCTTGGAGCTT-3`; COX5 5`, 5-AAGATT(T/C)G(T/C)TCAAACA(A/C)A(G/T)GC(T/C)CTTTC-3`; COX5 3`, 5`-T(T/C)ATTTAGATTG(G/A)AC(T/C)TGAGAATAACC(A/T)CC-3`. All genes were amplified by PCR from genomic DNA of YM4134. The products, which were of predicted size, were subsequently used as a template for probe synthesis by PCR incorporation of [alpha-P]dATP(31) . Exposed films were assayed for band intensity using a scanning laser densitometer (Molecular Dynamics).

Measurement of beta-Galactosidase Activity

All strains described were transformed with the integrating plasmid pYH10 containing the wild-type hemoglobin promoter driving the lacZ gene (32) and selected on minimal media (-ura). Two independent transformants were used for further study. beta-Galactosidase assays (33) were performed in duplicate on midlog-phase cells permeabilized with SDS and chloroform. The specific activity is expressed as (A times 3500)/(min times A of cells).

Physical and Genetic Mapping

The physical map location of YHG was determined using clone grid filters obtained from Linda Riles of Washington University(34) . The filters were used as described in the protocol accompanying the filter set. Genetic crosses between YD7 and yJC366 and subsequent sporulation and tetrad dissection of YGGM1 were performed using standard techniques(35) .

YHG Antibody Production and Western Blotting

The YHG gene was overexpressed in E. coli using the PET vector system (36) . After partial purification(8) , gel slices of the 47-kDa protein, confirmed as YHG by N-terminal amino acid sequencing, were used for rabbit immunization(37) . For Western analysis, total S. cerevisiae protein was fractionated by SDS-polyacrylamide gel electrophoresis(38) , transferred to nitrocellulose, incubated with primary antiserum (1:100) followed by alkaline phosphatase-conjugated secondary antibody (Bio-Rad), and developed as described(39) .


RESULTS

Hemoglobin mRNA Levels Decrease as the Cultures Exit Log Phase

Vitreoscilla and A. eutrophus hemoglobin levels increase as nutrient and oxygen concentrations become limiting when the cells exit the logarithmic growth period(11, 14) . S. cerevisiae also possesses gene products involved in oxygen metabolism whose levels increase after the cessation of log phase. These include the cytoplasmic catalase (CTT1) gene and the CYC7 gene, encoding iso-2 cytochrome c(40, 41) . To determine whether the S. cerevisiae hemoglobin follows this pattern, we inoculated a dilute sample of YM4134 into YPGal media and removed aliquots for steady-state mRNA isolation at the cell densities indicated in Fig. 1. The log-phase doubling time of 2 h in YPGal for this strain ceased at an A of about 2.5. Stationary phase, determined by the lack of any increase in A after 48 h of continuous shaking at 30 °C, was reached at an A of about 10.5. To be sure that cells were still viable, cultures were plated on solid YPD media, and subsequent colony counts were found to correlate well with cell densities. As shown in Fig. 1, the YHG message begins to decrease as the cells exit log phase and appears to be absent from stationary phase cultures. Densitometry analyses indicate a 25-fold decrease in YHG mRNA by an A of 5.5 and greater than a 100-fold decrease at higher optical densities. Levels of YHG mRNA also drop sharply upon exit from log phase when synthetic media or a non-fermentable carbon source is used (not shown), suggesting a global repression independent of nutrient source. The decrease in hemoglobin message upon exit from log-phase growth indicates that the hemoglobin is probably not required for oxygen-depleted respiration after the diauxic shift from log-phase growth in S. cerevisiae.


Figure 1: Repression of YHG mRNA with increasing cell density. Shown is the growth of strain YM4134 as a function of time after an initial inoculation of 10^4 cells. Cultures were grown in YPGal media with vigorous shaking at 30 °C. At the points indicated by the arrows, aliquots of culture were removed and total RNA isolated. The numbers above the inset indicate the A at the time of RNA isolation. 10 µg of total RNA was loaded into each lane. The RNA blot was probed with a radiolabeled YHG PCR product as described under ``Materials and Methods.'' Densitometry readings of the band intensities (setting A of 1.5 equal to 1.0) were, in order: 1.0, 1.0, 0.78, 0.69, 0.04, <0.01, and <0.01. Ethidium-stained intensity of the 18 S rRNA band was used as an internal RNA loading standard and gave intensities: 1.0, 1.0, 1.0, 1.2, 1.2, 0.93, and 0.81.



Effect of Oxygen Tension on the Transcription of YHG

The depletion of hemoglobin message as the cells exit log phase raises the possibility that intracellular oxygen levels may play a role in the expression of YHG. To examine this, we set up a variable-oxygen cell growth system. Cultures of YM4134 were inoculated into YPGal and continuously purged with defined gas mixtures (ranging from 100% O(2) to 0% O(2)/100% N(2)) until midlog phase was reached (A of 1-1.5). As shown in Fig. 2, the hemoglobin message is maximal in 100% oxygen and decreases as oxygen levels decline to about 0.1%. Compared with 21% O(2) by densitometry, a 4-fold lower but constant amount of message is detected from 0.1% oxygen down to strict anaerobiosis. Hemoglobin expression as a function of oxygen concentration was compared with the well characterized, oxygen-dependent message for translation initiation factor eIF-5a. In S. cerevisiae, eIF-5a is encoded by the aerobic/anaerobic gene pair TIF51A and TIF51B (also known as ANB1), which share 90% nucleotide sequence identity(42) . Fig. 2indicates that the switch from aerobic to anaerobic conditions for TIF51A and TIF51B occurs at about 0.1% oxygen, where both are similar in their expression. Above 0.1% O(2) no TIF51B mRNA is detected, and below 0.1% O(2) no TIF51A message is seen. A similar pattern was found for the COX5 aerobic/anaerobic gene pair when this blot was analyzed with a COX5-specific probe (not shown).


Figure 2: YHG gene expression at varied oxygen tensions. YM4134 was grown in YPGal at the defined oxygen concentrations shown above the figure. Oxygen tensions were maintained by bubbling defined gas mixtures continuously through the cultures. All cultures also contained 10 µg/ml ergosterol and 0.1% Tween 80 to allow anaerobic growth. After midlog phase was reached, cells were chilled on ice with continued bubbling for 20 min, and RNA was isolated. The blot was hybridized with a mixture of YHG and TIF51B probes prepared as described under ``Materials and Methods.'' TIF51A is the 90% identical aerobic gene partner of TIF51B, which cross-hybridizes to TIF51B probe. This blot was stripped and re-hybridized with a S. cerevisiae ACT1 probe to ensure equal loading of mRNA. Densitometry readings of the YHG message gave the following intensities (setting 21% as 1.0): 1.26, 1.0, 0.50, 0.28, 0.23, 0.32, and 0.23.



Deficiency of Heme and Heme-induced Transcription Factors Decrease Aerobic YHG Expression

A key intermediate in the pathway from oxygen levels to regulation of gene expression is heme, the biosynthesis of which requires molecular oxygen at two steps(19) . S. cerevisiae mutants that cannot synthesize heme mimic an oxygen-depleted state, repressing the expression of aerobic genes while activating anaerobic genes(16, 17) . The only known direct link between heme and oxygen-regulated gene expression is the interaction of heme with HAP1, a transcription factor that activates a variety of aerobic genes(16, 17) . Heme appears to function by facilitating the formation of an active HAP1/DNA complex or by altering the complement of proteins assembled at a HAP1-regulated promoter(41, 42) . The pathway leading to activation by HAP2/3/4, a heterotrimeric transcription factor complex involved in heme-induced transcription of CYC1, cytochrome oxidase subunit 5a (COX5a), and other aerobic genes, is currently unclear(16, 18) . Null alleles of the HAP2 polypeptide are sufficient to prevent complex assembly and subsequent activation at a HAP2/3/4-dependent promoter(43) .

Given the oxygen responsiveness of the YHG gene, we examined YHG mRNA levels in a S. cerevisiae strain (nem1) deleted in -aminolevulinic acid synthase, the enzyme catalyzing the first step in heme biosynthesis(19) . As shown in Fig. 3A, heme deficiency results in significantly reduced amounts of YHG mRNA (about 8-fold by densitometry). Levels return to those of wild type with the addition of 100 µg/ml -aminolevulinic acid, which circumvents the hem1 lesion in the pathway.


Figure 3: Effect of heme and heme-activated transcription factors on YHG mRNA levels. RNA was isolated from midlog phase cells grown in YPGal. The RNA blots were hybridized with the YHG PCR probe. All cultures were supplemented with 10 µg/ml ergosterol and 0.1% Tween 80. A, lanes show wild-type strain RZ53-6 (wt), RZ53-6hem1 (hem1), and RZ53-6hem1 supplemented with 100 µg/ml -aminolevulinic acid (hem1 + ALA). Setting wild type as 1.0 for band intensity, the following densitometry results were obtained: 1.0, 0.12, and 1.41. B, total RNA was isolated from strains BWG7-1a (wt), LPY22 (hap1), JO1-1a (hap2), and JS109 (hap1/hap2) grown under aerobic and anaerobic (N(2)) conditions. Both parts of the experiment are from the same blot, and the N(2) lanes have been placed underneath the aerobic lanes for comparison. Both aerobic and anaerobic conditions produce the identical 1.2-kb YHG mRNA. The ACT1 probe was used as an internal standard for mRNA loading. Densitometry results for the YHG band (with wild type in air set at 1.0) are: 1.0, 0.46, 0.29, and 0.02 in air and 0.25, 0.10, 0.10, and 0.11 in nitrogen.



Strains deficient in HAP1 or HAP2 also display a decrease in YHG message (2.2- and 3.4-fold by densitometry) compared with that of wild type (Fig. 3B, top). In a strain where both HAP1 and HAP2 have been mutated, YHG mRNA levels drop about 50-fold from wild type, suggesting that the heme/HAP pathway is predominant in the aerobic transcription of YHG. A strain deficient in the heme-induced repressor ROX1 (RZ53-6 rox1) had no change in aerobic YHG gene expression (not shown). The sharp decrease of transcription in the hap1/hap2 strain was surprising, given that there is some expression of YHG even under anaerobic conditions (Fig. 2). To determine if the anaerobic expression of YHG is HAP-independent, we isolated RNA from wild-type cells and from the HAP mutants grown anaerobically. The results, shown in the bottom of Fig. 3B, reveal persistent anaerobic production of YHG mRNA even in the hap1/hap2 double mutant.

To confirm that oxygen/heme control of YHG mRNA is at the level of transcription, 1.6 kb of the YHG promoter was fused to the lacZ reporter gene and integrated into the genome of wild-type, hem1, hap1, hap 2, and hap1/hap2 strains, and lacZ assays were performed (Table 2). A 4-fold decrease in lacZ activity was observed in heme-deficient cells compared with wild-type levels. Strains carrying a mutation in either HAP1 or HAP2 also showed about a 4-fold decrease from wild-type lacZ levels, whereas the hap1/hap2 double mutant produced about one-tenth of the beta-galactosidase produced by the wild-type strain. The YHG promoter/lacZ fusion construct integrated into the wild-type strain YM4134 also displayed a 3-fold decrease in YHG promoter activity as the cells exited log phase (Fig. 4) or were subjected to decreased oxygen tensions (not shown).




Figure 4: Effect of culture density on expression of the YHG promoter/lacZ construct. YM4134 carrying the integrated pYH10 construct was grown in YPGal media and assayed for lacZ activities at the optical densities shown. The lacZ activity points represent the averages of three separate cultures done in duplicate. Individual specific activities never varied by more than 15%.



Expression of Hemoglobin Is Independent of Carbon Source

All of the genes regulated by the HAP2/3/4 complex studied to date are subject to transcriptional repression in the presence of glucose, including those that are dually activated by HAP1(16, 18) . S. cerevisiae will preferentially ferment glucose even in the presence of oxygen, and many of the genes involved in utilization of other carbon sources are repressed by an order of magnitude or more when cells are grown in glucose-containing media(44) . Induction of HAP2/3/4-dependent promoters in media containing carbon sources other than glucose appears to be correlated with activation of HAP4 transcription(44) . To determine if the S. cerevisiae hemoglobin expression is glucose-repressed, cells were grown in media containing a variety of carbon sources. As shown in Fig. 5, no significant induction over glucose mRNA levels could be found with cells grown in galactose, raffinose, or the non-fermentable glycerol, lactate, and ethanol carbon sources. By both Northern analyses and hemoglobin promoter/lacZ activity assays (not shown), a similar lack of glucose repression in the YHG promoter was observed in synthetic media supplemented with various carbon sources.


Figure 5: Effect of carbon source on YHG expression. YM4134 cells were grown in YP medium supplemented with 2% glucose, 2% galactose, 2% raffinose, 4% glycerol, 4% lactate, or 4% ethanol. The latter three cultures were also supplemented with 0.1% galactose for enhanced early growth. Total RNA was isolated from midlog phase cultures, and the subsequent blot was hybridized with YHG. ACT1 probe was used as an mRNA loading standard.



Physical and Genetic Map Location of Flavohemoglobin

We physically mapped the YHG locus to determine if any genetic markers related to oxygen metabolism were in the region. The YHG PCR probe was hybridized to a set of clones that cover over 90% of the yeast genome(34) . The results identified a clone that contains a fragment of the right arm of chromosome VII within 40 kb of ADE3. A respiration-deficient mutant (PET54) is also in this region, encoding a previously described mitochondrial translational factor(45) . For genetic mapping and subsequent phenotypic characterization, the LEU2 gene was inserted into the hemoglobin locus by -recombination(23) . A site-specific disruption of the YHG locus was confirmed by Southern blot (not shown) as well as Northern and Western analyses (Fig. 6). The knockout strain, YD7, was crossed to yJC366, a strain containing a mutation in ADE3. The yhg locus was followed by scoring the LEU2 marker, and the ADE3 marker was followed by scoring red colonies(35) . Genetic mapping confirmed the presence of the flavohemoglobin locus on the right arm of chromosome seven, 15.4 centimorgans (19PD: 0NPD:8TT) from ADE3.


Figure 6: Northern and Western analyses of YHG knockout. A, total RNA was isolated and blotted from YM4134 (wt) and the hemoglobin knockout strain YD7 (-yhg). The membrane was hybridized to YHG probe and, after stripping, with ACT1 probe. B, total protein from 1.5 times 10^8 log-phase cells of YM4134 and YD7 was run on SDS-polyacrylamide gel electrophoresis and probed with rabbit alpha-YHG antiserum raised against recombinant YHG expressed in E. coli. Numbers corresponding to the 52- and 34-kDa molecular mass markers are shown. The 47-kDa band seen in the wt lane is the predicted size of the YHG protein(8) .



No phenotypic differences were found between the wild-type and the yhg strains in a variety of conditions tested, including growth on fermentable or non-fermentable carbon sources from 100% oxygen to strict anaerobiosis. When induced to lose its mitochondrial DNA by ethidium bromide treatment(46) , the knockout strain formed petite colonies with no effect on fermentative growth (not shown).


DISCUSSION

Regulation of the S. cerevisiae flavohemoglobin gene is clearly controlled by cell density and oxygen tension but in a manner different from that of the bacterial globins previously studied. During our efforts to correlate YHG transcriptional activation with oxygen concentration, we were able to determine at what oxygen tension the apparent switchover from aerobic to anaerobic gene expression in S. cerevisiae occurs. The messages for both the aerobic TIF51A and the anaerobic TIF51B are found at 0.1% oxygen. Above this concentration, only TIF51A is present; below this concentration, only TIF51B can be detected. A similar result was seen for the COX5a/b gene pair. This is also the oxygen tension where aerobic activation of the YHG promoter ends and the anaerobic expression of the YHG message begins. Thus, 0.1% likely straddles the concentration of oxygen necessary for heme biosynthesis.

Aerobic expression of YHG is predominantly activated by the HAP1 and HAP2/3/4 transcription factor complexes. An anaerobic system to produce YHG message independent of the HAPs is also present. Although there is a decrease in YHG gene expression as the cells approach anaerobiosis, the effect is not as dramatic as the depletion in YHG mRNA as the cells reach higher optical densities ( Fig. 1compared with Fig. 2). The additional repression of endogenous YHG gene expression upon exit from exponential phase may be due to a combination of factors such as hypoxic conditions, induction of a repressor, or the altered chromatin structure at higher culture densities that has been implicated in repressing most exponential phase mRNA species(47) .

Our results indicate that the YHG promoter/lacZ fusion construct is also regulated by heme and the HAPs, but interesting quantitative differences between Northern and transcriptional fusion analyses exist. The hem1 and hap1/hap2 strains display 4- and 10-fold decreases in lacZ activity relative to wild type, whereas quantitation of Northern analyses indicate a YHG mRNA decrease of 8- and 50-fold for the hem1 and hap1/hap2 strains, respectively ( Fig. 3and Table 2). The drop in lacZ activity as the cells exit log phase is also less dramatic than that of the native YHG message ( Fig. 4compared with Fig. 1). These results raise the possibility that sequences outside of the 1.6-kb upstream region governing the lacZ fusion influence transcription or stability of the endogenous YHG gene product.

The physiological function of hemoglobins in species other than vertebrate animals has not been well established. The present data suggest that, despite the high degree of homology between globins of prokaryotic and eukaryotic microorganisms, each species may have developed a discrete role for its hemoglobin. Through activation by bacterial FNR or related transcription factors, hemoglobins of the strictly respiring Vitreoscilla and A. eutrophus species are probably needed for metabolism when oxygen supplies are severely limiting or absent(9, 15) . Overexpression studies in E. coli indicate that the Vitreoscilla globin can increase oxygen uptake and can even act as a terminal oxidase(4, 5) . An A. eutrophus strain carrying a deletion of the flavohemoglobin gene could not transiently accumulate nitrous oxide during anaerobic growth using nitrate as the terminal electron acceptor, suggesting a role of this protein in the metabolism of a gas other than dioxygen(9) . A specialized role for the A. eutrophus globin is suggested by the positioning of the native flavohemoglobin gene on an episomal plasmid linked to denitrification processes (48) and by the inability of species that contain homologous globins, such as S. cerevisiae, to use nitrite as an anaerobic terminal electron acceptor(49) . Another area of variability between members of the flavohemoglobin family is in their affinities for oxygen. The dissociation constant for the C. norvegensis flavohemoglobin in solution is 2 times 10M, making it one of the most oxygen-avid hemoglobins known(50) . The dissociation constants for the Vitreoscilla and E. coli hemoglobins are 7.2 times 10 and 2 times 10M, respectively, a difference of at least 2 orders of magnitude from the C. norvegensis globin, despite nearly 50% sequence identity in the heme-binding domains of these proteins(51, 52) .

Clues to the possible function of the S. cerevisiae flavohemoglobin have been uncovered by the gene regulation results presented here. Unlike the Vitreoscilla globin, YHG is probably not involved in facilitating oxygen storage or diffusion during hypoxic electron transport. Contrary to many other genes involved in respiration, the YHG message represents the first known example of a HAP2/3/4-regulated gene that is not glucose-repressed, indicating that the gene may be required in both fermentable and non-fermentable carbon sources. There is an increase in YHG mRNA levels in cells grown under high oxygen tension. This phenomenon can be attributed to increased heme levels, which also stimulate superoxide dismutase and catalase transcription in S. cerevisiae(53, 54) . Flavohemoglobin in S. cerevisiae may also be involved in the detoxification of oxygen. It should be noted, however, that no detectable growth difference is seen between wild-type or yhg strains when grown in 100% oxygen or in the presence of hydrogen peroxide (not shown).

The capacity of the YHG promoter to remain active even during anaerobic growth suggests that the hemoglobin may be necessary in certain anaerobic conditions. A phenotype for the A. eutrophus strain lacking hemoglobin was found only when cells were grown anaerobically(9) . It can be postulated that low levels of YHG might be needed anaerobically to quickly initiate a signal cascade once oxygen becomes available. Activity of the C-terminal reductase domain in YHG could be altered with the binding of heme or heme and oxygen in the N-terminal portion of the molecule. A heme-containing oxygen sensor has been found in the Rhizobium bacteria, where an oxygen-regulated kinase in the N terminus is linked to a heme-binding domain in the C terminus(55) . Biochemical studies of the E. coli flavohemoglobin indicate that flavin reduction in the protein is increased upon deoxygenation, suggesting that the reductase activity may be altered by the availability of oxygen(52) . Further phenotypic studies of the YHG knockout strain and biochemical analyses of the YHG protein should provide insight into the role of flavohemoglobins from facultative eukaryotic microorganisms.


FOOTNOTES

*
This work was supported in part by a grant from the Lucille P. Markey Charitable Trust. 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.

§
Current address: PathoGenesis Corp., 201 Elliott Ave. W., Seattle, WA 98119.

To whom correspondence should be addressed: Dept. of Molecular Microbiology, Washington University School of Medicine, Box 8230, 660 S. Euclid Ave., St. Louis, MO 63110. Tel: 314-362-1514; Fax: 314-362-1232.

(^1)
The abbreviations used are: VtHb, Vitreoscilla hemoglobin; HAP, heme-activated protein; YHG, S. cerevisiae flavohemoglobin; TIF51, translation initiation factor 5; COX5, cytochrome oxidase subunit 5; ACT1, actin; kb, kilobase pair(s); PCR, polymerase chain reaction.


ACKNOWLEDGEMENTS

We would like to thank John Cooper, Leonard Guarente, and Richard Zitomer for the provision of strains and John Lawrence for use of the densitometer. We are indebted to Mark Johnston, Linda Riles, and Tina Hesman for the provision of yeast strains as well as for invaluable discussion.


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