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1995 Molecular Cloning, Sequencing, and Regulation of the Rhodobacter capsulatushemB Gene. Karl Joseph Indest Louisiana State University and Agricultural & Mechanical College
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MOLECULAR CLONING, SEQUENCING, AND REGULATION OF THE Rhodobacter capsulatus hemB GENE.
A Dissertation
Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy
in
The Department of Microbiology
by Karl Joseph Indest B. A., University of New Orleans, 1990 August 1995 UMI Number: 9609095
UMI Microform 9609095 Copyright 1996, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Aim Arbor, MI 48103 Dedication
This work is gratefully dedicated to my loving wife, Lisa, for inspiring me to pursue my dream. Acknowledgements
Words cannot express the deep appreciation and gratitude I have for my friend and wife, Lisa (the real hemB). Without her encouragement, emotional support, and love, I would have not been able to successfully complete this degree.
I would like to express a deep hearted thanks to my family for their encouragement and support (emotional & financial) throughout the years. They taught the importance of a good education, but now they want me to stop going to school and get a 'real' job.
I would like to express a sincere thanks to Alan J. Biel, my graduate advisor. His support (financial & financial) and encouragement have made my graduate career a successful one. I deeply appreciate the time and effort he has invested in training me, in the preparation of manuscripts (including this one), and in listening to me speak in incomplete sentences while nodding in approval.
I would like to thank my committee members for their advice over the years: Drs Battista, Achberger, Gayda, Shih, and Fuxa. A special thanks goes out to Dr. Battista. Without his recommendation, I would have never rentedThe
Evil Dead Part 1 & 2.
I would like to extend a large token of gratitude to my fellow colleagues in the lab: Georgia Ineichen, Kayta Kanazireva, Keith Canada, Alok Khanna, and
David Huang. Thanks for putting up with me on a day to day basis without killing me (especially Georgia).
Special thanks to my colleagues in the Battista lab: Valerie Mattimore,
Kumar Udupa, and Sharon Kelly.
Finally, a special thanks to Cindy Henk and Valerie Mattimore for helping me with slides and photographs.
KJI
iv Table of Contents
Page
Dedication ...... ii
Acknowledgments...... iii
Abstract ...... vii
Introduction...... 1 Formation of aminolevulinate and its conversion into protoporphyrin IX...... 2 Regulation of tetrapyrrole biosynthesis...... 13
Materials and Methods ...... 20 Strains and plasmids...... 20 Media ...... 20 Growth conditions...... 20 Nucleic acid isolation ...... 25 Restriction digest, ligations, nick translations, and other recombinant DNA techniques...... 27 Agarose gel electrophoresis...... 28 Transformation ...... 29 Electroporation ...... 29 Matings ...... 30 Southern analysis ...... 30 Dot blot analysis ...... 32 DNA sequencing...... 33 Pulsed Field Gel Electrophoresis (PFGE)...... 33 Protein determination...... 35 Porphobilinogen synthase assay ...... 35 Chloramphenicol acetyltransferase assay ...... 36 Porphyrin determination...... 36 Thin layer chromatography ...... 37
Results...... 38 Cloning of theR. capsulatus hemB gene...... 38 Restriction analysis of the hemB region...... 39 Expression of pCAP142(hemEP) in SB1003...... 42 Southern analysis ...... 43 Chromosomal location of theR. capsulatus hemB gene.. 45 Sequencing of theR. capsulatus hemB gene...... 48 Regulation of theR. capsulatus hemB gene...... 54
v Dot blot analysis ...... 54 Construction of a hemB-cat transcriptional fusion plasmid ...... 56 Effects of oxygen on transcription of thehemB-cat fusion plasmid ...... 56 Overexpression of thehemB gene in AJB456...... 58
Discussion...... 63
Literature Cited...... 71
Vita ...... 86
vi Abstract
The common tetrapyrrole pathway in the photosynthetic bacterium,
Rhodobacter capsulatus, is responsible for the synthesis of bacteriochlorophyll, heme, vitamin B-12, and siroheme. The common portion of this pathway is regulated up to 100-fold by changes in oxygen tension. This regulation is accomplished by controlling the intracellular level of porphobilinogen. One way in which porphobilinogen levels could be controlled is by regulating the synthesis of porphobilinogen. Porphobilinogen synthase, encoded by thehemB gene, is the second enzyme in the common tetrapyrrole pathway that catalyzes the dimerization of 5-aminolevulinic acid to form the monopyrrole porphobilinogen.
In order to further investigate the mechanism by which oxygen regulates porphobilinogen levels, theR. capsulatus hemB gene was cloned and sequenced.
The R. capsulatus hemB gene was cloned by complementation of an
Escherichia coli hemB mutant. Sequence analysis of the R. capsulatus hemB gene revealed that the putative porphobilinogen synthase has a metal-binding domain that more closely resembles that found in plant porphobilinogen synthases. The locations of thehemB and hemA genes on theR. capsulatus chromosome indicate that these gene do not form an operon.
Oxygen mediated transcriptional regulation of the R. capsulatus hemB gene was measured by dot blot analysis of mRNA from cells grown under 3% and 23% oxygen and by hemB-cat transcriptional fusion studies. Both of these methods reveal that the synthesis of porphobilinogen synthase does not change with shifts in oxygen tension. Oxygen tension also does not appear to regulate enzyme activity since the specific activity does not change significantly with changes in oxygen tension.
Overexpression of theR. capsulatus hemB, in the presence of exogenous aminolevulinate, in R. capsulatus can overcome the normal oxygen mediated regulatory mechanisms. While the mechanism of how oxygen modulates carbon flow down the common tetrapyrrole pathway remains elusive, it is clear that the level of porphobilinogen plays a crucial part in oxygen regulation. The possibility still exist that oxygen regulates the degradation of porphobilinogen. Introduction
The photosynthetic, purple, nonsulfur bacteria are a metabolically versatile group of organisms. One member of this group,Rhodobacter capsulatus, is able to obtain energy from many different sources. It can use light for photophosphorylation, or it can obtain energy from the oxidation of either organic compounds or inorganic compounds such as H2. The presence of the Calvin cycle (47), in addition to the Entner-Doudoroff and glycolytic pathways (30), allows this organism to use C02 or organic carbon compounds as a carbon source. In addition, R. capsulatus possesses a nitrogenase enzyme which allows it to fix atmospheric nitrogen. One factor in this organism's remarkable metabolic versatility is that it is one of a small group of organisms that can synthesize all four tetrapyrrole endproducts: heme, bacteriochlorophyll, vitamin B-12 and siroheme. Bacteriochlorophyll is the primary light-harvesting pigment involved in bacterial photosynthesis and is only produced under anoxic conditions. Heme is required under all growth conditions and is present as the prosthetic group in cytochromes, catalases and peroxidases. Vitamin B-12 has been shown to be a cofactor in methionine biosynthesis (24) and siroheme serves as a prosthetic group for the enzymes sulfite reductase (98) and nitrite reductase (99).
1 2
Formation of aminolevulinate and its conversion into protoporphyrin IX
Most of the information about the tetrapyrrole pathway in the
Rhodospirillaceae has come from studies usingR. sphaeroides. In 1964, while working with R. sphaeroides, Lascelles proposed that bacteriochlorophyll a and heme share a common pathway that bifurcates after the formation of protoporphyrin IX (88). This common portion of the tetrapyrrole pathway, from aminolevulinate to protoporphyrin IX, is found in most organisms (Fig. 1). There are only a few examples of organisms found in nature that have an incomplete tetrapyrrole biosynthetic pathway (55). Most of these organisms have complex nutritional requirements and require exogenous heme or a tetrapyrrole precursor for growth. As far as is known, all organisms either make or require tetrapyrroles. This suggests that the tetrapyrrole molecule is evolutionarily ancient, functioning in life's most important biochemical processes.
The aminoketone, aminolevulinate, is the first common intermediate found in the tetrapyrrole pathway. Synthesis of aminolevulinate can occur by two distinct routes: the C5 pathway, which uses the carbon skeleton of glutamate (8), or the C4 pathway, which involves glycine and succinyl-CoA (80). The C5 pathway is present in plants (8), algae (141), cyanobacteria (39), archeabacteria
(46) and eubacteria (41). Formation of aminolevulinate using the C5 pathway requires activation of glutamate by linking it to the tRNAglu. This reaction is catalyzed by glutamyl-1 -synthetase (104). Glutamyl-tRNA9lu is reduced to glutamate-1-semialdehyde by glutamyl-tRNAglu reductase, the product of the 3
Succlnyl CoA + Glycine hemA
Aminolevulinate hemB
Porphobilinogen hemC, hemD
Uroporphyrinogen III hemE
Coproporphyrinogen III hemF
Protoporphyrinogen IX hemG
Heme ^ Protoporphyrin IX ------> > Mg-Protoporphyrin hemH bchD bchH monomethyl ester bchl i Bacteriochlorophyll
Figure 1. The tetrapyrrole biosynthetic pathway in R. capsulatus. 4 hemA gene (3). Aminolevulinate is formed from glutamate-1-semialdehyde, following a transamination reaction, catalyzed by glutamate-1-semialdehyde aminotransferase, the product of the hemL gene (41).
The C4 pathway is found in the a-proteobacteria, which includes the
Rhodospirillaceae, but is mainly confined to animals (4,105). In the C4 pathway formation of aminolevulinate is catalyzed by aminolevulinate synthase, the product of the hemA gene. This enzyme forms aminolevulinate by the condensation of glycine and succinyl-CoA. This step is one of the most studied of the tetrapyrrole pathway. The R. capsulatus hemA gene has been cloned and sequenced from two independent sources. Biel et al. (143) isolated a cosmid, containing the R. capsulatus hemA gene, by complementation of aR. capsulatus hemA:Tr\5 mutant. Drews et al. (63) isolated the hemA gene fromR. capsulatus by probing a cosmid bank of R. capsulatus with the aminolevulinate synthase gene fromR. sphaeroides. When compared with otherhemA genes encoding aminolevulinate synthase, the predicted amino acid sequence is 69% to 73% similar, and 49% to 55% identical to other aminolevulinate synthases (38). The predicted molecular mass of the R. capsulatus aminolevulinate synthase is
50,491 Da, based on sequence information (38). Since there are no published reports on the purification of aminolevulinate synthase fromR. capsulatus, it is not known what the actual molecular mass is of the native enzyme. The native enzyme fromR. sphaeroides exist as a homodimer and has a molecular mass of 80,000 Da (103). The R. sphaeriodes enzyme requires pyridoxal phosphate for catalytic activity and is strongly inhibited by hemin, Mg-protoporphyrin or ATP
(43, 151). Multiple forms of aminolevulinate synthase fromR. sphaeroides have been isolated on DEAE-Sephadex (136). The different forms have varying degrees of activity and respond differently to environmental factors, such as light and oxygen (135). The enzyme forms have been found to be interconvertable, with the inactive form requiring cysteine trisulfide to become active (113).
Interestingly, R. sphaeroides has recently been found to encode two distinct aminolevulinate synthases, the products of thehemA and hemT genes (79).
These two genes are located on separate chromosomes and share homology with each other, as well as all previous characterized aminolevulinate synthases.
The protein products of thehemA and hemT genes, however, have not been correlated with the multiple forms of aminolevulinate synthase reported previously in R. sphaeroides (136).
The next step in the tetrapyrrole pathway is the formation of the monopyrrole, porphobilinogen. Porphobilinogen is formed by the condensation of two molecules of aminolevulinate with the loss of two molecules of water. This reaction is catalyzed by porphobilinogen synthase, the product of the hemB gene.
This reaction is the second most studied of the common tetrapyrrole pathway.
The R. capsulatus hemB gene has been cloned and sequenced and will be discussed in detail later. The R. sphaeroides hemB gene has also been cloned, but no sequence information is available (35). This makes the R. capsulatus hemB gene the only porphobilinogen synthase gene to have been sequenced from theRhodospirillaceae. Porphobilinogen synthase has been purified from R.
capsulatus (102) and R. sphaeroides (101). The native conformation of the
enzyme is believed to exist as a hexamer having a molecular mass of
approximately 260,000 Da for the R. capsulatus enzyme and 240,000 Da for the
R. sphaeroides enzyme (62). Porphobilinogen synthase fromR. sphaeroides
requires the presence of metallic cations and thiols for full activity and is strongly
inhibited by hemin (101). In contrast, the R. capsulatus enzyme has no metal
requirement, is less responsive to thiol compounds and is not inhibited by hemin
(102 ).
The first tetrapyrrole, uroporphyrinogen III, is formed by the sequential
action of two separate enzymes. Porphobilinogen deaminase, the product of the
hemC gene, catalyzes the deamination and polymerization of four molecules of
porphobilinogen to form the highly labile, linear tetrapyrrole, hydroxymethylbilane.
This linear tetrapyrrole is released into solution where it serves as the substrate for uroporphyrinogen III synthase, the product of thehemD gene. This enzyme catalyzes the rearrangement of ring four of hydroxymethlybilane followed by its subsequent cyclization into uroporphyrinogen III. In the absence of
uroporphyrinogen III synthase, hydroxymethylbilane is spontaneously converted into uroporphyrinogen I, a nonphysiological isomer. Porphobilinogen deaminase has been purified from R. sphaeroides (74) and R. palustris (82), as well as a variety of other prokaryotic and eukaryotic sources. In all cases, the deaminase exist as a monomer having a molecular mass ranging from 34,000 Da to 44,000 7
Da (71), with the exception of theR. palustris deaminase which has a molecular mass of 74,000 Da. Porphobilinogen deaminase requires a dipyrrolmethane cofactor which serves as a primer to which four substrate molecules are covalently attached (75). This cofactor is unique in that it is synthesized by the deaminase from two molecules of porphobilinogen. Now that theE. coli porphobilinogen deaminase has been crystallized and undergone preliminary X- ray diffraction studies greater insight into the mechanism by which this enzyme functions can be gained (76).
Information concerning uroporphyrinogen III synthase has lagged behind the other enzymes of the tetrapyrrole pathway due to the instability of the protein.
It was first demonstrated by Bogorad and Granick that heating spinach extracts prior to incubation with porphobilinogen led to the production of uroporphyrinogen
I (17). The authors later concluded that a labile protein, which they termed uroporphyrinogen isomerase (uroporphyrinogen III synthase) was responsible for the isomerization reaction (16). So far uroporphyrinogen III synthase has been purified to homogeneity fromE. coli (1), human erythrocytes (133), rat liver (125), and Euglena gracilis (60). The synthases appear in monomeric form having molecular masses ranging from 28,000 Da to 31,000 Da. There are scattered reports of the copurification of uroporphorinogen III synthase with porphobilinogen deaminase to form a complex called porphobilinogenase (2, 77, 112). Jordan maintains that these enzymes do not form a complex but rather function independently and sequentially (72). The cloning and sequencing of the genes 8 encoding porphobilinogen deaminase and uroporphyrinogen III synthase have been reported for E.coli (73, 130), human erythrocytes (109, 132), Bacillus subtilis (59), Pseudomonas aeruginosa (97) and other prokaryotic and eukaryotic sources. There is greater homology exhibited among the porphobilinogen deaminases than among the uroporphyrinogen III synthases. DNA sequence information from E. coli, B. subtilis, and P. aeruginosa further reveal that the hemC and hemD genes form an operon (59, 97, 117). This arrangement may be advantageous when considering that these enzymes function sequentially.
Uroporphyrinogen III is at a major branch point in the tetrapyrrole pathway.
Upon decarboxylation, uroporphyrinogen III gives rise to heme and bacteriochlorophyll. If uroporphyrinogen III is methylated, siroheme and vitamin
B-12 will be the final endproducts. Uroporphyrinogen decarboxylase, the product of thehemE gene, catalyzes the stepwise decarboxylation of the acetate groups on all four rings of uroporphyrinogen III to form coproporphyrinogen III. Currently, it is believed that the decarboxylation reaction occurs in an orderly, clockwise fashion starting with ring D (92). The enzyme has been purified to homogeneity from only one bacterial source, R. sphaeroides (70). A 600-fold purification revealed that the native enzyme exists as a monomer having a molecular mass of 41,000 Da. These findings are consistent with other reports regarding eukaryotic uroporphyrinogen decarboxylases, which have molecular masses ranging from 39,000 Da to 46,000 Da (44,126, 137). The enzyme is susceptible to inhibition by a variety of porphyrins; the more hydrophilic the side chain the greater the degree of inhibition. In addition, the enzyme is stimulated by thiol
compounds and inhibited by the metal ions, Hg+2 and Cu+2. The hemE gene has
been cloned fromR. capsulatus by complementing an E. coli hemE mutant (65).
DNA sequencing analysis has revealed that the gene encodes a 344 amino acid
protein having a predicted molecular mass of 43,611 Da (65). The putative
amino acid sequence shares an overall identity of 34% with uroporphyrinogen
decarboxylases from yeast (48) and B. subtilis (57) and is 36% identical to the
rat enzyme (111).
The formation of protoporphyrinogen IX occurs when the propionyl groups
on the A and B rings of coproporphyrinogen III are oxidatively decarboxylated
to vinyl groups. This reaction is catalyzed by coproporphyrinogen oxidase, the
product of thehemF gene. Eukaryotes and aerobically growing bacteria require
molecular oxygen in order to transform coproporphyrinogen III into
protoporphyrinogen IX (115), whereas anaerobically grown cells use a different
enzyme to complete this transformation. Using extracts ofR. sphaeroides, Tait
(127,128) showed that the anaerobic enzyme required NADH, ATP, L-methionine
or S-adenosyl-methionine for activity. He concluded that R. sphaeroides had two
different enzymes which possessed the ability to convert coproporphyrinogen III to protoporphyrinogen IX: an aerobic coproporphyrinogen oxidase, which required
0 2 as an electron acceptor, and an anaerobic coproporphyrinogen oxidase, which
used NADH as an electron acceptor. Neither one of these enzymes has been
purified to homogeneity from a bacterial source. The cloning of a putative anaerobic coproporphyrinogen oxidase gene fromR. sphaeroides has recently been reported (31). Using chemical mutagenesis, Hunter et al. (31) isolated a mutant that when grown anaerobically was unable to synthesize bacteriochlorophyll and excreted large amounts of coproporphyrin. This same mutant, under aerobic conditions, accumulated only trace amounts of coproporphyrin. By using conjugative gene transfer and transposon mutagenesis, the authors were able to clone the gene responsible for this phenotype which they believed was due to the lack of an anaerobic coproporphyrinogen oxidase.
Sequence analysis of this gene revealed the molecular mass of the enzyme to be 34,185 Da. A putative oxygen-independent coproporphyrinogen oxidase has also been reported in Salmonella typhimurium (145). Using chemical mutagenesis, the authors isolated mutant strains they believed were defective in both the aerobic and anaerobic coproporphyrinogen oxidase. By complementing these mutants, it was possible to clone both the aerobic (147) and anaerobic
(146) coproporphyrinogen oxidases, known as the hemF and hemN genes, respectively. ThehemN gene codes for a 52.8 kDa protein and has a 38% amino acid sequence identity to the putative anaerobic R. sphaeroides coproporphyrinogen oxidase. The aerobic coproporphyrinogen oxidase codes for a 31.6 kDa protein and is 90% identical to the E. coli enzyme (131) and 44% identical to the yeast enzyme (152). The yeast coproporphyrinogen oxidase has been purified to homogeneity and is a homodimer having a molecular mass of
70,000 Da (23). Some have suggested that the presence of these two enzymes may indicate that the branch point separating heme and bacteriochlorophyll
synthesis may not occur at the chelation step but higher up in the tetrapyrrole
pathway, at the level of coproporphyrinogen III (31).
The final intermediate that is common to heme and bacteriochlorophyll
biosynthesis is protoporphyrin IX. Protoporphyrinogen oxidase, the product of the
hemG gene, is a membrane bound protein, which catalyzes the six electron
oxidation of protoporphyrinogen IX to form protoporphyrin IX. The formation of
protoporphyrin IX can also proceed spontaneously, however, the enzymatic
reaction is significantly increased over the non-enzymatic reaction (115).
Protoporphyrinogen oxidase requires molecular oxygen for the oxidation of
protoporphyrinogen IX in higher eurkaryotes and aerobic bacterial systems.
Jacobs et al. (66, 67) were the first to report an oxygen-independent mechanism for protoporphyrinogen IX oxidation in extracts of E. coli, in which nitrate or fumarate served as the electron acceptor. The same authors reported protoporphyrinogen oxidase activity in R. sphaeroides under photosynthetic and aerobic growth conditions (68). The R. sphaeroides protoporphyrinogen oxidase activity was subject to inhibition by cyanide and azide, indicating the enzyme was coupled to the respiratory chain. Protoporphyrinogen oxidase has been purified and characterized from only one bacterial source, Desulfovibrio gigas (81). The enzyme is a membrane bound protein that has a molecular mass of 148,000 Da and consist of three nonidentical subunits having molecular masses of 12,000 Da,
18,500 Da, and 57,000 Da. These three subunits are believed to be held 12 together by sulfhydryl bonds. In contrast, the mammalian protoporphyrinogen oxidases that have been purified and characterized so far are monomers with a molecular mass of about 65,000 Da (34, 84, 124). The hemG gene has been cloned and sequenced from E. coli (116) and B. subtilis (56). The E. coli hemG gene is 546 nucleotides, coding for a protein with the predicted molecular mass of 21,202 Da, whereas the B. subtilis enzyme has a predicted molecular mass of 51,200 Da. The apparent disparity between the sizes of the mammalian and bacterial enzymes may indicate that these are two separate enzymes. Perhaps, there are two kinds of protoporphyrinogen oxidases, much like the two coprophyrinogen oxidases activities reported inR. sphaeroides.
The ultimate fate of protoporphyrin IX is determined by whether an Fe++ ion or a Mg++ ion is incorporated into the porphyrin ring. Bacteriochlorophyll will be the final endproduct if magnesium is incorporated into protoporphyrin IX, whereas, if a ferrous iron is incorporated into protoporphyrin IX, heme will be the final endproduct. Ferrochelatase, the product of thehemH gene, catalyzes the insertion of Fe++ into protoporphyrin IX. ThehemH gene has recently been cloned in R. capsulatus and codes for a protein with a predicted molecular mass of 46,000 Da (Kanazireva and Biel, unpublished). Ferrochelatase has been purified to homogeneity fromR. sphaeroides (32). The enzyme is an intrinsic membrane protein, consisting of a single polypeptide of molecular mass 115,000
Da. This value is rather high when compared to other bacterial ferrochelatases which have molecular masses of about 35,000 Da (58, 96). The R. sphaeroides 13 ferrochelatase has a broad substrate specifity and is capable of using Cu++ and
Zn++, in addition to iron, in the chelation reaction (33, 69). The enzyme is also sensitive to inhibition by heme, protoporphyrin, Mg-protoporphyrin, and N-methyl- porphyrins (32). The inactivating effects of N-ethylmalamide and iodacetamide suggest that the enzyme contains sulfhydryl residues essential for activity (33).
The enzyme(s) responsible for inserting magnesium into protoporphyrin IX are less characterized. The magnesium chelatase reaction was first reported in
R. sphaeroides by Gorchein (51), who demonstrated that the reaction was dependent on membrane integrity, low 0 2 tension, ATP, and coupling of magnesium insertion to methylation. Weinstein and Walker (140), working with pea chloroplast, proposed that magnesium chelation was a two step process. In the first step, magnesium chelatase components come together in the presence of ATP and undergo activation. This activated enzyme complex then catalyzes the magnesium insertion into protoporphyrin IX. Recently, Gorchein (52), using
R. sphaeroides, has assigned the magnesium chelatase activity to the bchD and bchl loci and the methyltransferase activity to the bchH locus.
Regulation of tetrapyrrole biosynthesis.
R. sphaeroides synthesizes tremendously different quantities of tetrapyrroles depending on its mode of growth. Cells ofR. sphaeroides growing photosynthetically produce an estimated 25 nmoles bacteriochlorophyll per mg dry weight, 0.3 nmoles heme per mg dry weight, and 0.07 nmoles vitamin B-12 per mg dry weight (86). Upon aeration bacteriochlorophyll synthesis ceases, while heme and vitamin B-12 synthesis continue unabated (86).
Bacteriochlorophyll levels in photosynthetically grown cultures reach 40 to 100 times that of bacteriochlorophyll levels in aerobically grown cultures (87), whereas hemoprotein content varies only two to four-fold with changes in oxygen tension
(107). The sudden shift from anaerobic, photosynthetic growth to aerobic, dark growth does not result in the accumulation of biosynthetic intermediates. This indicates that the cell has the ability to reduce carbon flow over the common tetrapyrrole pathway 100-fold while continuing to synthesize the other tetrapyrrole endproducts. Multiple mechanisms are in place that enable the cell to adapt its biosynthetic machinery to changes in the environment. So far at least three environmental factors have been identified that modulate carbon flow over the common tetrapyrrole pathway in R. capsulatus: oxygen, heme, and c-type cytochromes. While each of the factors appear to act independently, the mechanisms by which they alter carbon flow are poorly understood.
Cohen-Bazire et al. (29) were the first to present evidence of an efficient mechanism for the regulation of tetrapyrrole biosynthesis. Using extracts ofR. sphaeroides and R. rubrum they showed that introducing oxygen into cultures growing anaerobically in the light resulted in the abrupt cessation of bacteriochlorophyll synthesis. They proposed that the redox state of components of the respiratory chain could be influenced by oxygen and were critical in regulating bacteriochlorophyll synthesis. Marrs and Gest (94) have indicated these components may not be directly involved in regulating bacteriochlorophyll synthesis since mutants ofR. capsulatus with defects in the respiratory chain are still capable of preventing bacteriochlorophyll synthesis in the presence of oxygen. A genetic and physical map of the photosynthetic region ofR. capsulatus has made it possible to study bacteriochlorophyll synthesis at the molecular level (129). Biel and Marrs (11) examined oxygen regulation of bacteriochlorophyll gene expression by isolating and studying fusions between
Mud d1 (Apr, lac) and various bacteriochlorophyll biosynthetic ( bch) genes.
Transcription of the bch genes were two to three-fold higher in cultures grown with 2% oxygen than in those grown with 20% oxygen. Measurements bchof mRNA from cultures grown under high versus low oxygen tensions confirm these results (27). A two-fold change in bacteriochlorophyll gene expression may be significant, but it does not account for the 100-fold difference in bacteriochlorophyll levels between anaerobically and aerobically grown cultures.
In addition, studies with cells of R. sphaeroides indicate that the coupling of magnesium insertion and methylation to form magnesium protoporphyrin monomethyl ester is sensitive to oxygen (51). Biel (10) has demonstrated that a R. capsulatus bchE strain does not accumulate magnesium protoporphyrin monomethyl ester when grown under high oxygen tension. The fact that the organism is still synthesizing heme under these growth conditions indicates that oxygen controls the formation of magnesium protoporphyrin monomethyl ester from protoporphyrin. Exactly how oxygen is regulating this step is still unknown. 16
Mutational analysis of the photosynthetic gene cluster has revealed that
PufQ is essential for bacteriochlorophyll synthesis (149). While the role of PufQ remains obscure, strains deficient in PufQ exhibit a severe reduction in steady- state levels of bacteriochlorophyll under anaerobic conditions. PufQ expression is highly regulated by aeration, with a 30-fold increase in gene expression under anaerobic conditions (7). One model envisions PufQ acting as a carrier molecule that interacts with protoporphyrin IX (6). The resulting tetrapyrrole-protein complex serves as the substrate for magnesium chelatase leading to bacteriochlorophyll synthesis. While this model offers a plausible explanation for how oxygen regulates the magnesium chelatase reaction, it does not explain how carbon flow is diverted away from the common pathway to accommodate the overall decrease in tetrapyrrole biosynthesis. Kaplan (50), on the other hand, suggested that PufQ is specifically involved in the insertion of bacteriochlorophyll into developing spectral complexes and is not involved in the magnesium chelatase reaction.
A hint as to how carbon flow through the common pathway is reduced during aerobic growth was provided by a R. capsulatus bchH mutant. These mutants are unable to carry out the magnesium chelatase reaction and as a result cannot make bacteriochlorophyll. Interestingly, these mutants accumulate more protoporphyrin IX when grown under low oxygen than high oxygen, suggesting there is a point in the common tetrapyrrole pathway that is regulated by oxygen (11). Until recently, a likely candidate for this second control point was thought to be aminolevulinate synthase. The formation of aminolevulinate has been reported to be rate-limiting for the entire pathway and is generally considered to be the first committed step in tetrapyrrole biosynthesis (89).
Lascelles proposed that oxygen inhibits magnesium chelatase, resulting in the accumulation of protoporphyrin IX (22). Excess protoporphyrin IX would then be converted into heme by ferrochelatase. The rise in levels of heme would feedback inhibit aminolevulinate synthase reducing carbon flow over the common pathway. Evidence supporting this model has come from in vitro studies using purified aminolevulinate synthase from R. sphaeroides. The enzyme is inhibited by hemin, and to a lesser extent, magnesium protoporphyrin and protoporphyrin
(151). While aminolevulinate synthase activity in cultures ofR. sphaeroides vary five-fold with oxygen tension (85), the observed specific activity may not reflect the in vivo situation since the enzyme undergoes activation by endogenous trisulfides (113). However, aminolevulinate synthase specific activity in aerobically and anaerobically grown cultures of R. capsulatus is indistinguishable.
Transcriptional studies using a hemA-lacZfusion carried on a plasmid reveal only a two-fold change in hemA gene expression in response to changes in oxygen tension (144). These relatively small changes in enzyme activity and gene expression are not enough to account for the large increase in tetrapyrrole biosynthesis that accompanies anaerobic growth. The effects of exogenous aminolevulinate on a R. capsulatus bchH mutant further support the notion that aminolevulinate formation is not regulated by oxygen. Biel (10) has reported that 18
protoporphyrin levels are still subject to oxygen-mediated regulation in the
presence of exogenous aminolevulinate. However, whenbchH a mutant is
grown under high oxygen in the presence of exogenous porphobilinogen,
protoporphyrin accumulates to the level normally seen in anaerobically grown
cultures. This indicates that oxygen regulates the accumulation of
porphobilinogen. Perhaps the formation of porphobilinogen is the first true
committed step in tetrapyrrole biosynthesis. Supporting this idea is the
observation that only 20% of 14C aminolevulinate goes to make tetrapyrroles in
R. capsulatus (Biel, unpublished). While working with R. rubrum, Shigesada
(123) demonstrated that only 10% of the aminolevulinate is used for tetrapyrrole biosynthesis, while the rest is metabolized by a second metabolic pathway he termed the succinate-glycine cycle.
Recent reports inR. capsulatus and R. sphaeroides on the isolation of regulatory mutants deficient in photosynthetic gene expression has further complicated the picture as to the role oxygen plays in bacteriochlorophyll synthesis. Bauer (120) has isolated a regulatory protein, designated RegA, that positively activates gene expression of the light harvesting and reaction center polypeptides, but not the bacteriochlorophyll biosynthetic enzymes. Mutations in
RegA exhibit only a five-fold reduction in total bacteriochlorophyll content when growing anaerobically in the dark. Kaplan's group has isolated a similar protein, called PrrA, in R. sphaeroides (42). Interestingly, wild-type cells overproducing
PrrA actually induce the formation of photosynthetic clusters under aerobic 19
conditions, although not at the same level that occurs during anaerobic growth.
Penfold and Pemberton (106) have reported isolating a repressor of carotenoid
and bacteriochlorophyll synthesis they named PpsR. Mutations at this locus lead
to a five-fold increase in bacteriochlorophyll and carotenoid synthesis under
aerobic conditions. While these regulatory mutants are intriguing, they still exhibit
oxygen-mediated regulation of bacteriochlorophyll synthesis.
Experimental results indicate that porphobilinogen levels are subject to
oxygen regulation. In order to further investigate the mechanism by which
oxygen regulates porphobilinogen levels, theR. capsulatus hemB gene was
cloned and sequenced. Enzyme activity and gene expression were measured
under different oxygen tensions to evaluate the contribution that synthesis of
porphobilinogen may play in oxygen regulation. Expression of thehemB gene was measured by Dot blot analysis and by hemB-cat transcriptional fusion studies. The physical location of the hemB gene on theR. capsulatus chromosome was also determined. Materials and Methods
Strains and plasmids
Bacterial strains and plasmids used in this study are listed in Tables 1 and
2 .
Media
R. capsulatus was routinely grown in either a malate minimal salts medium
(RCV) (128) or 0.3% Bacto-Peptone-0.3% yeast extract(PYE; Difco Laboratories,
Detroit, Mich.). Escherichia coli was grown in Luria broth (9) modified by omitting glucose and decreasing the sodium chloride concentration to 0.5%. Solid media contained 1.5% agar (Difco). Antibiotics and other chemicals were added at the following concentrations (pg/ml): ampicillin 25; kanamycin 10; rifampicin 75; spectinomycin 10; streptomycin 10; chloramphenicol 25; tetracycline 10 forE. coli and 0.5 for R. capsulatus; 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside (X- gal) 40; lsopropyl-(3-D-thiogalactopyranoside 5. Stocks of bacterial strains were stored in 10% glycerol at -70°C.
Growth conditions
R. capsulatus and E. coli were routinely grown aerobically in the dark at
37°C with shaking. For growth of R. capsulatus under defined oxygen tensions, overnight cultures were subcultured into a 100 ml graduated cylinder containing
20 21
Table 1. Strains used in this study Designation Genotype Reference
E. coli
NM522 supE thi A(lac-proAB) Gough and Murray (53) hsd5 F[proA& lacF lacZLM15]
HB101 F' ara-14 proA2 lacY1 Boyer and Roulland- galK2 rpsL20 recA 13 Dussoix (18) xyl-5 mtl-1 supE44 hsdS20(rm)
SHSP1 thr-1 leuB6hemB1 ara- Sasarman and 13 thi-1 lacY1 gal-3 Horodniceanu (118) malA 1 xyl-7 myl-2 tonA2 supE44 Kr
R. capsulatus
PAS100 hsd-1 str-2 Taylor et al. (129)
SB1003 rif-10 Yen and Marrs (148)
Biel and Marrs (11) AJB456 CD (bcH'-lacZ)700 hsd-1 str-2 22
Table 2. Plasmids used in this study. Plasmid Relevent Characteristics Reference or discription
pBR322Q Apr, Tcr, Spr Omega cartridge cloned from pHP45Q (108) into Eco Rl site of pBR322.
pCM7 Apr, Tcr Close and Rodriguez (28) ,
pUC-4K Apr, Knr Vieira and Messing (138)
pKK223-3 Apr Brosius and Holy (21)
pUC18 Apr Vieria and Messing (138)
pRK2013 mob', tra+, Knr Ditta et al. (37)
pRK404 mob+, Tcr Ditta et al. (36)
pRPSB105 Apr, Knr Taylor et al. (129)
pCAP22 Apr,Tcr,Cmr 0.6kb Eco Rl generated DNA fragment from pCAP17 (13)cloned into pSUP202.
pCAP86 Apr pKK223-3 containing a 5.6 kb Eco Rl generated DNA fragment from R. capsulatus able to complement SHSP1. (This study)
Table 2 con't. 23 pCAP87 Apr pKK223-3 containing a 3.3 kb Pst i generated DNA fragment from f t capsulatus able to complement SHSP1. (This Study) pCAP103 Tcr 3.3 kb Pst I generated DNA fragment from pCAP87 cloned into pRK404. (This study) pCAP96 Apr, Strr 2.0 kb omega cartridge cloned into Eco RV site of pCAP87. (This study) pCAP118 Apr 3.3 kb Pst I generated DNA fragment from pCAP87 cloned into Pst I site of pUC18 (sequence generated from clone is the same direction as transcription). (This study) pCAP119 Apr 3.3 kb Pst I generated DNA fragment from pCAP87 cloned into Pst I site of pUC18 in the opposite orientation of pCAP118. (This study) pCAP142 Tcr, Smr, Spr Omega cartridge from pBR322Q cloned into the Hind III site of pCAP103. (This study)
Table 2 con't. 24 pCAP146 Apr, Cmr Chloramphenicol gene from pCM7 blunt-end ligated into Eco RV site of pCAP87. (This study)
pCAP147 Tcr Pst I generated DNA fragment from pCAP146 cloned into Pst I site of pRK404. (This study) pCAP148 Tcr, Smr, Spr Omega cartridge cloned into Hind III site of pCAP147. (This study) pCAP153 Chloramphenicol gene cloned into Bam HI site of pRK404. (This study) 25 fresh RCV media to an initial Klett value of approximately 20 (red filter) and grown for two generations under high versus low oxygen tension. Low oxygen conditions were obtained by sparging the culture with a mixture of 3% oxygen,
5% carbon dioxide, and 92% nitrogen, whereas high oxygen conditions were obtained by sparging the culture with compressed air supplemented with 3% oxygen. Cultures were incubated in a 37°C water bath.
Nucleic acid isolation
Plasmid minipreps were routinely isolated using QIAprep Spin Plasmid Kits
(Qiagen Inc., Chatsworth, CA). Large plasmid isolations were performed using the Wizard Maxiprep DNA purification system (Promega Inc., Madison, Wl). Both procedures are based on the modified alkaline lysis method of Birnboim and Doly
(139) and the adsorption of DNA to silica in the presence of high salt (26).
Chromosomal DNA was isolated from 100 ml overnight cultures ofR. capsulatus grown in PYE broth. Cells were harvested at 10,400 x g for 10 minutes and resuspended in 2.5 ml TES (50 mM Tris-CI, pH 8.0, 4 mM disodium ethylenediaminetetraacetic acid (EDTA), 1% NaCI). Proteinase K was added to a final concentration of 0.5 mg per ml. Ten percent sodium dodecyl sulfate
(SDS) in 1 M Tris-CI, pH 8.0 was added to a final SDS concentration of 1% and incubated 1 hour at 42°C. The suspension was repeatedly extracted with TES- saturated phenol followed by several extractions with chloroform-isoamyl alcohol
(24:1) until little or no white precipitate was seen. Pancreatic RNase (Sigma Co., 26
St. Louis, MO) was added to the aqueous phase at a concentration of 25 pg/ml
and incubated at 37°C for 30 minutes. Following incubation, the solution was
extracted once with an equal volume of chloroform-isoamyl alcohol (24:1) and
once with water-saturated diethyl ether until clear. An equal volume of
isopropanol was added and the solution was incubated 20 minutes at -20°C. The
solution was centrifuged at 9,750 x g for 20 minutes, and the remaining pellet was dried and resuspended in a minimal amount of water.
Total cellular RNA was isolated from 40 ml of R. capsulatus grown in RCV
broth to 80 Kletts. Cells were poured over an equal volume of -80°C buffer containing 50 mM Tris-CI, pH 7.5, 75 pg/ml rifampicin, 25 pg/ml chloramphenicol and harvested at 16,300 x g for 5 minutes. The cell pellet was resuspended in
4 ml of the same buffer containing 10 mg of lysozyme. After a 5 minute incubation at 4°C, 0.5 ml of 10% SDS was added and the solution was mixed by inversion. The suspension was extracted 2 to 3 times with an equal volume of
TES-saturated phenol followed by extraction 2 to 3 times with an equal volume of chloroform/isoamyl alcohol (24:1). Nucleic acids were precipitated by adding
0.5 ml 3 M sodium acetate, pH 4.8 and an equal volume of isopropanol and incubating the solution for 30 minutes at -20°C. After centrifugation at 9,750 x g for 30 minutes, the pellet was dried and resuspended in 1.5 ml 50 mM Tris-CI, pH 7.5, 1 mM EDTA, 10 mM magnesium chloride, 30 pg/ml DNase (Boehringer-
Mannheim, grade 1) and incubated at 37°C for 30 minutes. Following incubation, 27 the solution was extracted separately with an equal volume of water-saturated phenol and chloroform-isoamyl alcohol (24:1). The aqueous phase was divided into 3 equal portions and the RNA was precipitated from each by adding 3 M sodium acetate, pH 4.8, to a final concentration of 1 M and ethanol. RNA pellets were dried and stored at -80°C. All nucleic acids were quantified by measuring optical density at 260 nm using a PerkinElmer Lambda 3B UV/VIS spectrophotometer.
Restriction digests, iigations, nick translations, and other recombinant DNA techniques.
Restriction digests were routinely performed by digesting 1 pg of DNA in a 10 pi reaction consisting of 1pl (10U) of enzyme and 1 pi of the appropriate
10X buffer from New England Biolabs. Digest were incubated 1 to 2 hours at the optimal enzyme incubation temperature.
Ligation reactions were performed using T4 DNA ligase (New England
Biolabs, Beverly, MA) and incubated 4 to 16 hours at 16°C. A typical reaction consisted of 1 pg DNA, 2 pi 5X ligase buffer, 1 pi DNA ligase (400U), and water up to 10 pi.
Plasmid DNA was nick translated using the BRL nick translation kit (Life
Technologies, Grand Island, NY). Unincorporated nucleotides were removed by passing the DNA sample through a G-50 spin column (Worthington Biochemical
Corp., Freehold, NJ). A 2 pi sample was added to 5 ml of CytoScint scintillation 28 fluid and counts per minute were measured on a Beckman model LS6860 Liquid
Scintillation instrument.
Three prime recessed ends were made blunt using Klenow polymerase.
A typical fill-in reaction consisted of 1 pg DNA, 2 pi any 10X New England
Biolabs restriction enzyme buffer, 33 pM each deoxyribonucleotide, 1 unit Klenow polymerase (Promega Inc.), and water up to 20 pi. The reaction was incubated
15 minutes at 25°C and EDTA was added to a final concentration of 10 mM to stop the reaction. The polymerase was inactivated by heating the reaction to
75°C for 10 minutes.
Agarose gel electrophoresis
DNA was routinely fractionated through 0.8% agarose gels containing 0.5 pg/ml ethidium bromide using either the BRL Horizontal System Model H5 or the
Hoefer Gel Unit Model HE-33. A 50X stock solution of TAE (1X TAE = 0.04 M
Tris-acetate, 0.002 M EDTA) was diluted to either 1X or 4X to serve as electrophoresis buffer. Prior to electrophoresis, 1X loading buffer (0.25% bromothymol blue, 40% sucrose) was added to the DNA sample. 1X TAE agarose gels were run for 2 hours at 80 volts, whereas 4X TAE agarose gels were run overnight at 30 volts. Upon completion of electrophoresis DNA was visualized using a Fotodyne short wave UV light box and photographed with a
Polaroid camera. 29
Transformation
Transformations were performed based on the procedure by Kushner (83).
The E. coli strain was grown with vigorous shaking until a cell density of 5 x 107 cells/ml (O.D.5so«0.2) was achieved and 1.5 ml of culture was briefly spun in a microfuge tube. The cell pellet was gently resuspended in 1 ml of 100 mM morpholinopropane sulfonic acid, pH 6.5, 50 mM CaCI2, 10 mM RbCI and incubated on ice for 15 minutes. The solution was centrifuged for 30 seconds and the cell pellet was resuspended in 0.2 ml fresh buffer. Three microliters of dimethyl sulfoxide (DMSO) and up to 200 ng of DNA were added to the suspension. After 30 minutes incubation on ice, the cells were subjected to heat shock in a 43°C water bath for 1 minute. The suspension was immediately transferred to 1 ml of L-broth and incubated at 37°C for 1 hour without shaking.
A tenth of a milliliter of transformation mix was plated on the appropriate selective medium.
Electroporation
E. coli cells were grown in L-broth until early to mid log phase (O.D.66Q *
0.5-1.0) and transferred to a sterile 25 ml centrifuge tube where they were chilled on ice for 10 minutes. Following centrifugation for 10 minutes at 5,900g, x the cell pellet was washed 2 times with 10 ml of sterile cold water and once with 10 ml sterile cold 10% glycerol. The cells were resuspended in a final volume of
200 pi of 10% glycerol. Forty microliters of freshly prepared cells and 2 pg DNA 30 were added to a sterile 0.2 cm electroporation cuvette and mixed. The cell suspension was pulsed using the BioRad Gene Pulser set at 2.5 kV, 25 pF, and
200 ohms. Two milliters of L-broth were immediately added to the cuvette and transferred to a test tube to incubate for 1 hour at 37°C in a roller drum. Cells were plated out on the appropriate selective medium.
Matings
Triparental matings were performed as described below. E. coli donor strains and E. coli strain HB101, containing the helper plasmid pRK2013, were grown overnight in L-broth with slow shaking. RecipientR. capsulatus strains were grown overnight in PYE broth at 37°C with no shaking. One milliliter of the
R. capsulatus recipient strain and 200 pi of each of the E. coli strains were mixed together in a microfuge tube and centrifuged. The supernatant was decanted and the cell pellet was resuspended in 50 pi of medium which was spotted on a filter disc atop a PYE plate. The plate was incubated for 3-16 hours after which the plate was spread with the appropriate antibiotics and incubated at 37°C.
Southern analysis
DNA fragments were separated by electrophoresis through a 0.8% agarose gel after which the gel was trimmed to remove excess agarose. The gel was placed in a glass baking dish and covered with 1 M NaCI-0.5 M NaOH.
After soaking twice for 15 minutes with agitation, the solution was discarded and 31
the gel was rinsed with distilled water. The gel was then submerged in 1.5 M
NaCI-0.5 M Tris-CI, pH 7.0 and soaked twice for 15 minutes with agitation. After
being washed with distilled water, the gel was covered with 20X SSC (1X SSC
= 0.15 M NaCI, 0.015 M sodium citrate, pH 7.0) for 30 minutes. For transfer of
DNA fragments greater than 10 kb, the gel was first treated with 0.25 N HCI for
10 minutes prior to adding 1 M NaCI-0.5 M NaOH. DNA was transferred to
Nytran membranes using the S&S Turbo Blotter and Blotting Stack Assembly
(Schleicher & Schuell Inc., Keene, NH). Before transfer, Nytran membranes were
immersed in distilled water followed by a 5 minute soak in 20X SSC. Transfer
was complete within 3 to 7 hours. Following transfer, the blot was gently washed
in 2X SSC and baked for 1 hour at 80°C. Dried blots were stored in sealed
plastic bags. Prehybridizations and hybridizations were performed using the
Hybaid Micro-4 Hybridization Oven (National Labnet Company, Woodbridge, NJ).
Blots were first rolled in mesh and placed in glass bottles where they were briefly
rinsed with 2X SSC. Prehybridizations were carried out at 65°C for 4 hours to
overnight in 5X SSC, 1% SDS, 10X Denhardts (1 g ficoll, 1 g
polyvinylpyrrolidone, 1 g BSA, Pentex fraction V), and 0.5 mg/ml denatured salmon sperm DNA. Hybridizations were carried out for 18 to 24 hours at 65°C
in 5X SSC, 1% SDS, 2X Denhardts, and 0.1 mg/ml denatured salmon sperm
DNA. Both prehybridizations and hybridizations required 0.1 ml solution per cm2
blot. Washes were done for 30 minutes in 2X SSC-1% SDS at room temperature,
30 minutes in 0.5X SSC-1% SDS at room temperature, and 30 minutes in 0.5X 32
SSC-0.1% SDS at 65°C. The blots were allowed to air dry and exposed to
BioMax MR X-ray film overnight.
Dot blot analysis
RNA pellets were resuspended in 7.4 pi water and denatured in 5.4 pi 6
M glyoxal, 16 pi DMSO, 3.2 pi 0.1 M sodium phosphate for 1 hour at 50°C. After denaturation the solution was chilled on ice and 800 pi of cold 10 mM sodium phosphate, pH 7.0 was added to the samples. Serial dilutions of RNA samples were blotted onto Zeta-Probe (BioRad Laboratories, Hercules, CA) membranes using the BioRad Dot Blot apparatus. The membranes were baked at 80°C for
1 hour and stored in a sealed plastic bag. Prior to prehybridization, blots were washed in 0.1X SSC-0.5% SDS for 1 hour at 65°C. Prehybridizations were carried out at 65°C for 4 hours to overnight in 5X SSC, 50 mM sodium phosphate, pH 6.5,10X Denhardts, and 1 mg/ml denatured salmon sperm DNA.
Hybridizations were carried out for 18 to 24 hours at 65°C in 5X SSC, 25 mM sodium phosphate, pH 6.5, 2X Denhardts, and 0.1 mg/ml denatured salmon sperm DNA. Both prehybridizations and hybridizations were done in 0.1 ml of solution per cm2 of membrane. The blots were washed twice with 500 ml of 2X
SSC-0.1% SDS at room temperature, and once with 500 ml of 2X SSC-0.1%
SDS at 55°C for 1 hour. Blots were air dried and exposed to BioMax MR X-ray film overnight. 33
DNA sequencing
Nucleotide sequencing was performed using the New England Biolabs
Circumvent Kit based on the dideoxynucleotide chain termination method of
Sanger (114). Double stranded plasmid templates were extended using a highly thermostable Ventf (exo ) DNA polymerase. The dideoxy terminated chains were labeled by the incorporation of [a-35S]ATP into the nascent chains.
Sequencing reactions were incubated in a Hybaid Thermocycler (National Labnet
Laboratories, Woodbridge, NJ) for 25 cycles (1 cycle = 95°C for 1 min, 55°C for
1 min, 72°C for 1 min). The DNA products were separated by electrophoresis through a denaturing 6% polyacrylamide gel at 60°C using the Polar Bear
Isothermal Electrophoresis system (Owl Scientific, Woburn, NJ). Upon completion of electrophoresis, the gel was fixed in 20% ethanol-10% acetic acid and dried to 3M Whatman paper for 2.5 hours using BioRad gel drying system.
Sequencing gels were exposed 1 day using BioMax X-ray film. Unidirectional nested deletions used in sequencing were constructed with the Erase-a-Base system by Promega. This system is based on the procedure developed by
Henikoff (61) in which exonuclease III is used to digest DNA from a blunt or 5' protruding end while leaving the 3' protruding end intact.
Pulsed Field Gel Electrophoresis (PFGE)
Five milliliters of overnight cultureR. of capsulatus SB1003 (O.D.66Q® 1.0) was pelleted at 3,020 x g for 6 minutes and resuspended in 5 ml 50 mM EDTA, 34 pH 8.0. The cells were pelleted and resuspended in 0.5 ml 50 mM EDTA, pH 8.0 and 0.5 ml 1.6% low melting point agarose cooled to 55°C. A pasteur pipette was used to transfer the solution into a section of tubing (Nalgene; Rochester,
NY) approximately 24 inches long having an internal diameter of 1/16 of an inch. After the agarose solidified, the embedded cells were extruded from the tubing into 5 ml of 50 mM EDTA, pH 8.0 and allowed to incubate for 30 minutes at room temperature. The liquid was decanted and 5 ml of 3% lauryl sarcosine-
0.2 M EDTA, pH 8.5, and 5 mg proteinase K were added and the suspension was incubated overnight at 55°C . The agarose 'plugs' were washed 2 times with
5 ml of 50 mM EDTA, pH 8.0 for 30 minutes. Two hundred microliters of 50 mM
EDTA, pH 8.0 and 2 pi 100 mM phenylmethylsulfonyl fluoride (PMSF) were added and the solution was incubated for 1 hour. The plugs were rinsed twice with 5 ml of 50 mM EDTA, pH 8.0 for 30 minutes at room temperature to remove all traces of PMSF. One centimeter pieces of DNA plugs were placed in a microfuge tube containing 50 pi TE (10 mM Tris-CI, pH 8.0, 1mM EDTA, pH 8.0) and incubated on ice for 20 minutes. The TE solution was removed and the DNA was rinsed twice with 50 pi of the appropriate restriction enzyme buffer for 30 minutes on ice. The buffer was removed and 50 pi of buffer solution containing the appropriate enzyme was added to the plug and incubated on ice for 30 minutes. Prior to PFGE, the 1 cm DNA plug was sealed in the well of a 1% agarose gel with 1% low melting point agarose. The BioRad CHEF system was used for electrophoresis set at the following conditions: switching intervals 8 to 35
16 seconds, voltage 200, temperature 13°C, run time 24 hours in 0.5X TBE (1X
TBE = 0.089 M Tris base, 0.089 M boric acid, 0.002 M EDTA, pH 8.0) buffer.
Protein Determination
The protein concentration of cell extracts used for various enzyme assays was determined using the Bradford Dye-binding assay (BioRad) (19). A standard curve was constructed using purified bovine serum albumin (Pentex).
Porphobilinogen synthase assay
Porphobilinogen synthase activity was assayed based on the method developed by Shemin (122). One hundred milliliters ofR. capsulatus cells grown to 75 to 100 Kletts in RCV media were harvested and resuspended in 1 ml of 0.1
M potassium phosphate buffer, pH 7.6 with 0.1 M (3-mercaptoethanol. The cells were sonicated, under aerobic conditions, 2 to 4 times for 20 seconds with 30 second cooling periods using a Heat-Systems Ultrasonics sonifier (Model w-220).
The extract was centrifuged for 20 minutes at 27,000 x g. One hundred microliters of crude extract was added to 150 pi 1 M Tris-CI, pH 8.5, 75 pi 1 M
KCI, 50 pi 0.1 M aminolevulinate, pH 7.0, 0.6 pi 3-mercaptoethanol, 1.125 ml water and incubated for 30 minutes at 37°C. Following incubation, 2 ml of 0.5
M 20% TCA-0.1 M HgCI2 was added and the solution was spun for 2 minutes.
Two milliliters of modified Ehrlich's reagent (0.5 g p-dimethyllarninobenzaldehyde,
17 ml glacial acetic acid and 8 ml 70% perchloric acid) was added to the 36 supernatant and incubated at room temperature for 5 minutes. The optical density was determined at 553 nm. The specific activity was calculated using a molar extinction coefficient of 2.29 X 104 M'1 cm'1.
Chloramphenicol acetyltransferase assay
Chloramphenicol acetyltransferase assays were performed as described by Shaw (121). One hundred milliliters of R. capsulatus cells grown to 75 to 100
Kletts in RCV were harvested and resuspended in 1 ml of 50 mM Tris-CI, pH 7.8.
The cells were sonicated 3 to 4 times for 20 seconds with 30 second cooling periods. Cell debris was removed by spinning the extract at 27,000 x g for 20 minutes. Two hundred and fifty microliters of crude extract was added to 250 pi of 4 mg/ml 5,5'-dithiobis-(2-nitrobenzoic acid) in 1 M Tris-CI, pH 7.8, 50 pi 5 mM acetyl CoA, 1.950 ml water and the mixture was divided into two 1 ml samples.
The samples were blanked in the spectrophotometer and 10 pi of 5 mM chloramphenicol was added to one of the cuvettes. The change in optical density at 412 nm was determined. The specific activity was calculated using a molar extinction coefficient of 1.36 X 104 M'1 cm'1.
Porphyrin determination
Protoporphyrin and coproporphyrin were extracted from 1 to 5 ml of culture with 3 ml ethyl acetate-acetic acid (3:1). The bottom layer was removed and the remaining ethyl acetate layer was extracted twice with 3N HCI. Fluorescence of 37
the acid layer was determined using a Perkin-Elmer LS-3 fluorescence
spectrophotometer. Porphyrin concentrations were calculated by comparing fluorescence values to curves constructed using protoporphyrin and coproporphyrin standards (Porphyrin Products, Logan, Utah).
Thin layer chromatography
Total porphyrins were extracted into ethyl acetate-acetic acid (3:1) and spotted onto a Silica Gel G TCL plate (Fisher). Extracted porphyrins, coproporphyrin, and protoporphyrin standards (Porphyrin Products, Logan, Utah) were characterized by thin layer chromatography. The plates were developed in
2,6 lutidine-0.05N ammonium hydroxide (7:2) (54) and viewed under long-wave
UV light. Porphyrins were identified by their comigration with the appropriate standard. Results
Cloning of theR. capsulatus hemB gene
Chromosomal DNA from R. capsulatus strain PAS100 was isolated as described in Materials and Methods and digested to completion with either Eco
Rl or Pst I. The DNA was ligated into the expression vector pKK223-3, which contains the tac promoter and confers ampicillin resistance. The ligation mixture was transformed into E. coli strain SHSP1(/7emBf) by electroporation. Because
SHSP1 is Hem' it requires a fermentable carbon source, such as glucose, for growth. SHSP1 was routinely cultured in L-broth containing 0.2% glucose and
0.5% NaCI. It was, therefore, possible to select for HemB+, ampicillin-resistant colonies by plating the transformed cells on L-agar without glucose in the presence of 25 pg ampicillin per ml. HemB+, ampicillin-resistant transformants were obtained from both the Pst I and Eco Rl plasmid libraries. Hem+colonies, originating from either the Pst I or Eco Rl plasmid libraries, were picked and grown in broth for plasmid isolation. Those plasmids isolated from Hem+colonies, arising from the Pst I plasmid library, had identical restriction enzyme patterns when digested with Pst I, Eco Rl, or Bam HI and electrophoresed through a 0.8% agarose gel. One clone was chosen to work with and the plasmid from that clone was designated pCAP87. Those plasmids isolated from Hem+ colonies, arising from the Eco Rl plasmid library, had identical restriction enzyme patterns when digested with Eco Rl, Pst I, or Bam HI and electrophoresed through a 0.8%
38 39 agarose gel. One clone was selected to work with and the plasmid from this clone was named pCAP86. Plasmid DNA isolated from SHSP1(pCAP86) or
SHSP1 (pCAP87) was used to transform strain SHSP1 to ampicillin resistance.
All of the resulting colonies from either transformations were HemET, demonstrating that both plasmids carried the R. capsulatus hemB gene. In addition, SHSP1(pCAP86) and SHSP1(pCAP87) both exhibited porphobilinogen synthase activity, whereas SHSP1 showed minor levels of porphobilinogen synthase activity (Table 3).
Restriction analysisof the hemB region
Restriction digest of pCAP86 with Eco Rl revealed that pCAP86 contained a 5.6 kb DNA fragment cloned into pKK223-3. Restriction digest of pCAP87 with
Pst I revealed that pCAP87 contained a 3.3 kb DNA fragment cloned into pKK223-3 (Fig. 2). The restriction maps of the two plasmids indicate that they have a 3.0 kb Eco Rl-Pst I generated DNA fragment in common, suggesting they are from the same region of the chromosome. A restriction enzyme map of the
DNA inserts in either pCAP86 or pCAP87 indicated that both inserts had a different restriction enzyme map than theE. coli hemB DNA region.
The approximate location of thehemB gene was determined by subcloning various fragments from pCAP87 into pUC18. Plasmids containing either the Eco
Rl-CIa I generated DNA fragment (pCAP108) or the Nsp V-Bam HI generated
DNA fragment (pCAP111) were able to complement SHSP1, indicating that the 40
Table 3. Porphobilinogen synthase activity in strains containing hemB* plasmids.
Strain Porphobilinogen synthase Specific activity3 SHSP1 4.4 SHSP1(pCAP86) 100 SHSP1(pCAP87) 49 aSpecific activity expressed as nmoles of porphobilinogen formed per hr per mg protein at 25°C. Results are representative of at least two trials. pCAP86 EN v v B Bg L-L- -i—L- — L_
pCAP87
N VV C B pCAP111 i_____u i_____ i_____ I
EN VV C pCAP108 U ____ L-J___ ,___ 1
P EN VV C B P pCAP96 I L-J bed I I I Q
1 kb
Figure 2. Restriction map of plasmids containing the R.capsulatus hemB gene. Restriction enzyme cleavage sites are Bam HI (B), Cla I (C), Eco Rl (E), Eco RV (V), Nsp V (N), and Pst I (P). The omega cartridge (Q) is not drawn to scale. 42 hemB gene is located in the 1.4 kb region between the Nsp V site and the Cla
I site. This was confirmed by digesting pCAP87 with Eco RV and replacing the
Eco RV DNA fragment with the omega cartridge. The resulting plasmid, pCAP96, does not complement SHSP1 (Fig. 2).
The orientation of the DNA inserts in pCAP87 and pCAP86, with respect to thetac promoter, is also an important factor in determining whether or not the plasmid can complement SHSP1. The orientation of the DNA inserts in pCAP86 and pCAP87 put the Nsp V restriction enzyme site proximal to thetac promoter.
To further investigate this, the Pst I generated DNA fragment from pCAP87, was cloned into pUC18 in both orientations and named pCAP118 and pCAP119.
Only pCAP118, which contains the Pst I generated DNA fragment oriented such that the Nsp V site is proximal to the lac promoter, was able to complement
SHSP1. These results suggests that theR. capsulatus hemB gene is only expressed in E. coli with the help of an exogenous promoter, and thathemB transcripton proceeds from left to right as diagrammed in Fig. 2. The possibility exists that the Pst I generated DNA fragment from pCAP87 does not contain the promoter region of thehemB gene. Alternatively, theR. capsulatus hemB gene promoter may not be recognized by E. coli's DNA polymerase.
Expression of pCAP142(/)em£T) in SB1003
The 3.3 kb Pst I generated DNA fragment, from pCAP87, was subcloned into the broad host vector pRK404. The omega cartridge was placed upstream 43
from the Pst I generated DNA fragment to prevent readthrough transcription from the plasmid into the Pst I DNA fragment. The orientation of the omega cartridge, with respect to the Pst I generated DNA fragment, was such that the direction of transcription was extending away from the omega cartridge. This construct, pCAP142, was mated into SB1003 in order to determine whether the Pst I fragment carries the hemB control region. SB1003 and SB1003(pCAP142) were both grown under high and low oxygen tensions and porphobilinogen synthase specific activities determined as described in Materials and Methods. The results are summarized in Table 4. SB1003(pCAP142) has nearly a five-fold higher porphobilinogen synthase activity than the wild-type strain under both high and low oxygen tensions. These results indicate that the Pst I DNA fragment can express porphobilinogen synthase without the help of an exogenous promoter.
In addition, the results show that porphobilinogen synthase specific activity is 1.4- fold higher in cultures grown under low oxygen. The small change in enzyme activity is not enough to account for the large increase in porphyrin synthesis that accompanies anaerobic growth.
Southern analysis
To confirm that no rearrangements occurred during the cloning process and that there was only one copy of the hemB gene in theR. capsulatus chromosome, Southern analysis was performed. Chromosomal DNA fromR. capsulatus stain PAS100 was digested with either Eco Rl or Pst I, separated on Table 4. Overexpression of porphobilinogen synthase in SB1003.
Specific activity3 Strains 23% Oxygen 3% Oxygen SB1003 280 420 SB1003 (pCAP142) 1300 1900 aSpecific activity expressed as nmoles of porphobilinogen formed per min per mg protein at 25°C. Results are representative of at least two trials. a 4X TAE 0.8% agarose gel, and blotted to nitrocellulose. The blots were probed with either pCAP86 or pCAP87 that had been nick-translated. The blot of chromosomal DNA digested with Eco Rl, when probed with pCAP86, yielded a single band of 5.6 kb (Fig. 3 A). This size is consistent with the size of the insert in pCAP86. To confirm that the inserts in pCAP86 and pCAP87 overlap, pCAP86 DNA was digested with Eco Rl, blotted to nitrocellulose, and probed with pCAP87. The pCAP87 hybridized to the 5.6 kb band from pCAP86, indicating that the two plasmids carry DNA from the same region of the R. capsulatus chromosome (Fig. 3 B). In addition, pCAP87 hybridized to the 4.6 kb vector band and a large partially digested band corresponding to the whole plasmid.
Chromosomal location of theR. capsulatus hemB gene
A physical map of the genome ofR. capsulatus SB1003 constructed by
Fonstein et al. (45) made it feasible to map the location of the hemB gene.
Chromosomal DNA from strain SB1003 was digested en bloc with Ase I and Xba
I as described in Materials and Methods. The DNA fragments were separated using the BioRad CHEF Mapper Pulsed-Field Electrophoresis System (Fig. 4 A).
The DNA fragments were blotted to Nytran membranes using the Schleicher and
Schuell Turbo Blotter and probed with nick-translated pCAP87. The hemB gene was localized to Ase I fragment 2 and Xba I fragment 3' (Fig. 4 B). The chromosomal location of theR. capsulatus hemA gene was also determined using pCAP22 as a probe. The hemA gene was localized to Ase I fragment 5 46
A B
5.6kb o 4.6kb o
Figure 3. Southern analysis. A. PAS100 chromosomal DNA digested with EcoRI and probed with pCAP86. B. pCAP86 DNA digested with Eco Rl and probed with pCAP87. 47
A B
1 2 3 4
m im O270kb
Figure 4. A. PFGE of SB1003 chromosomal DNA digested with Ase I (lanel) and Xba I (Iane2). B. pCAP113(hemB) hybridized to a 370kb Ase I fragment (Ian3) and a 350 kb Xba I fragment (Iane4). 48
and Xba I fragment 3 (Fig. 5). The results of these mapping experiments,
summarized in Figure 6, indicate that the hemA and hemB genes do not
comprise an operon.
Sequencing of the R. capsulatus hemB gene
Nested unidirectional deletions were made from pCAP118 and pCAP119
using the Promega Erase-A-Base system. The nucleotide sequence of the Nsp
V-CIa I region was determined in both directions using the New England Biolabs
CircumVent kit (Fig. 7) (64). Analysis of this nucleotide region indicates that
there is a single open reading frame 995 nucleotides long. Codon preference
analysis, using the R. capsulatus codon preference table (150), shows that this
reading frame contains few rare codons (Fig. 8). The ATG codon beginning at
position 211 is the most likely candidate for a start codon, based on the codon
preference profile and the presence of a putative ribosome binding site upstream.
The ATG codons at positions 152 and 256 lack good ribosome binding sites, and the codon at position 256 is within a region conserved in the coding regions of several hemB genes. The open reading frame starting at position 211 encodes a protein with a molecular mass of 43,068 daltons, in agreement with a report of 40,000 daltons for the R. capsulatus enzyme (102). 49
C>350kb
<^220kb
Figure 5. A. PFGE of SB1003 chromosomal DNA digested with Ase I (lanel) and Xba I (Iane2). B. pCAP22(hemA) hybridized to a 220kb Asel fragment (Iane3) and a 350 kb Xba I fragment (Iane4). 50
gene was located was gene located bch hemE genes on a on genes of map physical hemE hemA 3.7 Mb and and R. R. capsulatus hemA hemB hemB restriction map along with the location of the bchregion was the bchregion of the location with along map restriction by Ineichen and Biel (63). Biel and by Ineichen SB1003. Ase I (a) and Xba I (x) restriction sites are labled. are labled. sites restriction (x) I Xba and The Ase (a) I SB1003. (45). et al. by Fonstein constructed The
CD CD Figure 6. Location of the the of 6. Location Figure 1 AGGCGATCCA ATACCAGAGG TTCGAAAACG ACCGCATGTC GATCAGCTCG AAGATCGTTT 61 GCAATGTCCA AGGTCTTGCC CGCCCGTTGC CTGCCCTTGG TTTCGCGCAT CGGCCGCCCG 121 GTTGCAAGCA TGCCTTCGCG CGGGCGCGAC AATGCGGGGC TTGAGCCGGT CTGCGATCCG 1 8 1 TGGCACCCAC GGCGACGAAG GAGACCGCCC ATGACCCTGA TCACCCCCCC CTTTCCCACC MTLI TPP FPT
2 4 1 AATCGCCTGC GCCGGATGCG' CCGCACCGAA GCCCTGCGTG ACCTCGCGCA GGAAAACCGG N R L R RMR RTE ALRD L A Q ENR
3 01 TTGTCGGTCA AGGACCTGAT CTGGCCGATC TTCATCACCG ACGTTCCCGG CGCCGATGTC LSVK D L I WPI FITD VPG ADV
361 GAGATCTCCT CGATGCCGGG GGTTGTCCGC CGCACCATGG ACGGCGCGCT GAAAGCGGCC EISS MPG V V R RTMD GAL K A A
421 GAGGGAAGCC GCGACGCTGG GCATTCCCGC GATCTGCCTG TTCCCCTCAC CGATCCGGCG EGSR DAG HSR DLPV PLT D P A
481 GTGAAGACCG AGACCTGCGA AATGGCCTGG CAGCCCGACA ACTTCACCAA CCGGGTGATC VKTE TCE MAW QPDN FTN RVI
541 GCGGCGATGA AGCAGGCGGT GCCCGAGGTC GCGATCATGA CCGATATCGC GCTCGATCCC AAMK Q A V PEV AIMT DIA LDP
601 TATAACGCCA ACGGCCATGA CGGGCTTGTC CGCGACGGCA TACTCCTGAA CGACGAAACC YNAN GHD GLV RDGI LLN DET
661 ACCGAGGCGC TGGTGAAGAT GGCGCTCGCC CAGGCCGCGG CCGGGGCCGA CATCCTTGGG TEAL VKM ALA Q A A A GAD ILG
721 CCGTCCGACA TGATGGATGG CCGCGTGGGC GCGATCCGGC AGGCGATGGA GGCCGCGGGT PSDM MDG RVG AXRQ AME A A G
781 CACAAGGATA TCGCCATTCT CAGCTATGCC GCGAAATATG CCTCGGCCTT CTATGGCCCG HKDI AIL S Y A AKYA SAF YGP
841 TTCCGCGATG CGGTCGGCGC CTCAAGTGCG CTCAAGGGCG ACAAGAAGAC CTATCAGATG FRDA V GA SSA LKGD KKT Y Q M •
901 AACCCCGCCA ACAGCGCCGA GGCCCTGCGC AATGTCGCCC GCGACATCGC CGAGGGCGCC NPAN SAE ALR NVAR DIA EGA
961 GACATGGTGA TGGTGAAACC GGGCATGCCC TATCTCGACA TCGTGCGTCA GGTGAAGGAT DMVM VKP GMP YLDI VRQ VKD
1021 GCCTTTGGCA TGCCGACCTA TGCCTATCAG GTCTCGGGCG AATATGCGAT GCTGATGGCG AFGM PTY A Y Q VSGE YAM L M A
1081 GCGGTGCAGA ATGGCTGGCT CAACCATGAC AAGGTGATGC TGGAAAGCCT GATGGCCTTC AVQN GWL NHD KVML ESL MAF
1141 CGCCGCGCCG GCTGCGATGG CGTTCTGACC TATTTCGCTC CCGCGGCGGC GAAGCTGATC RRAG CDG VLT YFAP AAA KLI
1201 GGCGCCTGAC CGCGCATCGG CTGCCCTTGG CCGCGGGCGC AAGTGACAAG CGCCCGCGCT G A
1261 GCGCTAATCT CGCCCCAACC
Figure 7. Nucleotide sequence ofR. the capsulatus hemB gene. 52
Figure 8. Codon Preference Plot of the region containinghemB the gene.
The codon preference profile hemBfor is reflected in the top panel.
Open reading frames are designated as uninterruped boxes beneath the plot in the panel. Rare codons are shown as vertical hatch marks in the box below each panel. The dashed line in each panel represents the rare codon threshold. A plot above this line signifies a region of DNA with few rare codons. The numbering system along the top and bottom of the figure coincide with the DNA sequence presented in Figure 7. 0 500 1,000
0.5 •
-0.0 « 1 —...... 2.0
1.5
1.0
0.5 «
-0.0
0.5
- 0.0
0 500 1,000
(Jl CO 54
Regulation of the R. capsulatus hemB gene
Dot blot analysis
Transcriptional regulation of the hemB gene by oxygen was examined by
Northern analysis. RNA was isolated from cultures of R. capsulatus strain
PAS100 grown under high and low oxygen tensions. Serial dilutions of the RNA samples were blotted onto Zeta-Probe membranes (BioRad), using a dot-blot apparatus, and hybridized to pCAP113. This construct contains the 0.6 kb Nsp
V-Eco RV generated DNA fragment from pCAP87, cloned into pUC18. This
DNA fragment is entirely within the coding region of thehemB gene. As a control for the amount of mRNA in the sample, the same serial dilutions were hybridized to pRPSB105. This plasmid carries the crtB gene which is not transcriptionally regulated by oxygen (27). Figure 9 is a representative example of the autoradiograms generated from the dot blot experiments. To quantitate hybridization, autoradiograms were scanned with a BioRad Video densitometer
Model 620. The differences in mRNA from high to low oxygen were normalized for the crtB gene and a correction factor was applied to hemB mRNA. For example, crtB mRNA was determined to be four-fold higher under high oxygen when compared to low oxygen. HemB mRNA was also determined to be four fold higher under high oxygen. After applying a correction factor of four tohemB mRNA under low oxygen, thehemB mRNA was the same under high and low oxygen. The results of the dot-blot experiments indicate thathemB mRNA do not change with oxygen tension. 55
crtB hemB
H L H L
• • WK % •
• • • * • • • • • •
M • •
Figure 9. Dot blot analysis. pRPSB105 {crtB) and pCAP113 (hemB) were hybridized to serial dilutions of total cellular RNA extracted from cells grown under high (H) oxygen and low (L) oxygen. 56
Construction of a hemB-cat transcriptional fusion plasmid
A hemB-cat transcriptional fusion plasmid was constructed in order to further study transciptional regulation (Fig. 10). The Eco RV generated DNA fragment in pCAP87 was replaced by the promoterless chloramphenicol acetyltransferase gene from pCM7 via a blunt-end ligation. The Eco RV sites are
both within the hemB coding region, therefore this placed the coding region of the
cat gene under control of thehemB promoter. The Pst I fragment, containing the hemB-cat fusion, was subcloned into the mobilizable plasmid pRK404. To prevent upstream transcriptional interference from the lac promoter, the omega cartridge was cloned into the Hind III site upstream of the hemB-cat fusion. This plasmid was designated pCAP148.
A second plasmid was constructed as a control for differences in plasmid copy number and supercoiling. The plasmid, designated pCAP153, is pRK404 with the chloramphenicol acetyltransferase gene cloned into the Hind III site.
Each plasmid was transformed into NM522 and moved into SB1003 via a triparental mating.
Effects of oxygen on transcription of thehemB-cat fusion
Strain SB1003(pCAP148) was grown in RCV media for two generations with initial oxygen tensions of 3% and 23%. Cultures grown with an oxygen tension of 3% exhibited a 1.4-fold higher chloramphenicol acetyltransferase .Hindi!! Pstl EcoRI .NspV
H ind lll. hemB' cat .Clal lacP
'hemB ..Bam HI
pCAP148 ,.Pstl 16900 bps
tet
Figure 10.HemB-cat fusion plasmid constructed to study transcriptional regulation by oxygen. activity than that found in the culture grown under 23% oxygen (Table 5). As a control for differences in the plasmid, e. g., copy number and supercoiling, that may accompany a shift in oxygen tension, chloramphenicol acetyltransferase activity was also determined in SB1003(pCAP153). Under low oxygen tension the chloramphenicol acetyltransferase specific activity was 1.4-fold higher than under high oxygen. After correcting for differences in the plasmid these results show that chloramphenicol acetyltransferase specific activity from pCAP148 is the same under high and low oxygen tensions.
Overexpression of thehemB gene in AJB456
It has been previously demonstrated that the addition of exogenous porphobilinogen to cultures of AJB456, growing under high and low oxygen, can eliminate oxygen-mediated regulation of protoporphyrin accumulation (10). These results suggest that the level of porphobilinogen is regulated by oxygen and that high levels of exogenous porphobilinogen can overcome this regulation. pCAP142 was moved into AJB456 in an attempt to mimic the above experiment in vivo by increasing endogenous levels of porphobilinogen. Strain
AJB456(pCAP142) was grown with initial oxygen tensions of 23% and 3% as described in Materials and Methods. At 150 Kletts Units, 5 ml samples were removed and porphyrins were extracted and quantified as described in Materials and Methods. As a control, AJB456 was also grown under high and low oxygen and porphyrins were extracted and quantified. These results (Fig. 11) illustrate 59
Table 5. Effects of oxygen on chloramphenicol acetyltransferase activity.
Specific activity3 Strains 23% Oxygen 3% Oxygen SB1003(pCAP148) 16 23 SB1003(pCAP153) 33 46 aSpecific activity expressed as nmoles of chloramphenicol acetylated per min per mg protein at 25°C. Results are representative of at least two trials. Figure 11. Figure [Protoporphyrin] (nmol) 2000 1000 1500 500 0 Protoporphyrin Shadedaccumulationbarsrefer toin AJB456. of AJB456(pCAP142)+ ALA whichis fromasingle determination. culturesgrown anwith initial oxygen tension of 3%, whereas,open barsrefer tocultures grown withan initial oxygen tensionof 23%. Results arerepresentative of leastat two trials,withexceptionthe J46 J46 J46 AJB456 AJB456 AJB456 AJB456 pA12 +L (PCAP142) +ALA (pCAP142) 1900 +ALA 1000
60 61
that pCAP142 has no effect of protoporphyrin accumulation in AJB456. To
ensure that pCAP142 was being expressed in AJB456, porphobilinogen synthase
assays were performed. Porphobilinogen synthase specific activity was
approximately five-fold higher under both high and low oxygen in the strain
containing pCAP142 (Table 6). Together these results suggest that the enzyme
is being expressed but there is no substrate present for it to act on. To test this
idea, AJB456(pCAP142) was grown in the presence of exogenous
aminolevulinate under high and low oxygen and porphyrins were extracted and
quantified. As a control, AJB456 was grown in the prepense of exogenous
aminolevulinate. In the prepense of exogenous aminolevulinate, AJB456
accumulates more protoporphyrin under low oxygen than high oxygen confirming
earlier results (Fig. 11) (10). Interestingly, exogenous aminolevulinate stimulates
porphyrin accumulation under both high and low oxygen in AJB456.
AJB456(pCAP142) in the presence of aminolevulinate accumulates even larger
amounts of porphyrins under high and low oxygen. In addition, oxygen-mediated
accumulation of porphyrins has changed, with even more porphyrins being accumulated under high oxygen than under low oxygen. Thin layer chromatography revealed that large amounts of coproporphyrin and
protoporphyrin were accumulating. These results support the notion that the level of porphobilinogen is affected by oxygen and that increasing the endogenous
levels of porphobilinogen overrides this effect. 62
Table 6. Overexpression of porphobilinogen synthase in AJB456.
Specific activity3 Strains 23% Oxygen 3% Oxygen AJB456 174 241 AJB456(pCAP142) 934 1078
aSpecific activity expressed as nmoles of porphobilinogen formed per min per mg protein at 25°C. Results are representative of at least two trials. Discussion
In 1957, Cohen-Bazire et al. (29) were the first to present evidence of an efficient mechanism for the regulation of tetrapyrrole biosynthesis. Since that time, a considerable effort has been dedicated to the identification of the major points of oxygen regulation in tetrapyrto.e biosynthesis. Oxygen is believed to control the pathway leading to bacteriochlorophyll at two points: the formation of magnesium protoporphyrin monomethyl ester from protoporphyrin IX, and the formation of porphobilinogen from aminolevulinate. So far, the mechanisms by which oxygen regulates tetrapyrrole biosynthesis at these points are unknown.
To investigate the mechanism by which oxygen regulates the formation of porphobilinogen, theR. capsulatus hemB gene was cloned and sequenced.
The codon preference profile of the 1281 nucleotide region sequenced suggests that there is only one possible open reading frame (Fig. 8, panel 1).
The putative amino acid sequence of porphobilinogen synthase from R. capsulatus shares an overall identity of 43% to 54% with porphobilinogen synthases from several different sources (15, 20, 25, 59, 90, 119). Within the coding region of theR. capsulatus protein, there are several regions that are highly conserved in relation to other known porphobilinogen synthases. In particular, the invariant lysine residue at position 256, proposed to be involved in
Schiff-base formation within the active site (49), and the arginine residue at position 306, thought to be important in catalysis (91), are present in all
63 porphobilinogen synthases genes sequenced. The presence of a metal-binding domain is another highly conserved feature among porphobilinogen synthases.
Most porphobilinogen synthases can be placed into two alternative groups depending on whether Zn++ or Mg++ is required for activity. The role of the metal ion is still unknown, but it is thought to have a structural and/or mechanistic function in protecting the sulfhydryl groups from oxidation or in catalysis (71).
The mammalian enzymes are zinc metalloenzymes (71). They possess a metal binding domain which consist of four cysteine residues, thought to be oxygen sensitive, and two histidine residues. (Fig. 12) (134). Bacteria also contain this motif except that one of the conserved cysteines is missing. In the caseE. ofcoli it has been shown that Zn++ is required for catalytic activity (95).
The corresponding metal binding domain in the plant enzymes resembles that of the nonplant enzymes except that the cysteines have been replaced with a threonine, alanine, and three aspartic acid residues thought to bind magnesium
(Fig. 12) (15). The lack of cysteine in the ion binding center correlates well with the fact that the enzyme is not blocked by sulfhydryl reagents (91).
The R. capsulatus enzyme metal binding domain closely resembles the magnesium binding domain found in plants, except an asparagine residue has replaced the conserved aspartate at position 134 (Fig. 12). Interestingly, it has been reported that theR. capsulatus enzyme does not have a metal requirement
(102). Perhaps by removing the negatively charged aspartate the metal ion is no longer needed. 65
Figure 12. Comparison of the metal-binding domain of R. capsulatus porphobilinogen synthase to those of plants and nonplants.
The R. capsulatus sequence shown corresponds to amino acids 120 to 141. Sequences from soybean (25), pea (15), spinach (119), Selaginella martensii (119), mouse (119), rat (14), human (142), S. cervisias (100), Methanothermus sociabilis (20), Bacillus subtilis (20), and E.coli (90) porphobilinogen synthase metal-binding domains are shown. Relevant amino acid residues are boxed. 66
Non-plants
Mouse L L V A V DV r L C P Y T S IT G H C G L L Rat LLV A C DV c L C P YTSHG HC GL L Human LLV A C DV c L C PYTSH GHC GL L Yeast LYI I £. DV c L C E YTSHG HC GV L M. sociabilis LVV IT DV c L C Q YT E HG HC GI V B. subtilis M VVV ADT c LC E Y T DHGH CG LV E. coli MI V M S DT c_ F C EYT S H G H C GV L
R. capsulatus VA I M t DI A L n PYN ANG H ET G L V
Plants
Soybean LV IYT D V A LD P YS SDG H DG IV Pea L I I YTD VALD PY S SDG HDG IV Spinach LII YTD VALD PYY YDGH DG IV S. martensii L VI Y X D V A L .Q PYS S n GH D. G I V 67
The effects of multiple copies of theR. capsulatus hemB gene on protoporphyrin accumulation in AJB456 was examined by moving pCAP142 into
AJB456. It was rationalized that AJB456(pCAP142) would generate an endogenous pool of porphobilinogen, capable of eliminating oxygen mediated accumulation of protoporphyrin. Previous results have demonstrated that exogenous porphobilinogen can eliminate oxygen mediated accumulation of protoporphyrin in AJB456 (10). However, AJB456(pCAP142), when grown under both high and low oxygen, accumulates protoporphyrin to the same extent as
AJB456 (Fig. 11), even though porphobilinogen synthase specific activity is five fold higher in the strain with the plasmid (Table 6). This suggests that the levels of aminolevulinate, and not porphobilinogen, are limiting. Interestingly, protoporphyrin accumulation in AJB456(pCAP142) is still regulated by oxygen.
When AJB456(pCAP 142) is grown in the presence of aminolevulinate, the accumulation of porphyrins is dramatically increased (Fig. 11). Porphyrin accumulation was so large under both conditions that porphyrins were excreted into the medium, reminiscent of AJB530 (12). Thin layer chromatography indicated that both coproporphyrin and protoporphyrin were being excreted. Most importantly, oxygen mediated accumulation of porphyrins has been altered from what is normally seen in AJB456. These results suggest that the overproduction of porphobilinogen can overcome the normal oxygen mediated regulatory mechanisms present in the cell. The excretion of excess porphyrins may be a safety measure to prevent photooxidative damage (110). 68
Expression of thehemB gene and porphobilinogen synthase specific activity were measured under different oxygen tensions to evaluate the contribution that synthesis of porphobilinogen may play in oxygen regulation.
Enzyme synthesis was measured by dot blot analysis and hemB-cat transcriptional fusions. Dot blot analysis (Fig. 8) shows that hemB steady-state mRNA levels are the same under high and low oxygen. The results from the hemB-cat fusion studies, presented in Table 5, show that the initiation of transcription of the hemB gene does not change with shifts in oxygen tension.
Oxygen tension also does not appear to regulate enzyme activity. Our measurements indicate that the specific activity of porphobilinogen synthase does not change significantly with changes in oxygen tension (Table 4). It has been reported that porphobilinogen synthase from bovine sources is oxygen labile and requires thiols for full activity (134). It is therefore possible, that the assay conditions employed may not reflect the in vivo situation. While the cultures used in this assay were grown under defined oxygen tensions, crude extracts and enzyme assay conditions were performed under aerobic conditions. However, the R. capsulatus enzyme may not be oxygen labile, like it's bovine counterpart, because it lacks the cysteine rich metal-binding domain believed to be responsible for oxygen sensitivity of the bovine enzyme.
While the mechanism of how oxygen modulates carbon flow down the common tetrapyrrole pathway remains elusive, it is clear that the level of porphobilinogen plays a crucial part in oxygen regulation. The addition of 69 exogenous porphobilinogen (10), as well as overexpression of porphobilinogen synthase can eliminate or alter oxygen regulation of the common tetrapyrrole pathway. The results of the gene expression studies and the enzyme assay studies overwhelmingly indicate that the synthesis of porphobilinogen synthase is not regulated by oxygen and suggest that oxygen does not regulate porphobilinogen synthase activity. A more likely possibility is that oxygen regulates the degradation of porphobilinogen. Porphobilinogen serves as the substrate for the enzyme porphobilinogen deaminase. This enzyme converts four molecules of porphobilinogen into the linear tetrapyrrole, hydroxymethylbilane.
This linear tetrapyrrole is released into solution where it serves as the substrate for uroporphyrinogen III synthase. Four molecules of porphobilinogen are, therefore, converted into one molecule of uroporphyrinogen. Preliminary results regarding the purification of the R. capsulatus porphobilinogen deaminase, reveal that the ratio of porphobilinogen used to uroporphyrinogen III formed is about 12 to 1, instead of the expected four to one (Canada and Biel, unpublished). This high ratio suggest that about 2/3 of the total carbon flow going down the common tetrapyrrole pathway is lost at this step. Perhaps oxygen regulates the pathway by influencing the ratio of porphobilinogen used to uroporphyrinogen III formed.
To determine if this is a real possibility, the R. capsulatus porphobilinogen deaminase needs to be assayed under strictly aerobic and anaerobic conditions and the ratios of porphobilinogen used to uroporphyrinogen III formed compared. 70
If the ratio of porphobilinogen used to uroprophyringen III formed decreases
under anaerobic conditions, this may explain how oxygen regulates the pathway.
Genetic mapping of thehem genes of bacteria have led to the discovery
of different gene arrangements in Gram-negative and Gram-positive organisms.
In the Gram-positive organism Bacillus subtilis, hem genes are clustered together
to form an operon (59). The hem genes in Staphlococcus aureus are also
clustered and thought to be in an operon (78). However, thehem genes in Gram-
negative organisms, including R. capsulatus, appear to be scattered along the
chromosome with the exception thehemC and hemD genes (5, 40, 78). The
isolation of an R' factor carrying the entire R. capsulatus photosynthesis region,
including the bacteriochlorophyll and carotenoid biosynthetic genes, prompted the
idea that the hem genes of the common tetrapyrrole pathway may also be
clustered in an operon (93). The results presented from the mapping experiments
indicate these genes are not arranged in an operon.
In conclusion, thehemB gene has been cloned and sequenced in an effort to resolve how porphobilinogen levels are regulated by oxygen. Dot blot analysis
and hemB-cat fusion studies indicate that transcription of the hemB gene is not
regulated by oxygen. These results suggest that the formation of
porphobilinogen is not regulated by oxygen. The mechanism by which
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152. Zagorec, M., J. Buhler, I. Treich, T. Keng, L. Guarente, and R. Labbe-Bois. 1988. Isolation, sequence, and regulation by oxygen of the yeast HEM 13 gene coding for coproporphyrinogen oxidase. J. Biol. Chem. 263:9718-9724. Vita
Karl Joseph Indest was born on October 20, 1968, in Fort Campbell,
Kentucky. He graduated from Brother Martin High School in May, 1986. In May,
1990 Karl was awarded the degree of Bachelor of Arts in Biology from the
University of New Orleans. In January of 1991, Karl entered the doctoral program in the Department of Microbiology at Louisiana State University, Baton
Rouge under the direction of Alan Jay Biel as his graduate advisor. Karl is currently a Ph.D. candidate in the Department of Microbiology at L.S.U.
86 DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate; Karl Joseph Indest
Major Field: Microbiology
Title of Dissertation: Molecular Cloning, Sequencing, and Regulation of the Rhodobacter capsulatus hemB Gene
Approved:
Major/Pr^essor and± Chairman
Dearr of the Graduate school
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Date of Examination:
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