Isolation and Characterization of a Rhodobacter Capsulatus Hemc Mutant

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LSU Historical Dissertations and Theses Graduate School

2001 Isolation and Characterization of a Rhodobacter Capsulatus hemC Mutant. Karen Coleman Sullivan Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Sullivan, Karen Coleman, "Isolation and Characterization of a Rhodobacter Capsulatus hemC Mutant." (2001). LSU Historical Dissertations and Theses. 251. https://digitalcommons.lsu.edu/gradschool_disstheses/251

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ISOLATION AND CHARACTERIZATION OF A RHODOBACTER CAPSULATUS HEMC MUTANT

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

The Department of Biological Sciences

by Karen Coleman Sullivan B.S., Nicholls State University, 1991 May 2001

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Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION

In Loving Memory of my Uncle Mike Sullivan

and

To my Wonderful Family,

Troy, Brandon, and Cole.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS

It is impossible to adequately express my deep appreciation for my best

friend, my husband, Troy. Without your love, support, and encouragement I

would never have completed this degree program. Thank you for being so

patient and understanding.

I would like to express my love and gratitude to my children, Brandon

and Cole, who have spent countless hours at LSU waiting for Mom to finish her

work. You can be proud now that Mom has finally completed “20th grade”.

Special thanks to my parents for their love and encouragement. I am

deeply indebted to you for taking care of Brandon and Cole when they had

chicken pox so that I could go to class and for babysitting whenever I needed

some time to regain my sanity. Also, thanks to my in-laws for their unending

encouragement. Your love and support mean the world to me.

I would like to thank my advisor, Dr. Alan Biel, for advice and

encouragement. I appreciate all the time and effort that you have put into my

graduate career. Thanks for putting up with my occasional tears. I will never be

able to eat at McDonalds without thinking of you. Thanks also to my other

committee members: Dr. LaBonte, Dr. Jarosik, Dr. Dimario, and especially Dr.

Achberger for his eternal patience and kindness.

There are several former members of our lab who will always have a

special place in my heart: Keith Canada, Alok Khanna, and Katya Kanazireva.

Thank you all for teaching me so much and for friendships I will never forget.

iii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

DEDICATION...... ii

ACKNOWLEDGMENTS...... iii

LIST OF TABLES...... vi

LIST OF FIGURES...... vii

ABSTRACT...... viii

INTRODUCTION ...... 1 Tetrapyrrole Pathway of R. cap su/a tu...... s 2 Tetrapyrrole Biosynthesis Regulation ...... 13 Oxygen Regulation of Bacteriochlorophyll Synthesis ...... 16 Oxygen Regulation of the Tetrapyrrole Pathway ...... 17

MATERIALS AND METHODS ...... 22 Strains and Plasmids ...... 22 M e d ia ...... 22 Growth Conditions...... 22 Chromosomal DNA Isolation ...... 25 Plasmid DNA Isolation ...... 25 Restriction Enzyme Digestion ...... 26 DNA Ligation ...... 26 Fill-in Reaction ...... 27 Nick Translation ...... 27 Agarose Gel Electrophoresis ...... 28 DNA Recovery from Agarose Gels ...... 28 Electroporation ...... 28 Conjugation ...... 29 Southern Hybridization ...... 30 Protein Determination ...... 31 Protein Determination by Dye Binding ...... 31 Lowry Protein Determination...... 31 Preparation of Cell Extracts ...... 32 Quantitation of Bacteriochlorophyll ...... 33 Aminolevulinate Synthase Assay ...... 33 Porphobilinogen Synthase Assay ...... 34 Porphobilinogen Deaminase Assay ...... 35 Measurement of Porphyrin Accumulation ...... 35 Measurement of Porphobilinogen Accumulation ...... 36

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS ...... 37 Crossfeeding Studies...... 37 Growth of E. coli hem Mutants Using Exogenous Protoporphyrin and H em in...... 37 Growth of an R. capsufatus hemA Mutant on Exogenous Hemin .... 42 Isolation of an R. capsufatus hemC M u ta n t ...... 42 Chromosomal Location of the Kanamycin Cartridge ...... 45 Bacteriochlorophyll Levels in Parental and Mutant Strains ...... 47 Enzymatic Activities in Parental and Mutant S trains ...... 47 Porphyrin Measurements in AJB530 ...... 49 Porphobilinogen Measurements in AJB565 ...... 51

DISCUSSION...... 53

REFERENCES...... 64

VITA ...... 82

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

Table 1. Bacterial strains ...... 23

Table 2. P la sm id s ...... 24

Table 3. Bacteriochlorophyll levels and enzymatic activities ...... 48

Table 4. Porphyrin measurements in AJB530 ...... 50

Table 5. Porphobilinogen measurements in AJB565 ...... 52

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

Figure 1. The tetrapyrrole biosynthetic pathway in R. cap sulatus...... 3

Figure 2. Crossfeeding s tu d y...... 38

Figure 3. Growth yield of E. coli S905 with varying porin ...... 40

Figure 4. Growth yield of E. coli S905 with varying protoporphyrin 41

Figure 5. Restriction maps of pCAP181 and pCAP168 ...... 44

Figure 6. Southern hybridization of R. capsulatus chromosomal DNA ... 46

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

Rhodobacter capsulatus, a purple, nonsulfur photosynthetic bacterium,

uses the tetrapyrrole pathway to synthesize four end products: heme,

bacteriochlorophyll, vitamin B12, and siroheme. This laboratory has focused on

the regulation of the common pathway leading from aminolevulinate to

protoporphyrin IX in R. capsulatus. The common portion of the pathway is

regulated up to 100-fold by changes in oxygen tension.

Research on the regulation of this pathway has been hampered due to a

lack of mutants. Until now, the only mutants isolated which block heme

production are hemA mutants, which lack aminolevulinate synthase. Since R.

capsulatus lacks a cytochrome-independent growth mechanism, mutations later

in the tetrapyrrole pathway would be lethal unless the mutant can use

exogenous hemin or protoporphyrin.

To overcome this lack of mutants, a method for isolating hem mutants

has been devised. First, a growth medium was developed so that the hemA

mutant, and presumably other hem mutants, grow well on exogenous hemin.

We then took advantage of the recent cloning of several of the R. capsulatus

hem genes to make a hem mutation in vitro and move it into R. capsulatus by

conjugation. Thus, we were looking for a high probability recombination event

rather than a much lower probability transposition event.

This study describes the development of this medium as well as the

isolation and characterization of a hemC mutant of R. capsulatus. This is the

viii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. first hem mutant in a photosynthetic bacterium, after hemA, which has a

complete block in the pathway. The mutant was constructed by inserting a

kanamycin-resistance cartridge into a plasmid containing the R. capsulatus

hemC gene. The mutated hemC gene was then recombined into theR.

capsulatus chromosome. The resulting mutant requires heme for growth, lacks

porphobilinogen deaminase activity and is unable to synthesize

bacteriochlorophyll. This mutant was also used to provide direct evidence that

the point of oxygen regulation in the tetrapyrrole pathway is located after the

formation of porphobilinogen, most likely the usage of porphobilinogen.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INTRODUCTION

Rhodobacter capsulatus is a purple, nonsulfur photosynthetic bacterium.

It is a remarkably diverse organism in its ability to obtain energy. Its ability to

grow by at least five different growth modes makes it one of the most

metabolically diverse organisms. Its preferred growth mode is

photoheterotrophically under anaerobic conditions in light using a variety of

organic compounds as carbon sources and as electron donors for NADH

production (35, 110). R. capsulatus is also capable of photoautotrophic growth

under anaerobic conditions in the light with C02 as the sole carbon source and

H2as the electron donor for NADH production (115) or as a chemoautotroph

(110) under anaerobic conditions in the dark using hydrogen as the energy

source (175). In addition, it is able to grow as a chemoheterotroph under

aerobic conditions in the dark using a variety of organic compounds for carbon

and energy and 0 2 as the final electron acceptor (110). R. capsulatus can

grow anaerobically in the dark using various sugars for carbon and either

nitrate, nitrous oxide, dimethyl sulfoxide (DMSO), or trimethylamine-N-oxide

(TMAO) as electron acceptors (108, 109, 115). In addition to these growth

modes, R. capsulatus can fix nitrogen in both light and dark conditions (7).

Another distinctive characteristic of R. capsulatus is its ability to

synthesize all four possible tetrapyrrole end products: bacteriochlorophyll,

heme, vitamin B12 and siroheme using the tetrapyrrole pathway.

Bacteriochlorophyll is the main light harvesting pigment and is synthesized only

when R. capsulatus is grown anaerobically in the light. Heme serves as a

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. prosthetic group in cytochromes, catalases and peroxidases and is always

required for electron transport regardless of growth mode. Studies in the

closely related organism, R. sphaeroides, has shown that vitamin B12 serves as

a cofactor of homocysteine methyltransferase which is necessary for

methionine biosynthesis (31). Other studies in this organism have

demonstrated that siroheme serves as a prosthetic group for sulfite reductase

(118) and nitrite reductase (119). When R. sphaeroides is grown

photosynthetically, bacteriochlorophyll accounts for 98% of all tetrapyrroles in

the cell (99). However, when grown aerobically, bacteriochlorophyll production

stops, but the other end products continue to be produced at the same levels

as when grown anaerobically (99).

All organisms that synthesize tetrapyrroles do so by using the

tetrapyrrole pathway. The few organisms with incomplete pathways require an

exogenous source of tetrapyrrole such as heme or a tetrapyrrole precursor

(54). Only the photosynthetic bacteria are capable of synthesizing all four

possible tetrapyrrole end products.

Tetrapyrrole Pathway of R. capsulatus

The common portion of the tetrapyrrole pathway (Figure 1) includes the

biosynthetic steps from aminolevulinate to protoporphyrin IX and is similar in all

organisms studied so far. The pathway branches after the formation of

protoporphyrin IX to form bacteriochlorophyll and heme. The first step in

tetrapyrrole biosynthesis is the formation of the aminolevulinate, a five-carbon

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Succinyl CoA + Glycine hemA

- Aminolevulinate I*hemB Porphobilinogen

^hemC

Hydroxymethylbilane SIROHEME ^hem D 4

VITAMIN B-12 «■— *■------’ Uroporphyrinogen ^hem E

Coproporphyrinogen

hemF ■ Protoporphyrinogen IX

hemG

■ Protoporphyrin IX / h e m H \ h e m e bacteriochlorophyll

Figure 1. The tetrapyrrole biosynthetic pathway in R. capsulatus.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aminoketone. There are two known ways in which aminolevulinate can be

formed. The first of these to be reported, the C4 pathway, involves the

condensation of glycine with succinyl CoA with the elimination of C-1 of glycine

as carbon dioxide (90). This reaction is catalyzed by the pyridoxal phosphate-

requiring enzyme aminolevulinate synthase. This mechanism of forming

aminolevulinate is found in animals (49), yeast (11), fungi (102), and purple

photosynthetic bacteria such as R. capsulatus (5).

A second aminolevulinate production pathway, called the C5 pathway,

was reported in 1974 (12). This pathway involves the conversion of glutamate

to aminolevulinate in a three step process. The reaction sequence consists of

three separate enzymes: glutamyl tRNA synthetase (127), an NADPH-

dependent glutamyl-tRNA dehydrogenase (6) and glutamate-1 -semialdehyde

aminotransferase (46). The C5 pathway is found in plants (12), algae (169),

cyanobacteria (44), archaebacteria (50) and eubacteria (46).

Although there are no published reports on the purification of

aminolevulinate synthase from R. capsulatus, the predicted molecular mass of

the enzyme is 50,491 Da based on sequence information (43).

Aminolevulinate synthase has been purified and characterized from R.

sphaeroides and found to be a homodimer with a subunit molecular mass of 80

kDa (123). The R. sphaeroides enzyme requires pyridoxal phosphate for

catalytic activity and is inhibited by hemin, Mg-protoporphyrin and ATP (47,

185). The R. capsulatus enzyme is also feedback inhibited by heme (86). R.

sphaeroides produces two different forms of aminolevulinate synthase which

permission of the copyright owner. Further reproduction prohibited without permission. are encoded by the hemA and hem Tgenes (155). These two genes are found

on separate chromosomes and have homology with each other and with other

characterized aminolevulinate synthases (155).

The hemA gene encoding aminolevulinate synthase has been

sequenced from many sources. The hemA gene of R. capsulatus was cloned

by two independent sources. Biel et al. (19) isolated a cosmid which

complemented a hemA:Tn5 mutant. Later, Hornberger et al. (62) isolated the

hemA gene from R. capsulatus by probing a cosmid bank of R. capsulatus with

the aminolevulinate synthase gene from R. sphaeroides.

The next step in the tetrapyrrole pathway is the formation of

porphobilinogen, a monopyrrole, by condensation of two aminolevulinate

molecules with the loss of two molecules of water. This reaction is catalyzed

by the product of the hemB gene, porphobilinogen synthase (54). The enzyme

has been studied from many organisms including plants (112, 143, 106),

human erythrocytes (4), bovine liver (174), and bacteria (61, 179, 128). The

porphobilinogen synthase enzymes from various sources differ in their metal

requirements. Porphobilinogen synthase from Escherichia coli was found to

have an octomeric structure with a native molecular mass of 290 kDa and

requires thiols and zinc (152).

The hemB gene encoding porphobilinogen synthase has been cloned

and sequenced from many organisms such as rat (22), human (170), yeast

(120) and bacteria (42, 56, 134, 84) including R. capsulatus (66). All

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sequences encode a protein of 35 to 45 kDa and were found to have 36 to 40%

identity to each other (105). Indest and Biel (67) sequenced the R. capsulatus

hemB gene and found that it contains a magnesium binding site but no zinc

binding site.

The next step in the tetrapyrrole pathway is the formation of

uroporphyrinogen III, the first cycl ic tetrapyrrole (81). Uroporphyrinogen III is

formed from porphobilinogen by ttie action of two enzymes, porphobilinogen

deaminase and uroporphyrinogera III synthase. Porphobilinogen deaminase,

the product of the hemC gene, is responsible for the polymerization of four

porphobilinogen molecules to form the linear tetrapyrrole hydroxymethylbilane

(10, 26). The hemC gene has been isolated and sequenced from a variety of

organisms including bacteria such as Escherichia coli (159), Bacillus subtilis

(56), Clostridium josui (52), Stapfrylococcus aureus (85), and Chlorobium

vibrioforme (111). It has also been sequenced from plants (171) and animals

such as mice (13) and human erythrocytes (133). The hemC gene from R.

capsulatus has been cloned and sequenced by Canada (28).

Porphobilinogen deaminase is a monomer with a molecular mass in the

range of approximately 35 kDa (8-0) and has been purified from many sources

including plants such as Arabidopsis (80), pea (151), and wheat (51) as well as

animals such as cow (139), human erythrocytes (116), and rat (114). It has

also been purified from many bacteria including Escherichia coli (83),

Rhodopseudomonas palustris (94), and Rhodopseudomonas sphaeroides (82).

It has recently been purified and characterized from R. capsulatus by Canada

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (28). There are no metal or other cofactor requirements for any of the

porphobilinogen deaminases which have been characterized to date.

Porphobilinogen deaminase has been found to contain a dipyrromethane

cofactor linked to a cysteine residue (59). This cofactor was found to be the

attachment site for the four porphobilinogen molecules during catalytic activity

(167). The assembly of the four porphobilinogen molecules into

hydroxymethylbilane occurs in a sequential order starting with ring A, followed

by rings B, C, and D (9, 145). In the absence of uroporphyrinogen III synthase,

hydroxymethylbilane spontaneously cyclizes to form the non-physiological

isomer uroporphyrinogen I. Uroporphyrinogen III is formed by cyclization of

hydroxymethylbilane by the product of the hemD gene, uroporphyrinogen III

synthase (8).

Uroporphyrinogen III synthase is a monomeric enzyme with a molecular

mass ranging from 28,000 Da to 38,500 Da (3, 60). It has been found to

facilitate the release of hydroxymethylbilane from porphobilinogen deaminase

(138). The enzyme is extremely heat sensitive, making its isolation difficult.

However, it has been successfully purified from several sources such as

bacteria (3, 153), cow liver (139), human red blood cells (163), Euglena (60),

mouse (104), and rat (150). No metal or cofactor requirements have been

identified. ThehemD gene, which encodes uroporphyrinogen III synthase, has

been sequenced from many organisms including E. coli (2, 142), B. subtilis

(56), C .josui (52), C. vibrioforme (117), cyanobacterium (77), human (162), and

S. aureus (85).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Uroporphyrinogen III is located at a branch point in the common

tetrapyrrole pathway. A small amount of uroporphyrinogen III is methylated

and goes through several steps to the synthesis of siroheme and vitamin B12.

The major amount of uroporphyrinogen III is decarboxylated by the enzyme

uroporphyrinogen III decarboxylase to form coproporphyrinogen III and is

eventually converted into heme or bacteriochlorophyll.

The enzyme uroporphyrinogen III decarboxylase catalyzes the

decarboxylation of all four acetate groups on the tetrapyrrole ring of

uroporphyrinogen III to methyl groups (55). The enzyme has been purified

from R. sphaeroides (79) and R. palustris (93) as well as from yeast (48), avian

erythrocytes (89), and mammalian sources (45, 136, 154). All

uroporphyrinogen decarboxylases purified to date are monomers of 38 to 46

kDa with the exception of the bovine form which is 57 kDa and the avian form

which is 79 kDa. Unlike most decarboxylation reactions, uroporphyrinogen

decarboxylase does not have a metal or coenzyme requirement.

The hemE gene, which encodes uroporphyrinogen III decarboxylase,

has been cloned and sequenced fromR. capsulatus by Ineichen and Biel (68).

It has also been sequenced from sources such as E. coli (125), B. subtilis (58),

human (135, 137), and yeast (41).

In the next step of the pathway, the enzyme coproporphyrinogen III

oxidase converts coproporphyrinogen III to protoporphyrinogen IX by oxidative

decarboxylation. The enzyme converts two propionic side chains on rings A

and B of coproporphyrinogen III into vinyls, releasing two molecules of C02and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. two H* atoms, producing protoporphyrinogen IX (129). Two distinct

coproporphyrinogen oxidase enzymes exist in several organisms such as E.

coli (160, 161), Salmonella typhimurium (177), Saccharomyces cerevisiae

(130), and R. sphaeroides (144, 156). The first, HemF, requires molecular

oxygen as an electron acceptor for the oxidative decarboxylation of

coproporphyrinogen III and, therefore, functions under aerobic conditions (130,

177). The second is designated HemN and functions under anaerobic

conditions. It requires Mg2+, methionine, ATP and either NAD* or NADP* for

activity (144,156).

The hemF analog of coproporphyrinogen III oxidase was purified from

bovine liver (92) and shown to exist as a homodimer. It was found to have a

molecular mass of 37,000 Da. The enzyme was also purified from yeast (27)

and the gene encoding it,HEM13 , was cloned and sequenced (186). It was

found to code for a protein with a molecular mass of 37,673 Da (27). The E.

coli hemF gene has been cloned by complementing a yeast HEM13 mutant

(161) and found to code for a protein with a molecular mass of 34,300 Da. The

E. coli hemN gene was later cloned by complementation of a hemF hemN

double mutant of Salmonella typhimurium (160). It was found to encode a

protein with a molecular mass of 52,800 Da. Both hemF (178) and the hemN

(176) genes have been cloned from Salmonella typhimurium and it was shown

that either one can support heme synthesis under aerobic conditions (177).

The hemF gene encodes a protein with a molecular mass of 34,000 Da while

the hemN gene encodes a protein with a molecular mass of 52,800 Da. Using

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the amino acid sequence of the bovine liver coproporphyrinogen III oxidase as

a probe, a mouse cDNA library was probed and the sequence of the mouse

hemF was determined (92). Thehem F sequence has also been determined

from human (157), tobacco (96), and barley (96). No hemN sequence has

been found in any of the eukaryotic sources since they grow only aerobically.

The final reaction in the common tetrapyrrole pathway is the formation of

protoporphyrin IX. This reaction is catalyzed by the membrane bound

protoporphyrinogen IX oxidase which removes six hydrogen atoms from the

protoporphyrinogen IX ring to form protoporphyrin IX (140).

Protoporphyrin IX oxidase has been purified from mammalian

mitochondria (131), bovine liver (148), mouse liver (38), yeast (132), barley

(71) and the anaerobic bacterium Desulfovibrio gigas (91). It is a membrane

associated protein (38, 71, 131, 132, 148). The eukaryotic proteins were found

to be monomers ranging from 57 to 65 kDa (38, 71, 131, 132, 148). Barley

protoporphyrinogen IX oxidase is chloroplast associated with a molecular mass

of 210 kDa, composed of 36 kDa subunits (70). The bacterial enzyme from

Desulfovibrio gigas is composed of three non-identical subunits (91). In

aerobic organisms, molecular oxygen serves as the electron acceptor for this

reaction (73, 72). In anaerobically grown cultures of E. coli, it has been shown

that nitrate and fumarate can serve as electron acceptors instead of oxygen

(74, 75). In photosynthetically grown cultures of R. sphaeroides,

protoporphyrinogen IX oxidation is closely linked to the respiratory electron

transport chain (76).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The hemG gene, encoding the enzyme protoporphyrinogen IX oxidase,

has been sequenced from B. subtilis (39, 57, 58), E. coli(141), human (126),

and tobacco (103). The hemG sequence of E. coli codes for a protein with a

predicted molecular mass of 21 kDa (141). All others code for proteins of 50 to

57 kDa.

The second branch point in the tetrapyrrole pathway is at protoporphyrin

IX. Insertion of a magnesium ion into the protoporphyrin IX ring is followed by

methylation which produces magnesium protoporphyrin monomethyl ester.

This compound is converted through several steps to bacteriochlorophyll.

However, if a ferrous ion is inserted into the ring, it leads to the formation of

heme. The enzyme ferrochelatase, encoded by the hemH gene, catalyzes the

insertion of Fe2+ into protoporphyrin IX to form protoheme.

The R. capsulatus hemH gene has been cloned by complementation

with an E. coli hemH mutant and sequenced (87). It encodes a protein with a

putative molecular mass of 46 kDa. The R. sphaeroides ferrochelatase has a

molecular mass of 115 kDa (37).

During aerobic growth, R. capsulatus has a membrane structure that is

similar to other gram-negative bacteria. When oxygen concentration drops

below 2.5%, many changes occur in the cell including invaginations of the

cytoplasmic membrane as well as bacteriochlorophyll and carotenoid

production. The photosynthetic apparatus, which is composed of three distinct

protein complexes that convert light to chemical energy, is formed within the

cytoplasmic membrane invaginations. The reaction center is where

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. photochemical charge separation occurs that leads to transfer of electrons

through the electron transport system, which produces a transmembrane

potential that drives ATP synthesis. Two light harvesting complexes LH-1

(B870) and LH-II (B800-B850) function to absorb visible and near-infrared

radiation and transfer the energy to the reaction center complex. Each of these

complexes has bacteriochlorophyll and carotenoids bound. In R. capsulatus,

the bacteriochlorophyll genes ( bch) encoding the enzymes that convert

magnesium protoporphyrin to bacteriochlorophyll have been identified by

complementation of bch mutants with R’-plasmids carrying various

bacteriochlorophyll genes (113). A physical map showing the location of the

bacteriochlorophyll and carotenoid genes was generated (158). Both the

bacteriochlorophyll genes and the carotenoid genes ( erf) encoding enzymes for

the biosynthesis of carotenoids are clustered in a 46 kb region of the genome

known as the photosynthetic gene cluster (183, 187). Within this region are

two operons, the puh operon which encodes polypeptides of the light

harvesting complex I and the puf operon which encodes the reaction center

polypeptides (184). The genes of the puc operon which encode the light

harvesting complex II polypeptides were not found to be located in this region

(181). The genes are arranged with the bch and crt genes located centrally

and flanked by the puh and puf operons (180). The entire R. capsulatus

photosynthetic gene cluster has recently been sequenced (1, 182). None of

the R. capsulatus hem genes which have been cloned to date are located near

the photosynthetic gene cluster (19, 28, 67, 68, 88). The University of Chicago

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. has a website which is dedicated to sequencing the complete genome of R.

capsulatus called the Rhodobacter capsuiapedia at www.rhodol.uchicago.edu.

Tetrapyrrole Biosynthesis Regulation

The end products of the tetrapyrrole pathway: heme, bacteriochlorophyll,

siroheme, and vitamin B12 have very different functions in the cell and are

produced in extremely different quantities depending on the growth mode of the

cell. When R. sphaeroides cells are growing photosynthetically,

bacteriochlorophyll accounts for 98% of all tetrapyrroles (99). Approximately

1% of tetrapyrroles is found as heme whereas siroheme and vitamin B12 are

only produced in trace amounts. If R. sphaeroides is grown aerobically,

bacteriochlorophyll is not produced but the other end products are synthesized

at the Same rate as when grown photosynthetically with no accumulation of

intermediates (99). For these reasons, the common tetrapyrrole pathway can

be expected to have many complex regulatory mechanisms. Four

environmental factors have been shown to influence the levels of tetrapyrrole

end products: c-type cytochromes, light, heme, and oxygen (20, 24, 32, 34).

These factors have been studied extensively in an attempt to understand the

regulation of this pathway.

Previous studies have implicated c-type cytochromes in the regulation of

tetrapyrrole biosynthesis based on the fact that c-type cytochrome mutants of

R. capsulatus accumulate photopigments (40, 95). Biel and Biel (20) isolated a

mutant of R. capsulatus, AJB530, which has a Tn5 insertion in the gene

encoding cytochrome c synthetase. This mutation completely prevents c-type

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cytochrome synthesis. Compared to the parental strain, AJB530 produces only

19% of the normal amount of bacteriochlorophyll but excretes large amounts of

coproporphyrin and protoporphyrin. The excretion of large amounts of

coproporphyrin and protoporphyrin led to the suggestion that the lack of c-type

cytochromes derepressed carbon flow over the common tetrapyrrole pathway

(16). However, total porphyrin levels, which includes bacteriochlorophyll,

coproporphyrin, and protoporphyrin, were found to be the same in wild-type R.

capsulatus as in the mutant AJB530 (64). It has been suggested that the lack

of c-type cytochromes in this strain interferes with the electron transport system

necessary for protoporphyrinogen oxidase activity, resulting in a partial block at

this step (64). This indicates that c-type cytochromes have no influence on

carbon flow over the tetrapyrrole pathway (64).

The influence of light on bacteriochlorophyll synthesis was first studied

by Cohen-Bazire et al. (34) in R. sphaeroides. They showed that highly

illuminated cultures of R. sphaeroides had low levels of bacteriochlorophyll,

while cultures grown in low light contained large amounts of photopigments.

This seemed to show that light regulates bacteriochlorophyll synthesis. Biel

and Marrs (18) showed that light has no effect on the transcription of the

bacteriochlorophyll genes of R. capsulatus, but that they are regulated by

oxygen. It was later shown that light causes the degradation of

bacteriochlorophyll and does not affect its synthesis (15).

One of the major factors that influences the regulation of the tetrapyrrole

pathway is heme. It has been shown to inhibit many enzymes of the common

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. tetrapyrrole pathway. In R. sphaeroides, aminolevulinate synthase was found

to be inhibited in vitro by hemin and results suggest a possible negative

feedback mechanism (24). In vitro inhibition of aminolevulinate synthase was

confirmed in several other studies (166, 185, 47). In R. sphaeroides,

porphobilinogen synthase was also found to be inhibited by hemin (24, 122).

However, porphobilinogen synthase in R. capsulatus was found to be

unaffected by hemin (121). In R. sphaeroides, the enzymes uroporphyrinogen

III decarboxylase and ferrochelatase were also shown to be somewhat inhibited

by hemin (79, 78). Several other studies indicate that heme-deficient cells

have higher carbon flow over the pathway due to lack of feedback inhibition of

aminolevulinate synthase (36, 63, 100). Lascelles found that adding ferric

citrate to media caused R. sphaeroides cells to produce 100 times less

coproporphyrin and protoporphyrin but 10 times more bacteriochlorophyll

(100). When R. sphaeroides or R. capsulatus cultures were grown

photosynthetically in iron-deficient media to limit heme production, large

amounts of coproporphyrin and Mg-protoporphyrin monomethyl ester were

produced (36, 100). In another study of heme deficient cells, R. capsulatus

and R. sphaeroides were grown in media with excess iron and the

ferrochelatase inhibitor, N-methylprotoporphyrin. This resulted in accumulation

of Mg-protoporphyrin monomethyl ester, while the level of bacteriochlorophyll

was not changed (Biel, unpublished results; 63). This regulation was simulated

in vivo by overexpressing a low copy number vector containing the hemH gene

of R. capsulatus (86). Overexpression of ferrochelatase caused increased

permission of the copyright owner. Further reproduction prohibited without permission. heme formation which inhibited aminolevulinate synthase and reduced the

synthesis of porphyrins (86). In R. capsulatus, heme and oxygen were found to

regulate tetrapyrrole synthesis by separate mechanisms (87).

In summary, oxygen tension is the major factor controlling synthesis of

the tetrapyrroles. In R. capsulatus, heme feedback inhibits aminolevulinate

synthase, which reduces carbon-flow over the common tetrapyrrole pathway.

These two mechanisms for regulating the common tetrapyrrole pathway are

completely separate from each other. Under high light intensities,

bacteriochlorophyll is still synthesized but the rate of degradation of the

photopigment increases. Thus, light does not regulate carbon-flow over the

common tetrapyrrole pathway. Similarly, this pathway is not regulated by the

by the presence or absence of c-type cytochromes. Mutations that prevent the

synthesis of c-type cytochromes appear to reduce the conversion of

protoporphyrinogen IX to protoporphyrin IX and coproporphyrinogen III. The

porphyrinogens are excreted and spontaneously oxidize to the corresponding

porphyrins.

Oxygen Regulation of Bacteriochlorophyll Synthesis

The exact mechanism of how oxygen controls bacteriochlorophyll

synthesis has been the subject of extensive research since Cohen-Bazire

showed that oxygen inhibits bacteriochlorophyll synthesis (34). They

suggested that regulation by oxygen is due to redox conditions of some carrier

of the electron transport system which could affect bacteriochlorophyll

synthesis. Biel and Marrs (18) constructed fusions of the lacZ gene to various

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bch genes of R. capsulatus in order to measure the transcription rates of these

genes. In these fusions, the lacZ gene was under the control of the bch gene

promoters. (3-Galactosidase activity was measured under different growth

conditions to assess the activity of the bch promoters. They found transcription

increased two to three fold with a drop in oxygen tension from 23% to 2%.

These results are confirmed by other studies of R. capsulatus and R.

sphaeroides which show that mRNA transcript levels of bch genes increases

two to three fold when switched from aerobic to anaerobic conditions (32, 65).

Although transcription of bch genes is regulated by oxygen two to three fold,

this alone cannot explain the halt in bacteriochlorophyll production when

switched from anaerobic to aerobic growth conditions. There must be other

factors that play a role in the regulation of bacteriochlorophyll biosynthesis.

Oxygen Regulation of the Tetrapyrrole Pathway

Lascelles and Hatch (97) proposed that oxygen inhibits the magnesium

chelatase and causes increased heme production due to a larger amount of

protoporphyrin IX available for conversion by ferrochelatase. They suggested

that the increased amount of heme would feedback inhibit aminolevulinate

synthase which would reduce carbon flow over the pathway without causing

overproduction of tetrapyrrole intermediates. Biel and Kanazireva (86) cloned

the R. capsulatus hemH gene and mobilized it back into R. capsulatus. They

found that ferrochelatase activity tripled under these conditions and

aminolevulinate synthase activity was lowered. This was the first in vivo

evidence to suggest that heme feedback inhibits aminolevulinate synthase in

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. R. capsulatus (86). Biel and Marrs (18) demonstrated that oxygen regulated

the synthesis of protoporphyrin IX by using an R. capsulatus bchH mutant

which cannot insert magnesium into the protoporphyrin IX ring. It was grown

under high and low oxygen tension and accumulation of protoporphyrin IX was

measured. Under low oxygen tension there was a dramatic increase in the

protoporphyrin levels, compared to high oxygen tension. This showed that

there is an oxygen-regulated step in the common portion of the tetrapyrrole

pathway.

In R. capsulatus there is no evidence that oxygen regulates

aminolevulinate synthase activity. There is no difference in aminolevulinate

synthase activity of cultures grown under high and low oxygen tension (17) and

there is only a two-fold change in transcription of the hemA gene as

determined by using a hemA-lacZ fusion vector (173). These changes are not

enough to explain the huge increase in tetrapyrrole synthesis during

photosynthetic growth.

In R. capsulatus, there is evidence that indicates that aminolevulinate is

not committed solely to tetrapyrrole synthesis. Biel (unpublished results)

showed that when R. capsulatus was grown in the presence of 14C-

aminoievulinate, 85% of the radioactivity was distributed to compounds other

than tetrapyrroles. This supports results from a previous study using

Rhodospirillum rubrum which showed that 14C-labeled aminolevulinate was

incorporated into tetrapyrroles as well as aminohydroxyvalerate,

hydroxygiutarate and glutamate (147). Recently in R. capsulatus, an

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. aminolevulinate dehydrogenase activity which converts aminolevulinate to

aminohydroxyvalerate in the presence of NADPH was detected (Khanna and

Biel, unpublished results).

Biel (17) showed that the major step regulated by oxygen is after

aminolevulinate synthesis. This he did by growing an R. capsulatus bchH

mutant with exogenous aminolevulinate under high and low oxygen tension.

There was no accumulation of protoporphyrin IX under high oxygen tension.

The bchH mutant was then grown with exogenous porphobilinogen under high

and low oxygen tension which resulted in an accumulation of protoporphyrin IX

under both high and low oxygen conditions. This suggests that oxygen

regulates porphobilinogen levels. Using a hemfi-chloramphenicol

acetyltransferase fusion vector and northern analysis of the R. capsulatus

hemB gene, it was shown that neither porphobilinogen synthase activity, nor

the transcription of the hemB gene is regulated by oxygen (66).

Overexpression of the R. capsulatus hemB gene in thebchH strain produced

similar results to those seen when exogenous porphobilinogen was added (66).

Canada (28) found that it may be the conversion of porphobilinogen to

hydroxymethylbilane that is regulated by oxygen. The stoichiometry of this

reaction is dramatically influenced by redox conditions. Ideally, it takes four

molecules of porphobilinogen to form one molecule of hydroxymethylbilane.

When cultures were grown and assayed under strict anaerobic conditions, it

took ten molecules of porphobilinogen to form one molecule of

hydroxymethylbilane. However, when cultures were grown aerobically it took

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. as many as 30 molecules of porphobilinogen to form one molecule of

uroporphyrinogen I. This suggests that as oxygen tension increases,

porphobilinogen is removed from the tetrapyrrole pathway possibly released as

dipyrrole and tripyrrole intermediates.

All of these results taken together suggest that the point of regulation by

oxygen lies between porphobilinogen and uroporphyrinogen III. Study of this

regulation has been hindered due to lack of mutants within the common

tetrapyrrole pathway. The only mutants isolated in photosynthetic bacteria

which block heme production are hemA mutants which lack aminolevulinate

synthase (101, 124, 172). Wright et al. (172) were able to demonstrate that a

hemA mutant will grow slowly when the medium is supplemented with

protoporphyrin IX or hemin. Since R. capsulatus lacks a cytochrome-

independent growth mechanism, mutations later in the tetrapyrrole pathway

would be lethal unless the mutant can use exogenous hemin or protoporphyrin.

Cardin and Biel (30) tried to isolate hem mutants by selecting for transposon

insertion mutants on a medium containing exogenous hemin. The insertion

mutants were then screened for a hemin requirement. No hem mutants were

found during this screen.

The purpose of this study was to further investigate the point of oxygen

regulation in the tetrapyrrole pathway. In order to do this, a medium was

developed that allows growth of hem mutants of E. coli. This medium was then

optimized to allow growth of the R. capsulatus hemA mutant. A plasmid was

constructed containing the hemC gene interrupted by a kanamycin cartridge.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This plasmid was moved into R. capsulatus by conjugation and kanamycin-

resistant colonies selected. These colonies were then screened for hemin-

dependence. AhernC mutant of R. capsulatus was isolated and characterized

by comparing enzymes of the tetrapyrrole pathway with parental levels.

Enzymes of the pathway located before the mutation were produced at wild

type levels while the HemC enzyme was not produced in any measurable

amount. The hemC mutant was grown under high and low oxygen tensions

and the porphobilinogen levels were measured. The amount of

porphobilinogen produced was similar regardless of whether the cells were

grown with or without oxygen. This shows that oxygen does not affect the

synthesis of porphobilinogen so the point of oxygen regulation must be at some

point after porphobilinogen synthesis, most likely the conversion of

porphobilinogen to uroporphyrinogen III.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MATERIALS AND METHODS

Strains and Plasmids

Bacterial strains and plasmids used in this study are listed in Tables 1

and 2.

Media

E. co//was grown in L-broth (14) modified by omitting glucose and

decreasing the sodium chloride concentration to 0.5%. R. capsulatus strains

were grown in either RCV, a malate minimal salts medium (168), or PYE, 0.3%

Bacto-Peptone-0.3% yeast extract (Difco Laboratories, Detroit, Ml). Solid

media contained 1.5% agar (Difco). Antibiotics and other supplements, when

necessary, were added to the following final concentrations: kanamycin, 10 fj.g

per ml; ampicillin, 25 ^g per ml; streptomycin, 75 pg per ml; aminolevulinate,

0.1 mM; hemin (dissolved in NaOH), 0.5 mM; cysteine, 0.5 mM; methionine, 0.5

mM. R. capsulatus hem mutants were grown on RCV plates supplemented

with 0.5 mM hemin, 10 pg per ml kanamycin, 0.5 mM cysteine and 0.5 mM

methionine. Both E. co//and R. capsulatus strains were stored in 10% glycerol

at -85°C.

Growth Conditions

E. coli strains were grown aerobically at 37°C with shaking. R.

capsulatus strains were grown aerobically in the dark at 37°C with shaking or

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1. Bacterial Strains.

Strain designation Genotype Reference

R. capsulatus

PAS 100 hsd-1 str-2 Taylor et al. (158)

AJB529 hemA::Tn5 hsd-1 str-2 W right et al. (172)

AJB565 hemCr.kan This study

AJB530 hsd-1 str-2 ccl-1 Biel and Biel (20)

E. coli

NM522 supE thi A(lac-proAB) Gough and Murray (53) hsd5 FJproAB* lacF facZ AM15]

S17-1 294 recA (RP4 Tc::Mu Simon et al. (149) Km::Tn7 ATn1)a

‘The RP4 derivative is inserted into the chromosome at an unknown location.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2. Plasmids

Plasmid Relevant Characteristics Reference or Description

PCAP154 Apr, hemC* 4 kb R. capsulatus genomic BamHI fragment in pUC18 (28)

PCAP155 Apr, hemE* 2 kb R. capsulatus genomic EcoRI fragment in pUC18 (69)

PCAP168 Apr 0.5 kb Hincll-EcoRI fragment of pCAP155 in pUC18 (28)

PCAP180 Apr, hemC" 4 kb BamHI fragment of pCAP154 cloned into pSUS202 (This study)

PCAP181 Apr, Kmr, hemCv.kan Kanamycin gene from pUC4K cloned into Stul site of pCAP180 (This study)

PCAP183 Apr, Kmr, hemEv.kan Kanamycin gene from pUC4K cloned into Mscl site of pCAP155 (This study)

pSUP202 Apr, Cmr, Tcr, mob+ Simon et al. (149)

pUC18 Apr Vieria and Messing (164)

pUC-4K Kmr Vieria and Messing (164)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without shaking, if used as the recipient for conjugation. When used for

enzyme assays, strains were grown on solid media with 100-200 colonies per

plate until colonies reached approximately three mm in diameter.

Chromosomal DNA Isolation

R. capsulatus chromosomal DNA was isolated from one ml overnight

cultures grown in RCV medium using the Puregene DNA isolation kit (Gentra

Systems, Inc., Minneapolis, MN). The cells were harvested by centrifugation at

16,000 x g for one minute. The cell pellet was then suspended in 300 (A cell

lysis solution. The samples were incubated for five minutes at 80°C to allow

cell lysis to occur. RNase A solution (1.5 //I) was added to the cell lysate

which was then incubated at 37°C for 30 minutes. After samples cooled to

room temperature, 100 iA of protein precipitation solution was added followed

by centrifugation at 16,000 x g for three minutes. The supernatant containing

the DNA was transferred to a 1.5 ml tube with 300 (A of 100% isopropanol.

After centrifugation at 16,000 x g for one minute, the DNA was visible as a

small white pellet. The supernatant was discarded and the DNA pellet was

washed with 300 [A of 70% ethanol. After centrifugation at 16,000 x g for one

minute, the supernatant was poured off and the DNA pellet was air dried for 15

minutes. DNA hydration solution (50 /J) was then added and the DNA was

rehydrated overnight. DNA solutions were stored at -20°C.

Plasmid DNA Isolation

Plasmid DNA was isolated using the QIAprep Spin Plasmid Kit (Qiagen

Inc., Chatsworth, CA). The isolation procedure is based on the modified

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. alkaline lysis method of Birnboim and Doly (21). The procedure consisted of

preparation of a cleared lysate, adsorption of DNA to a silica gel membrame in

the presence of high salt, washing away the salt and eluting the plasmid DNA

with 50 (A Tris-CI buffer, pH 8.5. The concentration of DNA in each sample

was calculated by measuring the absorbence at 260 nm using a Lambda 3B

UV/VIS spectrophotometer (Perkin-Elmer Corp., Norwalk, CT). At 260 nm, 1

optical density unit equals 50 £*g DNA per ml. Plasmid DNA solutions w ere

stored at -20°C.

Restriction Enzyme Digestion

DNA was typically digested by mixing 1 ^9 of DNA in a 10 /A reaction

volume containing five to ten units of restriction enzyme and 1 A of the

appropriate 10X reaction buffer. All restriction enzymes and buffers were

supplied by New England Biolabs, Beverly, MA. The enzyme digestions were

incubated in a dri-bath incubator for one to three hours at the recommended

incubation temperature.

DNA Ligation

DNA was prepared for ligation by precipitation with one-tenth volume of

3 M sodium acetate, pH 4.8 and three volumes of cold 95% ethanol. After

incubation at -80°C for twenty minutes, the sample was centrifuged for five

minutes. The pellet was washed with cold 70% ethanol then dried in a vacuum

desiccator and resuspended in sterile deionized water. Ligation reactions

consisted of 1 /ug of DNA, 1 A of 10X T4 DNA ligase buffer, 1 A (400 U) of T4

permission of the copyright owner. Further reproduction prohibited without permission. DNA ligase (New England Biolabs, Beverly, MA), and water up to 10 A final

volume. The mixtures were incubated overnight at 16°C.

Fill-in Reaction

DNA fragments with 5' protruding ends were filled in by mixing 1 ^g of

DNA, 1 [A (5 U) Klenow Polymerase, 1 A 10X Buffer, 33mM dNTP mix

(Promega Inc., Madison, Wl) and water up to 10 nI final volume. Following

incubation at25°C for 15 minutes, 10 mM EDTA was added to terminate the

reaction. The polymerase was inactivated by incubation at 75°C for 10

minutes.

Nick Translation

Plasmid DNA was nick translated by using a BRL nick translation kit

(Life Technologies, Inc., Gaithersburg, MD). The reaction mixture contained

0.02 mM dGTP, dTTP, and dCTP, 0.05 M Tris-CI (pH 7.8), 5 mM magnesium

chloride, 10 mM (3-mercaptoethanol, 3 nuclease-free bovine serum albumin,

1 yjg of DNA, 20 ^Ci a 32P dATP (Amersham Life Science, Inc., Arlington

Heights, IL). Five A of DNA polymerase I and 20 units of DNase I were added

and the reaction was incubated at 15°C for one hour. Five A of stop buffer

(0.3 M EDTA, pH 8.0) was added and the mixture was filtered through a

Sephadex G-50 mini-spin column (Worthington Biochemical Corporation,

Freehold, NJ) to remove the unincorporated nucleotides. Two A of labeled

plasmid DNA was spotted on a filter disc placed in a scintillation vial and five

ml of Cytoscint scintillation fluid (ICN Biomedicals, Costa Mesa, CA) was

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. added. The counts per minute of the labeled DNA was quantified using a

Liquid Scintillation Counter (Beckman model LS 6800).

Agarose Gel Electrophoresis

Agarose gel electrophoresis of chromosomal and plasmid DNA was

carried out using 0.7% agarose gels containing 0.5 ^g/ml ethidium bromide.

The electrophoresis was performed using either the BRL Horizontal System,

Model H5 or the Hoefer Gel Unit, Model HE-33. A 50X stock solution of TAE

(1X TAE = 40 mM Tris-acetate, pH 8.0-2 mM EDTA, pH 8.0) was used as

electrophoresis buffer. The buffer was diluted to 4X for overnight runs at 25

volts or to 1X for short runs (under two hours at 90 volts). DNA samples were

prepared for electrophoresis by adding one-tenth volume of 10X loading buffer

(0.25% bromothymol blue, 40% sucrose). After electrophoresis, the DNA

bands were visualized using a shortwave UV light box (Fotodyne, Inc., New

Berlin, Wl). Gels were photographed with a Polaroid camera. The size of the

DNA fragments was estimated using molecular length markers of lambda DNA

digested with Hindlll.

DNA Recovery from Agarose Gels

DNA was routinely recovered from agarose gels using the GENECLEAN

II Kit (BIO 101, Inc., La Jolla, CA). The GENECLEAN procedure is based on

the data of Vogelstein and Gillespie (165).

Electroporation

E. coliceUs were grown in L-broth until O.D.660 in the range of 0.5 to 1.0

was reached. Approximately 30 ml of cell culture was centrifuged at 5,900 x g

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in a Sorvall SS34 rotor for eight minutes. The ceil pellet was washed with 30

ml of cold sterile water followed by five washes with 1 ml cold sterile 10%

glycerol. The cell pellet was resuspended in a final volume of 1 ml of cold

sterile 10% glycerol. Forty /A of the freshly prepared competent cells and up to

3 ixg of DNA were transferred to a sterile 0.2 cm cuvette and mixed. The cells

were subjected to electroporation in a BioRad Gene Pulser (BioRad

Laboratories, Hercules, CA) set at 2.5 kV, 25 /x f, and 200 ohms. The cell

suspension was immediately transferred to a tube with two ml of L-broth. The

cell suspension was incubated at 37°C for one hour in a roller drum and 0.1 ml

was plated onto a selective medium.

Conjugation

The R. capsulatus recipient strain PAS100 was grown in 10 ml of PYE

medium at 37°C overnight without shaking. E. coli strain S17-1, containing the

donor plasmid, was grown in 10 ml of L-broth supplemented with appropriate

antibiotics overnight at 37°C with shaking. One ml of the R. capsulatus

recipient culture was mixed with 0.1 ml of the E. coli donor culture in a sterile

tube. After centrifugation and removal of most of the supernatant, the cells

were gently resuspended in the remaining liquid and transferred to a

nitrocellulose disc placed on the surface of an RCV plate. After incubation at

room temperature overnight, the nitrocellulose disc was transferred to an RCV

plate with the appropriate supplements. The cells were spread with 0.1 ml of

RCV medium and the plate was incubated at 37°C for two to three days.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Southern Hybridization

After agarose gei electrophoresis, gels were placed in denaturing buffer

(1.5 M NaCI-0.5 M NaOH) for 30 minutes with slow shaking. The gels were

then rinsed with distilled water and placed in neutralizing buffer (0.5 M Tris-CI,

pH 7.0-1.5 M NaCI) for 30 minutes with slow shaking. Next, gels were washed

in 20X SSC transfer buffer (3 M NaCI-0.3 M sodium citrate, pH 7.0) with slow

shaking for 30 minutes. The denatured DNA was transferred to Nytran

membranes using the S & S Turboblotter and Blotting Stack Assembly

(Schleicher & Schuell Inc., Keene, NH). The Nytran membranes were prepared

by rinsing with distilled water briefly then soaking five minutes in 20X SSC.

The transfer time was three hours for small fragments and overnight for large

fragments. After transfer, the blots were washed with 2X SSC for five minutes

then baked at 80°C for one hour. Dried blots were sealed in plastic bags.

Blots were prehybridized at 65°C for one hour in five ml of prehybridization

solution (6X SSC-1% SDS-10X Denhardt’s [1 g ficoll-1 g polyvinylpyrrolidone-1

g bovine serum albumin]-2.5 mg/ml denatured salmon sperm DNA).

Prehybridizations and hybridizations were carried out in glass bottles using

Hybaid Micro-4 Hybridization Oven (National Labnet Company, Woodbridge,

NJ). Following the removal of the prehybridization solution, blots were

incubated in five ml of 6X SSC-1% SDS-2X Denhardt’s-0.5 mg denatured

salmon sperm DNA-106 counts per minute of denatured probe/ml of solution at

65°C overnight. The hybridization solution was then replaced with five ml of

2X SSC-1% SDS and blots were washed for 30 minutes at room temperature.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. They were subsequently washed twice in 0.5X SSC-1% SDS for 30 minutes at

room temperature and once at 65°C. The blots were air dried, taped to filter

paper, and used to expose Kodak XRP-5 X-ray film (Eastman Kodak Company,

Rochester, NY) for one to two days.

Protein Determination

Protein Determination by Dye Binding

The protein concentrations in cell extracts were determined using the

BioRad Protein Assay (BioRad Laboratories, Hercules, CA) which is based on

the method of Bradford (23). Bovine serum albumin (BSA) was used to create

a standard protein curve. Four dilutions of between 10 ^g and 70 ^g BSA were

placed in test tubes. The cell extract to be tested was placed in a separate test

tube. Deionized water was added to all tubes to bring the total volume to four

ml. One ml of BioRad Dye Reagent Concentrate was then added to each tube

and the mixture was vortexed. After five minutes incubation at room

temperature the absorbence at 595 nm was measured. The protein

concentration of the cell extract was determined by comparing its absorbence

to the BSA standard curve.

Lowry Protein Determination

The Lowry protein determination method (107) was used in a modified

version to estimate the protein concentration in cell extracts used for

bacteriochlorophyll measurements. Cell pellets were dissolved in one ml of 0.2

N sodium hydroxide and boiled for two minutes. One tenth ml of that sample

was transferred to another tube and deionized water added up to 0.5 ml.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bovine serum albumin (BSA) was used as a standard. Five tubes containing 0

mg to 0.4 mg BSA per ml were mixed with 0.1 ml 0.2 N sodium hydroxide and

deionized water was added up to 0.5 ml. Five ml of reagent A (1 ml 1% copper

sulfate-1 ml 1% sodium potassium tartrate-98 ml 2% sodium carbonate in 0.1 N

sodium hydroxide) was added to each 0.5 ml sample tube. After incubation for

ten minutes at room temperature, 0.5 ml of reagent B (1 N Folin’s reagent) was

added to each tube. Samples were incubated at room temperature for 30

minutes and the absorbence at 660 nm was measured. The protein

concentration was determined by comparison of the samples’ absorbence to

the BSA standard curve.

Preparation of Cell Extracts

R. capsulatus was grown either in 200 ml of RCV overnight or spread

onto RCV plates containing hemin, cystine and methionine so that

approximately 300 colonies grew on each plate and colonies were 1-3 mm in

diameter. When necessary, the medium contained appropriate antibiotics.

Strain AJB529 was grown in the presence of 0.1 mM 5-aminolevulinate. The

cells were harvested by centrifugation at 10,400 x g for ten minutes. Cell

pellets were suspended in either two ml of 0.1 M Tris-CI, pH 7.5 or two ml of

0.1 M potassium phosphate buffer, pH 7.6-0.01 M p-mercaptoethanoi. Cells • were then sonicated three to four times for 20 seconds each with one minute

cooling periods using a Heat Systems Ultrasonic Sonicator, model W-220

(Ultrasonics, Inc.). The cell suspension was then centrifuged in a Sorvali SS34

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. rotor at 27,000 x g for 20 minutes and the extract transferred to another tube

and stored at 4°C.

Quantitation of Bacteriochlorophyll

The bacteriochlorophyll concentration in R. capsulatus cultures was

measured by centrifuging one ml of cell culture then resuspending the cell

pellet in one ml of deionized water and extracting the bacteriochlorophyll with

one ml of acetone-methanol (7:2). After incubation in the dark for 15 minutes,

the suspension was centrifuged and the absorbence of the supernatant was

measured at 770 nm in a Perkin-Elmer Lambda 3B UVA/IS Spectrophotometer

(Perkin-Elmer Corporation, Norwalk, CT). The pellet was used for protein

concentration determination by the modified Lowry method. The

bacteriochlorophyll concentration of the sample was determined using the

millimolar extinction coefficient of 76 (33).

Aminolevulinate Synthase Assay

Cell extracts were prepared in 0.1 M Tris-CI, pH 7.5 as described above

and stored at 4°C for two hours to allow activation of the 5-aminolevulinate

synthase (98) then assayed by the procedure described by Burnham (25). The

assay mixture contained 75 /u\ of cofactor mixture (2 ml 0.2 M ATP, pH 7.0-

1.75 ml 0.01 M CoA-1.35 ml 0.01 M pyridoxal phosphate-2 ml deionized water),

175 (A of substrate mixture (5 ml 1 M glycine-5 ml 1 M succinate, pH 7.0-5 ml

0.1 M MgCI2-2.5 ml 1 M Tris-CI, pH 7.5), and 250 jul of crude extract and

deionized water. The blank contained the same ingredients except the

substrate mixture was replaced with deionized water. Samples were incubated

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. for 30 minutes at 37°C. The reaction was stopped by adding 250 ^I of 10%

trichloroacetic acid. The mixture was centrifuged for two minutes and the

supernatant was transferred to another tube containing 1 ml of sodium acetate,

pH 4.7 and 25 iA of 2,4-pentanedione. The tubes were capped and placed in a

boiling water bath for 15 minutes. The samples were then cooled to room

temperature and 1.75 ml of freshly prepared modified Ehrlich’s reagent (0.5 g

p-dimethylaminobenzaldehyde-21 ml glacial acetic acid-4 ml 70% perchloric

acid) was added. Samples were incubated at room temperature for 20 minutes

and the optical density at 556 nm was measured. The aminolevulinate

synthase specific activity was determined as nanomoles aminolevulinate

formed per hour per mg protein.

Porphobilinogen Synthase Assay

Porphobilinogen synthase activity was measured using the procedure

developed by Shemin (146). Cell extracts were prepared in 0.1 M potassium

phosphate buffer, pH 7.6 with 0.01 M P-mercaptoethanol as described above.

One hundred(A of crude extract was added to 150 iA 1 M Tris-CI, pH 8.5, 75 /A

1 M KCI, 50 ^l 0.1 M aminolevulinate, pH 7.0, 0.6 y\ P-mercaptoethanol, and

1.125 ml water and incubated for 30 minutes at 37°C. The reaction was

stopped by adding 0.5 ml of 20% trichloroacetic acid and 0.1 M mercuric

chloride. Samples were centrifuged for two minutes and two ml of modified

Ehrlich’s reagent (0.5 g p-dimethylaminobenzaldehyde-21 ml glacial acetic

acid-4 ml 70% perchloric acid) was added to the supernatant. Samples were

incubated at room temperature for five minutes. The optical density at 555 nm

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. was measured. The porphobilinogen synthase specific activity was determined

as nanomoles porphobilinogen formed per hour per mg protein.

Porphobilinogen Deaminase Assay

Porphobilinogen deaminase activity was measured using the procedure

described by Jordan and Shemin (82). Cell extracts were prepared in 0.1 M

Tris-CI, pH 8.0 as described above. Porphobilinogen deaminase was assayed

by following the disappearance of the substrate porphobilinogen. The assay

mixture contained 50 mM Tris-CI, pH 8.0, 0.2 mM porphobilinogen (Porphyrin

Products, Logan, UT) and crude cell extract in a total volume of one ml. The

mixture was incubated at 37°C . At intervals, aliquots were removed for

monitoring porphobilinogen usage. To assay the disappearance of

porphobilinogen, aliquots of 100 were transferred into 1.4 ml of 10%

trichloroacetic acid. To this, 1.5 ml of modified Ehrlich’s reagent (0.5 g p-

dimethylaminobenzaldehyde-21 ml glacial acetic acid-4 ml 70% perchloric

acid) was added and the samples incubated for ten minutes at room

temperature. The absorbence was then measured at 554 nm in a Perkin-Elmer

Lambda 3B UV/VIS spectrophotometer (Perkin-Elmer Corp., Norwalk, CT).

The porphobilinogen deaminase specific activity was determined as nanomoles

porphobilinogen consumed per hour per mg protein.

Measurement of Porphyrin Accumulation

Cultures of AJB530 were grown on solid RCV media for two days then

harvested and diluted to obtain approximately 5.6 X 107 cells/ml. One-tenth ml

of cells were spread onto solid RCV media containing no carbon source. They

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were then incubated both at atmospheric oxygen conditions and under

anaerobic conditions at 37° C. They were also incubated on media with and

without porphobilinogen. After an incubation period of from one to four hours,

the ceils were harvested and assayed for porphyrin accumulation using the

method described previously (17).

Measurement of Porphobilinogen Accumulation

Cultures of AJB565, the hemC mutant, were grown for two days on solid

RCV media with hemin, cysteine, and methionine added. Cells were then

harvested and diluted to approximately 5.6 X 107 cells/ml. One-tenth ml of cells

were then spread onto RCV media without a carbon source. Cells were

incubated for one to four hours both under atmospheric oxygen conditions and

under anaerobic conditions. Cells were harvested and sonicated as described

previously. Porphobilinogen accumulation was measured by the method of

Shemin (146). The cells were sonicated, then 0.5 ml of 20% trichloroacetic

acid and 0.1 M mercuric chloride was added. Samples were centrifuged for two

minutes and two ml of modified Ehrlich’s reagent (0.5 g p-

dimethylaminobenzaldehyde-21 ml glacial acetic acid-4 ml 70% perchloric

acid) was added to the supernatant. Samples were incubated at room

temperature for five minutes. The optical density at 555 nm was measured.

Results were reported as micromoles porphobilinogen per liter.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS

Crossfeeding Studies

It was previously observed that R. capsulatus strain AJB530 excretes a

soluble protoporphyrin-porin complex (20). A series of studies were initiated to

determine whether this complex might be used to grow E. coli and R.

capsulatus hem mutants. As a first step, the ability of AJB530 to crossfeed E.

coli hem mutants was determined. E. coli hemA, hemB and hemD mutants

were cross-streaked with the R. capsulatus strain AJB530 on PYE medium.

This medium does not contain a fermentable carbon source and will not

support the growth of E. coli hem mutants. These studies showed that the E.

coli hem mutants can be crossfed by AJB530. As can be seen in Figure 2, an

E. coli hemA mutant, S905, is shown growing as a vertical streak due to the

* porphobilinogen-porin complex secreted by AJB530.

Growth o f E. coli hem Mutants Using Exogenous Protoporphyrin and Hemin

Having determined that the R. capsulatus protoporphyrin-porin complex

can promote the growth of E. coli hem mutants, the next step was to develop a

medium to grow these mutants. In addition to being able to grow using the

protoporphyrin-porin complex secreted by AJB530, E. coli hem mutants are

also able to grow without a fermentable carbon source when exogenous

protoporphyrin and porin alone from R. capsulatus strain PAS100 are added to

liquid medium. To make this media, the supernatant from a culture of R.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2. Crossfeeding study AJB530 is shown as a horizontal streak and S905, anE. coli hemA mutant, is shown growing vertically.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. capsulatus was autoclaved and protein assays performed. Protein levels

ranged from 1.5 to 2.0 ^g/ml. Seventy-five percent of protein in the

supernatant was found to be the outer membrane porin of R. capsulatus (20).

This medium was optimized by adding different amounts of porin and

protoporphyrin and growth rate as well as growth yield was measured. Varying

amounts of supernatant were added to PYE media containing 10 mg

protoporphyrin. Growth rates were unaffected, with generation times of

approximately 50 minutes (data not shown). Hovuever, growth yield in media

containing 0.6 /^g/ml of porin (total protein 0.8 //gVml) was found to increase

approximately 19 fold compared to media without porin (Figure 3). Growth

yield increased with increasing amounts of porin up to 0.6 then declined

sharply. Next, 0.6 fj.glm\ porin was added to PYE media along with different

amounts of protoporphyrin and growth yields measured (Figure 4). Growth

yields again increased steadily as the protoporphyrin amount increased up to 1

mg/ml protoporphyrin. Amounts above 1 mg/ml produced decreasing growth

yields. Optimum growth yield was obtained using 1 mg/ml of protoporphyrin

and 0.6 ^g/ml of porin. These results are representative of several trials.

Similar results were obtained using hemin instead of protoporphyrin.

Based on the E. coli growth results above, the ability of the R.

capsulatus hemA mutant to grow on this medium -was assayed. Surprisingly, it

was found that 99 % of the cells were killed after Just 1.5 hours. Different

amounts of porin and protoporphyrin were tried with similar results.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Growth yield

t u

_ I 60

0 I ' .

0.2 0-4 0.6 Q-3 1.0

|jg/ml porin

Figure 3. Growth yield of E. coli S905 with varying porin. The indicated amounts of porin were added to PYE medium. The medium was inoculated with E. coii strain S905 cells. After 24 hours incubation, cfu/ml were measured.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Growth yield

50 -

° 40 x E 2o 30

i • 2 0 - t i ;

10 i ; f» ■ * t ♦ 11 n □ 0.025 0.05 0.1 0.15 025 035 0.5 0.75 10 125 15

mg/ml protoporphyrin IX

Figure 4. Growth yield of E. coli S905 with varying protoporphyrin. The indicated amounts of protoporphyrin were added to PYE medium. The medium was inoculated with E. coli strain S905 cells. After 24 hours incubation, cfu/ml were measured.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. When protoporphyrin was added to the medium but no porin, the cells were not

killed as quickly as when porin was added however, the cell numbers increased

by only one generation in 24 hours. This is comparable to the results obtained

by using media without porin or protoporphyrin included. Although this medium

can be used to grow E. coli hem mutants by respiration, it can not be used to

grow R. capsulatus hem mutants.

Growth of an R. capsulatus hemA Mutant on Exogenous Hemin

Since R. capsufatus is unable to grow fermentatively, hem mutants would

be expected to require exogenous protoporphyrin or hemin for growth. We

used the hemA mutant, AJB529 (172) to develop a medium that would allow

growth of a hem mutant on hemin. Our laboratory has previously demonstrated

(172) that this mutant can form 1 mm diameter colonies in two days on a malate

minimal salts medium (RCV; 16) containing 0.1 mM aminolevulinate, and can

form similar sized colonies on RCV media containing 0.1 mM hemin (dissolved

in 0.1 M sodium hydroxide) in three to four days (172). Growth on this medium

was not sufficient to allow the isolation of R. capsulatus hem mutants (29).

However, it was found that by increasing the hemin concentration to 0.5 mM,

AJB529 was able to form 1 mm diameter colonies within two days.

Interestingly, this mutant grows very poorly in liquid media (172).

Isolation o f an R. capsulatus hemC Mutant

The R. capsulatus hemC gene was previously cloned into pUC18 (164)

to yield pCAP154 (Canada and Biel, unpublished; Ineichen and Biel,

unpublished). The 4.0 kb BamHI fragment from pCAP154 was then inserted

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. into the vector pSUP202 (149), yielding pCAP180. This vector was chosen

because it can be mobilized into R. capsulatus, but replicates only in E. coli

(149). The 1.3 kb kanamycin cartridge was removed from pl)C-4K (164) by

cutting with HincW and blunt-end ligated into theStul site within the hemC gene

in pCAP180. The resulting plasmid, pCAP181, has a kanamycin cartridge

within the hemC gene and contains approximately 0.9 kb of R. capsulatus DNA

on one side of the cartridge and 3.1 kb on the other side (Figure 5).

This plasmid was mobilized from E. coli strain S17-1 (164) to R.

capsulatus strain PAS100 (158) using the procedure described by Marrs (113).

After overnight mating, the cells were spread onto RCV plates containing 0.5

mM hemin, 10 //g per ml kanamycin, 0.5 mM cysteine and 0.5 mM methionine

to select for kanamycin-resistant colonies. The resulting colonies were then

screened for hemin-dependence, which could only arise by recombination of

the mutated hemC gene into the R. capsulatus chromosome. A kanamycin-

resistant, hemin-requiring strain, designated AJB565, was isolated and used

for all subsequent experiments. This strain forms 1 mm diameter colonies

within two days on plates containing hemin, cysteine and methionine. As

expected, AJB565 required cysteine and methionine in addition to hemin.

Since siroheme and vitamin B-12 are required for the production of the amino

acids cysteine and methionine and the mutation in this strain is before the

branch needed to make these amino acids, they must be included in the

medium. As is the case for the hemA mutant AJB529, the hemC mutant would

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. kan

B pCAP181 hem C hem E E H pCAP168

Figure 5. Restriction map of pCAP181 and pCAP168. B=BamHI; E-EcoRI; H=Hindlll

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. only grow on solid media containing hemin, not in liquid media containing

hemin.

Chromosomal Location of the Kanamycin Cartridge

In order to confirm that the kanamycin cartridge had recombined into

the chromosome within the hemC gene, Southern blot analyses were

performed. Chromosomal DNA from strains PAS100 and AJB565 were

digested with EcoRI and separated by agarose gel electrophoresis. After

blotting to a Nytran filter, the blots were incubated with ^P-labeled probes

made from pUC-4K, which contains the kanamycin-resistance gene. As

expected, pUC-4K hybridized to a 9.0 kb fragment of AJB565 DNA, but did not

hybridize to PAS100 DNA (Figure 6, lanes 3 & 4). These results indicate that

the 1.3 kb kanamycin cartridge was inserted into a chromosomal EcoRI

fragment of approximately 7.7 kb. Chromosomal DNA from the two strains was

also probed with pCAP168 (Canada and Biel, unpublished) which contains a

portion of the hemC gene (Figure 5). This probe hybridized to a 7.5 kb

fragment of PAS100 DNA and to a 9.0 kb fragment in AJB565 (Figure 6, lanes

5 & 6), confirming that there is an insertion of the kanamycin cartridge in the

hemC gene of AJB565. In order to confirm that the entire plasmid had not

inserted into the chromosome, both PAS100 and AJB565 were probed with

pSUP202, the vector used to construct pCAP181. As can be seen (Figure 6,

lanes 1 and 2), pSUP202 did not hybridize to DNA from either of these strains.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 2 3 4 5 6

Figure 6. Southern hybridization of R. capsulatus chromosomal DNA

1 - PAS100 probed with pSUP202 2 - AJB565 probed with pSUP202 3 - PAS100 probed with pUC-4K 4 - AJB565 probed with pUCMK 5 - PAS100 probed with pCAP168 6 - AJB565 probed with pCAP168

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Bacteriochlorophyll Levels in Parental and Mutant Strains

AJB565 was first characterized by measuring the amount of

bacteriochlorophyll produced compared to the parental strain, PAS100, and the

hemA mutant, AJB529 (Table 3). Since the hemC mutant will not grow in liquid

media, all measurements were performed on cultures grown on RCV hemin

agar plates. PAS100 was found to produce 3.9 fzg of bacteriochlorophyll per

mg of protein when growing on RCV hemin plates, as compared to AJB529,

which produces 0.4 mg of bacteriochlorophyll per mg of protein. It was

previously demonstrated that the hemA mutant, AJB529, is somewhat leaky,

and produces approximately 10% of the normal amount of bacteriochlorophyll

(172). As would be expected for a hemC mutant, AJB565 did not produce a

measurable amount of bacteriochlorophyll.

Enzyme Activities in the Parental and Mutant Strains

To further characterize AJB565, the activities of several enzymes in the

tetrapyrrole pathway were assayed and the results compared to the parental

strain, PAS100 and the hemA mutant, AJB529. Since AJB565 could not be

grown in liquid media, PAS100 and AJB529 were grown both in liquid media

and on solid media and enzyme activities measured to determine if the different

growth conditions affected the specific activities of the enzymes. As shown in

Table 3, the different growth conditions had little, if any, effect on the specific

activities of the enzymes in these two strains.

As expected, the specific activity of aminolevulinate synthase in AJB529

was dramatically lower than that of the parental strain, while the specific

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3. Bacteriochlorophyll levels and enzymatic activities.

Strain Growth Bchla ALA PBG PBG Condition Synthaseb Synthase6 Deaminased PAS 100 broth 74 220 130 PAS 100 agar 3.9 78 220 110 plates AJB529 broth 9.0 210 160 AJB529 agar 0.4 9.1 180 120 plates AJB565 agar 0.0 91 210 NDe plates

a Bacteriochlorophyll concentration was measured as described by Clayton (33), using a millimolar extinction coefficient of 76 (770 nm) and is expressed as nmol per mg protein.

b Aminolevulinate synthase specific activity was determined as described by Burnham (25) and is expressed as nanomoles aminolevulinate formed per hour per mg of protein.

c Porphobilinogen synthase specific activity was determined as described by Shemin (146) and is expressed as nanomoles porphobilinogen formed per hour per mg of protein.

d Porphobilinogen deaminase specific activity was determined by measuring the disappearance of porphobilinogen, as described by Jordan and Shemin (82) and is expressed as nanomoles porphobilinogen consumed per hour per mg of protein.

° Not detected. Minimum detection level is 4 nanomoles porphobilinogen consumed per hour per mg of protein.

Above results are representative of several trials.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activities of porphobilinogen synthase (hemB) and porphobilinogen deaminase

(hemC) were comparable to those of the parental strain. The specific activities

of aminolevulinate synthase (hemA) and porphobilinogen synthase (hemB) in

AJB565 were comparable to those of the parental strain and the hemA mutant,

indicating that the enzymes in the tetrapyrrole pathway prior to porphobilinogen

deaminase were unaffected by the insertion of the kanamycin cartridge.

AJB565 did not produce a measurable amount of porphobilinogen deaminase,

indicating again that the kanamycin cartridge inserted into the hemC gene and

disrupted it, preventing the synthesis of porphobilinogen deaminase.

Porphyrin Measurements in AJB530

Since the hemC mutant can’t be grown in liquid media, experiments to

determine whether porphobilinogen formation is regulated by oxygen had to be

carried out on solid media. To show that cells can be grown aerobically on

solid media if the cell concentration is low, porphyrin levels in AJB530 were

measured from cells incubated with and without oxygen on solid media.

AJB530 was used in these measurements because it accumulates large

amounts of porphyrins which would be easy to monitor. AJB530 was grown

overnight, then harvested and spread onto plates without a carbon source for

incubation. Porphyrin levels were measured every hour for four hours. Cells

were incubated on plates with and without porphobilinogen in the medium

under atmospheric oxygen conditions and in anaerobic containers. As can be

seen in Table 4, when incubated without porphobilinogen in an anaerobic

environment, the amount of porphyrins increased 4.5 fold over four

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4. Porphyrin measurements in AJB530

-PBG +PBG Time(hr) +0, *2 +0, ^ 2

0 4.0a 4.0 12 12

2 4.0 4.0 28 32

4 4.0 18 32 40

aUnits are presented as (M. Results are representative of several trials. PBG=porphobilinogen

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. hours. When incubated in the presence of oxygen, the porphyrin level did not

increase. These results are consistent with previous experiments conducted in

liquid media (16). When the cells were incubated in the presence of

porphobilinogen, with or without oxygen, the porphyrin levels rose 2.7 and 3.3

fold, respectively. There was no increase in cell number during the incubation

period as determined by plate counts. These results are in agreement with

previous studies in liquid media (17). These results are representative of

several trials.

Porphobilinogen Measurements in AJB565

The hemC mutant, AJB565, accumulates porphobilinogen due to the

block in its tetrapyrrole pathway. To determine whether the point of oxygen

regulation of the tetrapyrrole pathway occurs before or after porphobilinogen,

AJB565 was incubated with and without oxygen for four hours and

porphobilinogen levels were measured every hour. AJB565 was first grown on

RCV media containing hemin, cysteine, and methionine for two days. The cells

were then harvested, diluted, and spread onto RCV plated without a carbon

source. As can be seen in Table 5, porphobilinogen levels were similar

regardless of whether AJB565 was incubated with or without oxygen. There

was a 1.4 andl .5 fold increase, respectively, in porphobilinogen levels after the

four hour incubation. This indicates that the formation of porphobilinogen is not

regulated by oxygen. As a control, this assay was also performed on the

parental strain, PAS100. The strain had a small amount of porphobilinogen,

17.6 mM, which did not increase during the four hour incubation period.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5. Porphobilinogen measurements in AJB565

Time(hr) +Q? -O?

0 17.0a 17.0

1 20.5 19.5

2 25.7 23.7

3 29.6 26.9

4 25.5 23.7

aUnits are presented as //M. Results are representative of several trials.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DISCUSSION

R. capsulatus has the ability to grow under diverse environmental

conditions. It also has the ability to synthesize all possible tetrapyrrole end

products and regulate their synthesis based on environmental conditions. This

has made it a very useful organism for studying the tetrapyrrole pathway.

Bacteriochlorophyll levels vary greatly with growth conditions while heme levels

are relatively constant. This regulation has been the focus of extensive

studies.

The focus of our research has been to study the oxygen regulation of

the common tetrapyrrole pathway in R. capsulatus. Members of our lab have

attempted to do this by the cloning, sequencing, and characterization of the

hem genes of the common tetrapyrrole pathway. Members of this lab have

cloned and sequenced several of the hem genes such as hemA (19), hemB

(67), hemC (28), hemE (68), and hemH (88). In addition, several attempts

have been made to produce hem mutants of R. capsulatus.

Since R. capsulatus has no heme-independent growth mode, a mutation

in a hem gene would be lethal unless the mutant was able to use exogenous

tetrapyrrole intermediates. Wright et al. (172) were able to isolate a hemA

mutant using transposon mutagenesis. The mutant is able to grow using

exogenous aminolevulinate and will also grow slowly if 0.1 mM hemin or

protoporphyrin IX is added to the medium. Cardin and Biel (30) tried to isolate

hem mutants by selecting for transposon insertion mutants on medium

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. containing exogenous hemin. The insertion mutants were then screened for a

hemin requirement. Approximately 4400 colonies were screened without

isolating any hem mutants (29). They concluded that growth of R. capsulatus

on the medium containing hemin was too poor to allow for selection of hem

mutants.

Another approach was taken in an attempt to isolate hem mutants by

developing a medium which allows growth of hem mutants. R. capsulatus

strain AJB530 was found to excrete large amounts of protoporphyrin and

coproporphyrin bound to a protein (20). The protein was found to be the major

outer membrane porin of R. capsulatus (20). Attempts were made to find out

whether hem mutants could use this protoporphyrin-porin complex in order to

grow by respiration. Since E. coli can grow by fermentation, there are several

E. coli hem mutants. Crossfeeding studies were done which showed that E.

coli hem mutants were able to use the protoporphyrin-porin complex to grow.

The R. capsulatus outer membrane porin was also found to be excreted

by w ild type R. capsulatus without protoporphyrin or coproporphyrin attached.

Therefore, the supernatant from the R. capsulatus strain PAS100 was used to

make a liquid medium to which protoporphyrin could be added. This allowed

us to optimize the medium by varying the concentration of porin and

protoporphyrin in the medium in order to find which concentrations allow

maximum growth. For E. coli hem mutants, approximately a 20 fold increase in

growth yield was obtained using a medium containing 1 mg/ml protoporphyrin

and 0.6 ^g/ml porin compared to a medium

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. without the porin. It appears likely that the porin in the medium allows the

protoporphyrin to remain soluble so that it can be taken up by the cells.

The next step was to use this medium to grow the R. capsulatus hemA

mutant and possibly use this medium to isolate other R. capsulatus hem

mutants. Surprisingly, this medium would not support the growth of the hemA

mutant. In fact, when the R. capsulatus hemA mutant was inoculated in this

medium it lived a much shorter time than when it was inoculated into media

without protoporphyrin or porin. Perhaps since the R. capsulatus hemA m utant

already contains the porin which was added to the medium and excretes it into

the medium itself, the additional porin overloads the cells and is lethal to them.

While this medium would not support the growth of R. capsulatus hem

mutants, and so can not be used for our purposes, it should prove useful in

growing hem mutants of E. coli or other organisms. E. coli hem mutants must

grow by fermentation or have a second mutation that allows them to take up

hemin from the medium. The inclusion of the R. capsulatus porin in the

medium apparently solubilizes the protoporphyrin or hemin and allows its

uptake by E. coli. This obviates the need for the isolation of a hemin transport

derivative of the E. coli hem mutants. Since the R. capsulatus porin is a very

stable protein, the medium is relatively easy to make and use. We have not

investigated the mechanism by which E. coli takes up the hemin or

protoporphyrin. For example, it might be interesting to determine whether or

not the porin binds to E.coli, and what E. coli proteins might be involved in this

process.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A new approach was taken to develop media which would allow growth

o f the R. capsulatus hemA mutant. Since Wright et al. (172) had some success

in growing the hemA mutant on media containing 0.1 mM hemin dissolved in

NaOH, we decided to try to optimize this media to get better growth. We found

that increasing the amount of hemin to 0.5 mM produced much faster growth.

The hemA mutant can form 1 mm colonies in just two days as opposed to four

days on media with 0.1 mM hemin. Interestingly, thehemA mutant will not

grow in liquid media containing hemin. Similar results were obtained by Wright

et al. (172).

Once a medium was obtained which would support growth of the R.

capsulatus hemA mutant, and presumably other hem mutants, we used it to

isolate more mutants. Since several R. capsulatus hem genes have now been

cloned, this would allow us to make a hem mutation in vivo by inserting an

antibiotic resistance cartridge into a plasmid borne copy of the hem gene o f

interest and moving it intoR. capsulatus by conjugation. This would allow us to

select for a high probability recombination event instead of a lower probability

transposition event.

A previous member of our lab, Dr. Georgia Ineichen, constructed a

plasmid, pCAP154 (Canada and Biel, unpublished; Ineichen and Biel,

unpublished), which contains the hemC gene. The fragment containing the

hemC gene was moved into pSUP202 (149) to make pCAP180. This formed a

plasmid which can be mobilized into R. capsulatus but can’t replicate there,

allowing us to select for cells in which the hemC gene from the plasmid had

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. recombined into the chromosome. A kanamycin cartridge was then inserted

into the middle of thehemC gene on pCAP180, forming pCAP181. This

allowed selection for kanamycin resistance. After mating, the kanamycin-

resistant colonies were screened for hemin-dependence, resulting in one

hemin-dependent colony, AJB565. This strain grows well on solid RCV media

containing hemin, cysteine and methionine. Siroheme and vitamin B-12 are

required for production of the amino acids cysteine and methionine. Therefore,

since the mutation in the pathway is before the branch needed to make

siroheme and vitamin B-12, the strain requires both to be included in the

medium. As reported for the hemA mutant, the hemC could not be grown in

liquid media.

Southern blot analyses showed that the kanamycin cartridge was

inserted into thehemC gene of AJB565, the hemC mutant. Strains AJB565

and PAS100 were also probed with a plasmid containing a portion of the hemC

gene. It hybridized to a 7.5 kb fragment in PAS100 but to a 9 kb fragment in

AJB565. This shows that the hemC gene in AJB565 does in fact have an insert

the size of the kanamycin cartridge, 1.5 kb. To show that this mutant was

formed by homologous recombination between the plasmid pCAP181 and the

chromosome, the blots were also probed with pSUP202 which is the vector

used to construct pCAP181. It did not hybridize to either thehemC mutant or

the parental strain.

An R. capsulatus hemC mutant would be unable to produce

bacteriochlorophyll when grown on exogenous hemin. Since the hemC m utant

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. does not grow in liquid media, a method had to be devised to measure

bacteriochlorophyll levels in cells grown on solid media. Dilutions of R.

capsulatus cells were plated so that 200 to 300 colonies grew on each plate.

After incubation for two days, the cells were washed off the plates and the

bacteriochlorophyll levels were determined. The values obtained for strains

PAS100 and AJB529 were similar to those obtained from cultures grown in

liquid media (168). As found previously, the bacteriochlorophyll level in

AJB529 was approximately 10% of the level found in PAS100 (168). This is

due to the fact that the hemA mutation is slightly leaky (168). As predicted, the

hemC mutant, AJB565, did not produce a measurable amount of

bacteriochlorophyll.

Other enzyme assays were also performed to further characterize the

hemC mutant. Several of the enzymes in the tetrapyrrole pathway were

measured in the hemC mutant and compared to the parental strain as well as

the hemA mutant. PAS100 and AJB529 were grown in liquid as well as on

solid media to determine whether the different growth conditions had any effect

on the assay results. Aminolevulinate synthase as well as porphobilinogen

synthase levels were similar regardless of growth conditions. Porphobilinogen

deaminase levels were slightly lower when grown on solid media compared to

cells grown in liquid media. In AJB529, the HemA protein, aminolevulinate

synthase, was produced in much lower amounts than in the parental strain.

Porphobilinogen synthase and porphobilinogen deaminase levels in AJB529

were comparable to the parental strain. In AJB565, aminolevulinate synthase

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and porphobilinogen synthase levels were similar to the parental strain which

shows that the enzymes of the tetrapyrrole pathway before porphobilinogen

deaminase were unaffected by the mutation. AJB565 produced no measurable

amount of porphobilinogen deaminase, confirming that the hemC gene was

disrupted by the kanamycin cartridge making it unable to produce the hemC

enzyme.

All these results together indicate that strain AJB565 has an insertion on

its hemC gene, making it the first hem mutant with a complete block in the

tetrapyrrole pathway in a photosynthetic bacterium. The next step was to use

this mutant to measure the effect of changes in oxygen tension on the levels of

porphobilinogen in the cell. This would allow us to determine if the point of

oxygen regulation in the tetrapyrrole pathway occurs before the hemC gene or

after.

After more than forty years, the mechanism by which oxygen regulates

the common tetrapyrrole pathway is still not understood. In fact, even now, it is

not yet clear which step in the pathway oxygen regulates. In R. capsulatus, it

has been demonstrated that oxygen does not regulate aminolevulinate

formation (17), nor does it regulate transcription of the hemA gene (172).

Aminolevulinate synthase is feedback inhibited by heme, but that control

mechanism is independent of regulation by oxygen (86). All of these

observations are more understandable in light of the fact that only a small

fraction of the aminolevulinate formed is converted into tetrapyrroles. Most of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the aminolevulinate is reduced to aminohydroxyvalerate by aminolevulinate

dehydrogenase (Khanna and Biel, unpublished observations).

A study in which porphyrin accumulation from exogenous

porphobilinogen was measured indicated that exogenous porphobilinogen can

cause high levels of porphyrin accumulation even in cultures grown under high

oxygen (17). This result was confirmed by moving a plasmid containing the R.

capsufatus hemB gene into R. capsulatus strain AJB530. In this strain, the

increased levels of porphobilinogen synthase resulted in the accumulation of

high levels of porphyrins even in cultures grown under high oxygen (66).

These results suggest that oxygen either regulates the synthesis of

porphobilinogen from aminolevulinate, or the conversion of porphobilinogen

into uroporphyrinogen III.

The existence o f an R. capsulatus hemC mutant would greatly help in

determining which of these two steps is regulated by oxygen. A hemC m utant

lacks porphobilinogen deaminase and as such is unable to convert

porphobilinogen to uroporphyrinogen III. Such a mutant could be grown under

high and low oxygen tensions and the intracellular levels of porphobilinogen

could be determined. If oxygen regulates the synthesis of porphobilinogen

from aminolevulinate, then the intracellular level of porphobilinogen would be

high when a hemC mutant was grown under low oxygen and would be low

when the mutant was grown under high oxygen. If, on the other hand, oxygen

regulates some step after the synthesis of porphobilinogen, then the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. intracellular levels of porphobilinogen in a hemC mutant would be expected to

be high regardless of the oxygen tension under which the mutant was grown.

Since the hem C mutant, like the hemA mutant, will not grow on hemin in

liquid media (171), the experiments seeking to measure porphobilinogen levels

had to be carried out on solid media. As a control, porphyrin levels in AJB530

incubated anaerobically and aerobically on solid media were measured. Most

of the cells in a colony will be anaerobic. When incubated in the presence of

oxygen, porphyrin levels did not increase. However, when incubated

anaerobically, porphyrin levels increased 4.5 fold. These results are in

agreement with those obtained when AJB530 was grown under high and low

oxygen in liquid media (17). The agreement between these results

demonstrate that it is possible for cells on solid media to remain aerobic if the

cell concentration is low enough. By not including a carbon source in the

medium, it was possible to avoid problems due to differences in growth rate

that might occur under different growth conditions. As a further control,

AJB530 was grown aerobically and anaerobically in the presence of exogenous

porphobilinogen. Under these conditions, porphyrin levels were elevated

under both high and low oxygen tensions, confirming that exogenous

porphobilinogen can alleviate oxygen-mediated regulation of the common

tetrapyrrole pathway. Again, these results are in agreement with those

obtained when AJB530 was grown in a liquid medium (17).

Next, the hem C mutant, AJB565, was incubated aerobically and

anaerobically to determine whether porphobilinogen accumulation is regulated

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. by oxygen tension. Both aerobic and anaerobic incubation resulted in

increased porphobilinogen accumulation (Table 5). These results suggest that

oxygen does not regulate the synthesis of porphobilinogen. It seems most

likely therefore that oxygen regulates the conversion of porphobilinogen to

uroporphyrinogen III.

It has been demonstrated that oxygen does not regulate transcription of

the hem C gene (28). Thus, it is unlikely that oxygen controls the common

tetrapyrrole pathway by regulating the synthesis of porphobilinogen

deaminase. It is possible that the activity of this enzyme is somehow regulated

by oxygen. Seen in this light, the observations of Canada (28) are most

interesting. Part of his research involved purifying and characterizing

porphobilinogen deaminase. This enzyme polymerizes four molecules of

porphobilinogen to form the linear tetrapyrrole, hydroxymethylbilane. In the

assay for this enzyme, the hydroxymethylbilane formed is spontaneously

converted to uroporphyrinogen I, which is then oxidized to uroporphyrin I.

Canada (28) demonstrated that porphobilinogen deaminase is much more

efficient in converting porphobilinogen to hydroxymethylbilane when anaerobic

conditions were maintained throughout cell growth and enzyme assay. Under

these conditions, approximately 13 porphobilinogen molecules were consumed

for every molecule of uroporphyrin I formed. When the cells were grown

aerobically and the enzyme assayed under aerobic conditions, the number of

porphobilinogen molecules used to form one molecule of uroporphyrin I

increased to 24. Only when the reducing agent sodium borohydride was added

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to the reaction mixture did the ratio of porphobilinogen consumed to

uroporphyrin I formed drop to the expected 4:1 (28)t

All evidence to this point suggests that the rmajor control point of oxygen-

mediated regulation in the common tetrapyrrole pathway is the conversion of

porphobilinogen to hydroxymethylbilane. We hypothesize that under aerobic

conditions porphobilinogen deaminase produces muostiy dipyrrole and tripyrrole

intermediates instead of the tetrapyrrole that can ciarcularize to form

uroporphyrinogen III. Under anaerobic conditions, rthe enzyme most often

forms tetrapyrroles with fewer dipyrrole and tripyrroMe intermediates being

formed. Such shifts in the ability of the enzyme to successfully polymerize

porphobilinogen into hydroxymethylbilane has been noted in purified

porphobilinogen deaminase from other organisms, sand mechanistic studies

have been performed on the enzyme from E. coli (review ed in 28).

Considerable work remains to be done to confirm this hypothesis. The

isolation o f a hem E mutant would allow us to measure the effect of oxygen

tension on uroporphyrinogen III accumulation. The ? hypothesis would also be

greatly strengthened if the levels of dipyrroles and tiripyrroles formed could be

measured in vivo, perhaps by feeding a culture radioactive porphobilinogen

and following the production of radioactive derivatiwes under high and low

oxygen tensions.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. VITA

Karen Coleman Sullivan was bom on August 20, 1969, in Orange,

Texas. She graduated from Hanson Memorial High School in Franklin,

Louisiana, in May 1987. In December 1991, she was awarded the degree of

Bachelor of Science in biology from Nicholls State University in Thibodeaux,

Louisiana. In January 1996, Karen entered the doctoral degree program in the

Department of Microbiology at Louisiana State University, Baton Rouge, under

the direction of Alan J. Biel, her graduate advisor. Karen is currently a doctoral

candidate in the Department of Biological Sciences at Louisiana State

University, Baton Rouge. In May of 2001 the degree of Doctor of Philosophy

will be granted at the spring commencement.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DOCTORAL EXAMINATION AND DISSERTATION REPORT

Candidate: Karen Coleman Sullivan

Major Field: Microbiology

Title of Dissertation: Isolation and Characterization of a Rhodobacter capsulatus hemC Mutant

Approved:

EEL*

EXAMINING COMMITTEE:

Date of Examination:

12 - 8 -0 0

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