ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS of the TUT FDGH OPERON of THAUERA AROMATICA STRAIN T1 a Dissertation Presented T

Home , Alanine, Benzylsuccinate synthase

ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE

TUT FDGH OPERON OF THAUERA AROMATICA STRAIN T1

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Reena Bhandare

November 2007 2

This dissertation titled

ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE

TUTFDGH OPERON OF THAUERA AROMATICA STRAIN T1

REENA BHANDARE

has been approved for

the Department of Biological Sciences

and the College of Arts and Sciences by

Peter W. Coschigano

Associate Professor of Microbiology

Benjamin M. Ogles

Dean, College of Arts and Sciences

ABSTRACT

BHANDARE, REENA, Ph.D, November 2007, Biological Sciences

ANAEROBIC TOLUENE DEGRADATION: GENETIC ANALYSIS OF THE

TUTFDGH OPERON OF THAUERA AROMATICA STRAIN T1 (134 pp.)

Director of Dissertation: Peter W. Coschigano

Toluene is an aromatic hydrocarbon that is widely used in our everyday life. It is a

major water-soluble constituent of petroleum and can pollute surface as well as ground

waters. The toxic nature of toluene is responsible for causing severe health hazards. The

study of toluene degrading bacteria has attracted attention because of their potential to

clean up spills. Thauera aromatica strain T1 is one such bacterium capable of degrading toluene under anaerobic conditions.

The tutE tutFDGH gene cluster is essential for the first step of anaerobic toluene degradation in T. aromatica strain T1. The tutF, tutD and tutG genes are proposed to

code for the three subunits of the enzyme benzylsuccinate synthase, which is involved in the initial step of anaerobic toluene degradation pathway. The tutE gene is proposed to code for the enzyme benzylsuccinate synthase activase. The precise role of the tutH gene in toluene degradation is currently unknown, but it is proposed to have an ATP/GTP binding domain and is assumed to be involved in benzylsuccinate synthase complex formation. This is consistent with its proposed role as a chaperone of “ATPases

Associated with a Variety of Cellular Activities” (AAA) class.

Work presented here demonstrates that the gene tutH is essential for toluene metabolism. A plasmid carrying an in-frame tutH deletion was unable to produce wild- type TutH protein in a tutG chromosomal deletion background (chromosomal deletion in tutG does not result in production of TutH due to a polar effect on downstream genes).

The resultant construct was unable to complement a polar tutG chromosomal mutation, indicating the importance of tutH in toluene degradation. Further, site-directed mutagenesis was used to identify amino acids in TutH that are essential for toluene metabolism. The TutH putative ATP/GTP binding domain was disrupted by changing glycine, lysine and serine at positions 52, 53 and 54 to alanine, arginine and alanine respectively. Additionally, other amino acids which are found to be highly conserved across closely related bacteria were also targeted for mutagenesis. Leucine and asparagine at positions 158 and 159 of TutH were changed to serine and alanine, glycine at position 161 was changed to alanine and arginine and phenylalanine at positions 177 and 178 were changed to alanine and serine, respectively. The resultant constructs were unable to complement a polar tutG chromosomal mutation, indicating that these amino acids are essential for toluene metabolism and might play important functional role.

Additionally, using ProteoEnrich™ ATP Binders™ Kit (Novagen), it was demonstrated that the wild-type TutH protein can bind ATP, thereby supporting the possibility that it has a role in complex formation of benzylsuccinate synthase.

Furthermore, attempts were made to identify amino acids in TutF and

TutG proteins that are essential for toluene metabolism. Using site-directed mutagenesis cysteine and alanine at positions 9 and 10 of TutF were changed to tyrosine and serine respectively and cysteine at position 29 of TutG was changed to serine. The resultant mutant constructs were unable to complement relevant chromosomal deletions, indicating that the substituted amino acids are important for toluene metabolism.

Approved:

Peter W. Coschigano

Associate Professor of Microbiology

To my parents, for their love and support.

ACKNOWLEDGMENTS

It is a known fact that “Dissertation = 10% inspiration + 90% perspiration”. I would like to thank all those who have helped me to balance this equation.

Firstly, I would like to thank my advisor Dr. Peter Coschigano, for his excellent guidance, immense support and help. This four year journey in science would have been possible without him, but definitely not worth it!

I would like to thank my PhD committee members, Dr. Don Holzschu, Dr. Calvin

James and Dr. Guy Riefler for their valuable suggestions, help and support. My special thanks go out to Dr. Tomohiko Sugiyama and Dr. Noriko Kantake for their guidance and help with some of my experiments. Further, I would like to thank my current and ex lab members, Bethany Dean-Henderson, Mark Calabro, April Lust and Paul Wiehl, for their much required help and suggestions. I would also like to thank NSF, Ohio University

Department of Biological Sciences, Biomedical Sciences, and Graduate Student Senate for financial support.

At last but not the least, I would like to thank my family and friends for their love, support and encouragement, which always kept me rolling.

TABLE OF CONTENTS

Abstract ...... 3

Acknowledgments...... 7

Table of Contents...... 8

List of Tables ...... 12

List of Figures...... 13

Note ...... 15

1 Introduction...... 16

1.1 Objective of this research...... 16

1.2 Importance of toluene degradation ...... 17

1.3 Aerobic toluene degradation...... 18

1.4 Anaerobic toluene degradation...... 20

1.4.1 Importance of anaerobic toluene degradation...... 20

1.4.2 Bacteria involved in anaerobic toluene degradation...... 20

1.4.3 Mechanism of anaerobic toluene degradation ...... 21

1.5 Anaerobic degradation of other aromatic compounds...... 24

1.6 Degradation of halogenated aromatic compounds...... 26

1.7 Comparison of phylogenetically related toluene degrading bacteria.... 27

1.7.1 Comparison at morphological and physiological levels ...... 27

1.7.2 Comparison at genetic level...... 28

1.8 Role of tutE tutFDGH operon in toluene degradation...... 31

1.9 Benzylsuccinate synthase...... 34

1.9.1 Mechanism...... 34

1.9.2 Homology with other enzymes ...... 36

1.9.3 Substrate range for benzylsuccinate synthase...... 36

1.10 Regulation of toluene degrading pathway in T. aromatica strain T1 ... 37

1.10.1 Regulatory two component system...... 37

1.10.2 Regulation by benzylsuccinate...... 38

1.11 β-oxidation pathway ...... 40

1.12 Benzoyl-CoA pathway...... 44

1.12.1 Biochemistry of Benzoyl-CoA pathway...... 44

1.12.2 Genes involved in the benzoyl-CoA pathway ...... 47

2 Materials and methods ...... 50

2.1 Plasmids and Strains ...... 50

2.2 Media ...... 54

2.3 Anaerobic cultivation...... 55

2.4 DNA preparation and subcloning ...... 55

2.5 Selection of amino acids for site-directed mutagenesis...... 56

2.6 Site-directed mutagenesis ...... 60

2.7 Triparental Mating...... 64

2.8 Testing for complementation ...... 65

2.8.1 Cell growth and sample preparation ...... 65

2.8.2 HPLC analysis for detection of toluene and phenylitaconic acid..... 66

2.9 Cell growth and induction...... 67

2.9.1 RNA isolation...... 67

2.9.2 RT-PCR...... 68

2.9.3 Protein extraction...... 70

2.9.4 Protein quantitation...... 70

2.9.5 Coomassie staining...... 70

2.9.6 Analysis for ATP binding ...... 71

2.9.7 Western blot analysis ...... 72

3 Results...... 73

3.1 Mutations in tutF...... 73

3.1.1 Construction and complementation of tutF mutation ...... 73

3.1.2 Protein production by tutF mutants ...... 76

3.1.3 RT-PCR analysis of tutF mutants...... 78

3.2 Mutations in tutG and tutH ...... 80

3.2.1 Construction and complementation of tutG mutation...... 80

3.2.2 Role of tutH in toluene metabolism...... 82

3.2.3 Construction and complementation of tutH mutations ...... 83

3.2.4 Protein production by tutG and tutH mutants...... 89

3.2.5 RT-PCR analysis of tutH and tutG mutants...... 92

3.3 Analysis of the TutH putative ATP binding domain ...... 94

3.4 Induction with benzylsuccinate...... 96

4 Discussion...... 100

4.1 Mutations in tutF and tut G...... 100

4.2 Role of tutH gene in toluene metabolism ...... 101

4.3 Mutations in tutH ...... 103

4.4 Induction by benzylsuccinate...... 106

5 Future Prospects...... 108

5.1 Site-directed mutagenesis ...... 108

5.2 Induction by benzylsuccinate...... 108

References...... 110

Appendix...... 125

LIST OF TABLES

Table 1: Bacteria capable of degrading toluene under anaerobic conditions ...... 21

Table 2: Bacteria capable of degrading aromatic compounds to benzoyl–CoA...... 25

Table 3: Bacterial plasmids used for study...... 52

Table 4: Bacterial strains used for study...... 53

Table 5: Primer sequences used for site-directed mutagenesis...... 62

Table 6: Generation of desired amino acid changes using site-directed mutagenesis with

simultaneous engineering of restriction sites in the mutant constructs...... 63

Table 7: Primer sequences used for RT-PCR analysis ...... 69

Table 8: Test for the ability of site-directed mutations of the tutF gene to complement the tutF::kan chromosomal deletion mutation...... 75

Table 9: Test for the ability of site-directed mutation of the tutG gene to complement the tutG::kan chromosomal deletion mutation...... 81

Table 10: Test for the ability of plasmids carrying tutH mutation (in ATP binding domain) to complement a (polar) tutG::kan chromosomal mutation ...... 87

Table 11: Test for the ability of plasmids carrying tutH mutations to complement a

(polar) tutG::kan chromosomal mutation...... 88

LIST OF FIGURES

Figure 1: Aerobic toluene degradation pathways in different Pseudomonas strains...... 19

Figure 2: Proposed pathway for anaerobic toluene oxidation to benzoyl-CoA...... 23

Figure 3: Degradation of aromatic compounds to benzoyl-CoA...... 26

Figure 4: Organization of genes involved in anaerobic toluene degradation in T. aromatica strains...... 30

Figure 5: Role of the tutE tutFDGH operon in anaerobic toluene degradation in T. aromatica strain T1...... 33

Figure 6: Mechanism of benzylsuccinate synthase...... 35

Figure 7: Proposed pathway of anaerobic toluene oxidation of toluene to benzoyl-CoA in

T. aromatica K172...... 42

Figure 8: Organization of bbs genes involved in β-oxidation pathway...... 43

Figure 9: Comparison of benzoyl-CoA metabolism by R. palustris and T. aromatica strain K172...... 46

Figure 10: Genes involved in benzoyl-CoA metabolism...... 49

Figure 11: A. Multiple sequence alignment using CLUSTALIX (1.83) ...... 58

Figure 12: B. Multiple sequence alignment using CLUSTALIX (1.83) ...... 59

Figure 13: Mobilization of the plasmid carrying tutH site-directed mutations into ΔtutG chromosomal mutant background...... 61

Figure 14: Western blot examining TutH protein production from toluene induced cells with tutF::kan chromosomal mutation...... 77

Figure 15: RT-PCR analysis examining tutD message production from toluene induced

cells with tutF::kan chromosomal mutation...... 79

Figure 16: Western blot examining TutH protein production from toluene induced cells

with tutG::kan chromosomal mutation...... 90

Figure 17: Western blot examining TutH protein production from toluene induced cells

with tutG:: kan chromosomal mutation...... 91

Figure 18: RT-PCR analysis examining tutH message production from toluene induced

cells with tutG::kan chromosomal mutation...... 93

Figure 19: Analysis of the TutH protein for its ability to bind ATP...... 95

Figure 20: Western blot examining TutH protein production from benzylsuccinate

induced cells with tutG:: kan chromosomal mutation...... 98

Figure 21: Western blot examining TutH protein production from benzylsuccinate

induced cells with tutG:: kan, tutF:: kan, and tutD:: kan chromosomal mutations...... 99

NOTE

Part of the information in this dissertation has been published (Bhandare et al., 2006.

Biochemical Biophysical Research Communications. 346:992-998.) and other major parts have been submitted for publication.

1 INTRODUCTION

1.1 Objective of this research

Thauera aromatica (T.aromatica) strain T1 is a facultative anaerobic, denitrifying bacterium with the potential to degrade toluene under anaerobic conditions (36). This project involves the genetic analysis of the toluene utilization genes tutE tutFDGH in T. aromatica strain T1, which are involved in the first step of anaerobic toluene degradation

(25). Genes tutF, tutD and tutG are proposed to code for the three subunits of the benzylsuccinate synthase enzyme, a key enzyme participating in the initial step of anaerobic toluene degradation (11, 17, 23, 25, 26, 59, 60). The role of tutH in toluene metabolism is currently unknown. However, TutH is predicted to have a putative ATP binding domain and is proposed to act as a chaperone, assisting in complex formation of benzylsuccinate synthase (26, 47, 55, 67). Hence, the present research is an effort to

determine the role of tutH in toluene degradation. Additionally it is intended to identify

amino acids in the TutF, TutG and TutH proteins that are essential for toluene

degradation and that might play important structural or functional roles in

benzylsuccinate synthase (encoded by tutF, tutD and tutG) complex formation.

1.2 Importance of toluene degradation

Toluene is an important constituent of various commercial products including

paints, dyes, cosmetics, pharmaceuticals, various chemicals and plastic articles (2, 3).

Toluene is a natural component of petroleum and the petroleum industries are the major

source of benzene, toluene, ethylbenzene and xylene (BTEX) pollution in the

environment (2, 3). Toluene, because it is water soluble, can contaminate both surface

and ground waters (63). The toxic nature of toluene is responsible for causing severe health hazards including suppressed colored vision, hearing impairment, kidney damage, sleeping disorders, depression of the central nervous system, attention disorders, and reproductive anomalies (2, 3). Further, the US Environmental Protection Agency (US

EPA) has classified toluene as a priority pollutant (2, 3).

In the past few years, toluene degrading bacteria have attracted attention because of their potential to clean up spills (2, 3, 63). Both aerobic as well as anaerobic bacteria are capable of degrading toluene (63). However, aerobic degradation is not effective under anoxic environmental conditions, making anaerobic degradation of toluene an important treatment alternative (63). Both aerobic and anaerobic toluene metabolisms are reviewed below.

1.3 Aerobic toluene degradation

Aerobic toluene degradation has been studied in various bacteria, especially those

belonging to the genus Pseudomonas (95). Five different pathways have been proposed

for different strains of Pseudomonas, P. putida mt-2, P. mendocina KR., P. cepacia G4,

P. picketti PK01 and P. putida F1 (see Figure 1). These pathways involve an initial

reaction comprising the oxidation of toluene catalyzed by oxygenases. The aerobic

toluene degradation pathway in P. putida mt-2 involves oxidation of the methyl group of toluene to produce benzoic acid. Subsequently, cis-benzoate dihydrodiol is produced

which is further dehydrogenated to produce catechol (95). Aerobic toluene degradation in

P. mendocina KR, P. cepacia G4 and P. picketti PK01, involves oxidation of toluene to

produce cresol isomers (p-cresol, o-cresol and m-cresol, respectively) as intermediate

products. p-cresol is further converted into protocatechuate, while o-cresol and m-cresol

are transformed into 3-methylcatechol. P. putida F1 can metabolize toluene by producing

cis-toluene dihydrodiol, which is further converted into 3-methylcatechol. Catechol,

protocatechuate and 3-methylcatechol can further undergo aromatic ring cleavage by

either ortho-cleavage or meta-cleavage pathways, to produce non-aromatic intermediates

(like acetate, pyruvate and succinate) which enter and are metabolized by the

Tricarboxylic Acid Cycle (TCA cycle) (44, 57, 68, 95).

Figure 1: Aerobic toluene degradation pathways in different Pseudomonas strains

(95). (A) P. putida mt-2 ; (B) P. mendocina KR; (C) P. cepacia G4; (D) P. picketti PK01; (E) P. putida F1.

1.4 Anaerobic toluene degradation

1.4.1 Importance of anaerobic toluene degradation

The amount of oxygen penetrating subsurface environments decreases as the

distance from the surface increases (63). In addition, aerobic bacteria present in the

environment can utilize available oxygen, making conditions anoxic. Once the available

oxygen is depleted, only anaerobic bacteria can carry out toluene degradation (63).

1.4.2 Bacteria involved in anaerobic toluene degradation

Anaerobic toluene degradation has been reported in a wide range of bacteria including methanogenic bacteria, iron reducing bacteria, sulfate reducing bacteria and

phototrophic bacteria (seeTable 1).

Table 1: Bacteria capable of degrading toluene under anaerobic conditions

Nutritional mode Organism References Methanogenic Members of Methanosaeta and (10) Methanospirillum genera (31) Denitrifying T. aromatica strain T1 (35, 36) T. aromatica strain K172 (6, 80) Azoarcus sp. strain T (29) Azoarcus sp. strain EbN1 (73) Sulfate-reducing strain PRTOL1 (9) Desulfobacula toluolica (74) Desulfobacterium cetonicum (66)

Iron reducing Geobacter metallireducens (62, 64)

Phototrophic Blastochloris sulfoviridis strain ToP1 (94)

1.4.3 Mechanism of anaerobic toluene degradation

Several pathways have been proposed for anaerobic toluene degradation, while

lacking the sufficient experimental evidence to support them (4, 37). The most widely

accepted pathway for anaerobic toluene degradation has been proposed based on

biochemical studies with T. aromatica strain K172 (17) and Azoarcus sp strain T (14).

The first step in anaerobic toluene degradation involves the addition of fumarate to the methyl group of toluene to form benzylsuccinate (see Figure 2) (17), and it is catalyzed by a glycyl radical enzyme, benzylsuccinate synthase (11, 17, 59, 60). Benzylsuccinate is

further oxidized to benzoyl-CoA, via a ß-oxidation pathway and ultimately degraded to

non-aromatic end products (like acetyl CoA) that are metabolized via the TCA cycle (43,

45, 59). Conversion of toluene to benzylsuccinate has also been observed in sulfate reducing bacteria (12), phototrophic bacteria (94) and a methanogenic culture (10), indicating that toluene degradation in other phylogenetically diverse bacteria might initially proceed via a similar mechanism (see Table 1).

(a, b)

Figure 2: Proposed pathway for anaerobic toluene oxidation to benzoyl-CoA

(1) Benzylsuccinate synthase (2) a. benzylsuccinate CoA-transferase, b. benzylsuccinyl- CoA dehydrogenase (3) phenylitaconyl-CoA hydratase (4) 3-hydroxyacyl-CoA dehydrogenase (5) benzoylsuccinyl-CoA thiolase. (Modified from the original source (59))

1.5 Anaerobic degradation of other aromatic compounds

Bacteria can degrade a large number of aromatic compounds to benzoyl-CoA (see

Table 2). Certain modifications of aromatic rings are carried out to activate aromatic

compounds before entering the pathway, e.g. hydroxylation, oxidative addition oxidation,

lyase reactions, decarboxylation, o-demethylation, α-oxidation and reductive dehydroxylation (6, 18, 20, 30, 34, 45, 64). The majority of these activated compounds undergoe various reaction steps and are ultimately converted into a central intermediate called benzoyl-CoA (see Figure 3), which undergoes a series of degradation steps and is finally broken into non-aromatic end products (43, 45) (see section 1.12). Alternatively,

certain aromatic compounds can undergo degradation by using other pathways that

involve central intermediates like, resorcinol, phloroglucinol and hydroxyquinones (41,

70, 79).

Table 2: Bacteria capable of degrading aromatic compounds to benzoyl–CoA

Nutritional Bacteria Aromatic substrate References mode Denitrifying T. aromatica strain K172 Toluene, phenol, 2- (6, 18, 20, bacteria aminobenzoate,benzoate 80, 82) Azoarcus strain EBN1 Ethylbenzene, toluene (56, 73) Phototrophic R. palustris 4-hydroxybenzoate, 3- (30, 42) bacteria chlorobenzoate, benzoate Fermenting Syntrophus Benzoate (34, 81) bacteria gentianae/Syntrophus aciditrophicus in consortium with methanogenic/sulfate reducing bacteria. Iron reducing Geobacter metallireducens Phenol, p-cresol, toluene (62, 64) bacteria

Figure 3: Degradation of aromatic compounds to benzoyl-CoA

Source: (45)

1.6 Degradation of halogenated aromatic compounds

Halogenated aromatic compounds are mostly man-made and toxic in nature. This has lead to the pursuit for microbial communities that can dehalogenate and detoxify these compounds (76). Pathways employed by bacteria for aromatic degradation of

halogenated compounds are similar to those employed for degradation of other aromatic

compounds (76). Under aerobic conditions degradation reactions of these compounds are

catalyzed by oxygenases, leading to ring cleavage (76), while under anaerobic conditions

degradation is often characterized by hydrolytic or reductive pathways leading to the loss

of aromaticity (65, 76). Chlorinated compounds can be degraded by the breakdown of

carbon skeleton and mineralization of chlorine substituents. Halogen group removal is a

key step in the degradation of these compounds, which can occur before attack on the

aromatic ring or attack on the aromatic ring followed by dehalogenation (76).

Dehalogenating microorganisms produces enzymes called dehalogenases, which can

cleave C-halogen bonds (50). Pseudomonas strain CBS3 encodes dehalogenases

(encoded by genes dehCI and dehCII), that are involved in the degradation of 4-

chlorobenzoate (50).

1.7 Comparison of phylogenetically related toluene degrading bacteria

1.7.1 Comparison at morphological and physiological levels

T. aromatica strain T1 is a member of the beta subclass of proteobacteria (87). It

is phylogenetically related to other toluene degrading denitrifiers such as T. aromatica

strain K172, and Azoarcus sp. strain T (87). Although, T. aromatica strain T1 shares

76.5% total genomic similarity with T. aromatica strain K172 at the nucleotide level, the

two strains exhibit significant differences at the morphological, nutritional and molecular

levels (87). It displays only 0-5% total genomic similarities with Azoarcus at the

nucleotide level. T. aromatica strain T1 is peritrichously flagellated whereas T. aromatica

strain K172 exhibits a degenerate peritrichous flagellar arrangement (35, 87). T. aromatica strain T1 and T. aromatica strain K172 are able to utilize toluene as the sole carbon source, but are not able to utilize m-xylene (35, 87). Azoarcus strain T can utilize both toluene and m-xylene as sole carbon sources (92). T. aromatica strain K172 can break down phenol and 4-hydroxylcinnamate, while T. aromatica strain T1 cannot (87).

Further, T. aromatica strain T1, T. aromatica strain K172 and Azoarcus sp. strain T. can metabolize both m-cresol and p-cresol (87, 92).

1.7.2 Comparison at genetic level

Genes responsible for toluene degradation are found to be conserved across T. aromatica strain T1 and the phylogenetically related denitrifying bacteria T. aromatica strain K172 and Azoarcus sp. strain T (1, 26, 47). The genes involved in the conversion of toluene to benzylsuccinate are designated as the tutE tutFDGH genes in T. aromatica strain T1 and the corresponding benzylsuccinate synthase genes are designated bssDCABE genes in T. aromatica K172 and Azoarcus sp. strain T (1, 26, 47). In T. aromatica strain T1, these genes are organized into two distinct operons while in T. aromatica strain K172 and Azoarcus sp. strain T, they are organized in a single operon

(see Figure 4), (1, 11, 26, 47). The tutE tutFDGH gene cluster has been found to be induced by toluene and by benzylsuccinate, whereas the homologous bssDCABE operons

29 of T. aromatica strain K172 and Azoarcus sp. strain T have not been reported to be induced by benzylsuccinate (22, 26). Computer analysis has indicated the presence of a corresponding operon in T. aromatica strain EbN1 consisting of eight genes

(bssDCABEFGH) which is predicted to be organized in a single transcriptional unit (see

Figure 4) (55).

(A)

tutE tutF tutD tutG tutH

(B) 1 kb

bssD bssC bssA bssB bssE

(C)

bssD bssC bssA bssB bssE bssF bssG bssH

Figure 4: Organization of genes involved in anaerobic toluene degradation in T. aromatica strains

(A) tut operon of T. aromatica strain T1 (26). (B) bss operon of T. aromatica strain K172 and Azoarcus sp. strain T (1, 47). (C) bss operon of T. aromatica strain EBN1 (55). Arrow indicates transcriptional start site.

1.8 Role of tutE tutFDGH operon in toluene degradation

The genes encoding benzylsuccinate synthase and benzylsuccinate synthase

activating enzyme have been cloned and characterized in T. aromatica strain T1, T.

aromatica strain K172 and Azoarcus sp. strain T (1, 26, 47). Computer analysis has

indicated the presence of homologous genes in Azoarcus sp. strain EbN1 (55). The tutE

tutFDGH gene cluster is found to be essential for toluene metabolism in T. aromatica

strain T1 (22, 25).

The tutFDG genes from T. aromatica strain T1 and the homologous bssCAB

genes from T. aromatica strain K172, Azoarcus sp. strain T and Azoarcus strain EbN1 are

proposed to encode the three subunits of benzylsuccinate synthase (1, 26, 47, 55).

Products of tutD and bssA are found to have a high degree of similarity with pyruvate

formate lyase enzyme, and are proposed to be activated by formation of a glycyl free

radical (1, 26, 47, 55). tutE from T. aromatica strain T1 and the homologous gene bssD

from T. aromatica strain K172, Azoarcus sp. strain T and Azoarcus sp strain EbN1 are proposed to code for the “S-Adenosyl Methionine” (SAM) dependent benzylsuccinate synthase activating enzyme, required for production of the glycyl free radical (necessary for enzyme activity) (23, 25, 26, 47, 54, 58, 60, 86). This gene is located upstream of the benzylsuccinate synthase coding genes and is transcribed in the same direction (1, 26, 47,

55). Finally the tutH gene in T. aromatica strain T1 is co-transcribed with the tutFDG

gene cluster and is presumed to code for an ATP/GTP binding protein. Further, tutH

32 resembles the bssE gene from the bssDCAB gene cluster of T. aromatica strain K172 and

Azoarcus sp strain T, which are also proposed to have a putative ATP/GTP binding domain (1, 26, 47, 55, 67). TutH (and the homologous BssE proteins) may function in the assembly, activation, or disassembly of benzylsuccinate synthase (1, 26, 47, 55, 67) (see

Figure 5).

tut tutE tutF tutD tutG tutH

Tut TutE TutF TutD TutG TutH Chaperone,function unknown (Involved in assembly/ disassembly of Benzylsuccinate Benzylsuccinate synthase synthase activase: (Tut FDG)) (Activates TutD by forming a glycine free radical)

Complex formation of Benzylsuccinate synthase (Tut FDG) subunits

Figure 5: Role of the tutE tutFDGH operon in anaerobic toluene degradation in T. aromatica strain T1.

1.9 Benzylsuccinate synthase

1.9.1 Mechanism

Benzylsuccinate synthase is an oxygen sensitive, glycyl-radical enzyme that

catalyzes the first step of anaerobic toluene degradation by adding fumarate to the methyl

group of toluene (11, 12, 17, 54, 59, 60). The enzyme has been purified from T.

aromatica strain K172 and Azoarcus sp. strain T (54, 60). Electronic paramagnetic spectroscopy (EPR) has indicated the presence of an oxygen-sensitive, stable organic

radical in benzylsuccinate synthase of Azoarcus sp. strain T (54). Benzylsuccinate

synthase is comprised of three subunits (α2β2γ2) which are responsible for its

heterohexameric structure (54, 60). Glycyl radical enzymes require an adenosylmethionine dependent activating enzyme, to generate a free radical at the glycine

residue (86). Spectroscopic and molecular analysis suggests a possible mechanism of

action for benzylsuccinate synthase. In the initial step of the reaction, the glycyl radical

abstracts hydrogen from the adjacent cysteine to form an intermediate thiyl radical. This, in turn, abstracts hydrogen from the methyl group of toluene to produce a benzyl radical.

The benzyl radical reacts with the double bond of fumarate to form a benzylsuccinyl radical, which eventually abstracts hydrogen from cysteine to yield benzylsuccinate and regenerates a thiyl radical (see Figure 6) (11, 13, 23, 54, 60).

Thiyl Benzyl radical radical

Glycyl radical (Benzylsuccinate Synthase)

Thiyl Benzylsuccinyl radical radical

Figure 6: Mechanism of benzylsuccinate synthase

(modified from original source :(48))

1.9.2 Homology with other enzymes

Benzylsuccinate synthase shows a significant homology with other well

characterized enzymes viz. pyruvate formate lyase (PFL) and anaerobic ribonucleotide

reductase (ARR) from E.coli (11, 13, 23, 54, 60). PFL is a key enzyme that is involved in

anaerobic glucose metabolism (53). ARR is required for bacterial DNA replication under

anaerobic conditions (88).

Benzylsuccinate synthase, PFL and ARR are found to have conserved amino acid

residues which are critical for their function (54). Benzylsuccinate synthase and ARR are

both known to have a single conserved cysteine residue, while most of the PFL enzymes

are known to have two conserved cysteine residues. Catalytically active forms of

benzylsuccinate synthase, PFL, and ARR contain a conserved glycine residue required

for formation of a free radical that catalyzes initial substrate transformation (53, 54, 58,

60, 71, 77, 88, 93).

In T aromatica strain T1, site-directed mutagenesis of the tutD gene has indicated

the presence of a conserved glycine residue at the position 828 and a conserved cysteine residue at the position 492 that are essential for toluene metabolism (23).

1.9.3 Substrate range for benzylsuccinate synthase

Benzylsuccinate synthase is found to be the key enzyme responsible for the first step of anaerobic toluene degradation in a wide range of bacteria (see Figure 2).

Biochemical analysis of the purified enzyme from T. aromatica strain K172 and

Azoarcus sp. strain T suggest that benzylsuccinate synthase is involved in anaerobic degradation of toluene (54, 60). Further, benzylsuccinate synthase is also thought to be involved in the degradation of m-xylene and cresol isomers in Azoarcus sp. Strain T and cresol isomers but not xylene in T. aromatica strain K172 (87, 92). Analysis of a bssA defective mutant in Azoarcus sp. strain T indicated that bssA is essential for metabolism of both toluene and m-xylene (1). The difference in substrate specificity of the enzyme can be attributed to the genetic differences among the bacteria in spite of a high degree of similarity at nucleotide levels (1, 26, 47, 55, 87). However, there is no direct experimental evidence (using purified benzylsuccinate synthase), indicating the involvement of benzylsuccinate synthase in the later (m-xylene and cresol) reactions.

1.10 Regulation of toluene degrading pathway in T. aromatica strain T1

1.10.1 Regulatory two component system

Regulatory genes in the toluene degradation pathway have been identified and characterized in T. aromatica strain T1 (tutBC) (27) and T. aromatica strain K172

(Toluene degradation induction (tdiSR)) (61). In T. aromatica strain T1 tutC is located upstream of tutB, while in T. aromatica strain K172, the homologous gene tdiS is located upstream of tdiR. Proteins TutC and TdiS show significant homology to sensor members of the two-component family of signal transduction proteins. Proteins TutB and TdiR

exhibit homology to DNA binding protein members of the two-component sensor-

regulator family (27, 61). Sensor proteins detect a signal from the environment of an

organism, autophosphorylate and transmit a signal (phosphate group) to a response

regulator protein (having both phosphorylation and DNA binding sites) (27, 61).

Computer analysis indicated the presence of another operon (tutB1C1) in T. aromatica

strain T1, which is upstream of and is oriented in the opposite direction to tutBC (61).

The exact role of these genes in toluene degradation has not been clearly identified.

1.10.2 Regulation by benzylsuccinate

The tutE tutFDGH gene cluster of T. aromatica strain T1 is found to be induced by both toluene and benzylsuccinate (22, 25, 26). It has been previously been reported that the tutF, tutD and tutG genes are essential for toluene metabolism in T. aromatica

strain T1 (25). When induced with toluene, mutations in the strains carrying

chromosomal deletions in the tutF, tutD and tutG genes were not complemented by the plasmids carrying in-frame deletions in the corresponding genes (tutF, tutD and tutG

genes respectively), as indicated by the inability of these mutants to metabolize toluene.

Further, Northern analysis indicated the absence of wild-type levels of tutE message in

these mutants (22). The wild-type plasmid (carrying tutE tutFDGH gene cluster) was able

to complement the tutF, tutD and tutG chromosomal deletion, restore toluene metabolism

and produce comparably high levels of tutE message (22, 25) Additionally, Northern

analysis of the tutF and tutD chromosomal deletion mutants carrying: (a) the vector

39 alone, (b) a plasmid with an in-frame deletion of the tutF gene, (c) a plasmid with an in- frame deletion of the tutD gene, failed to produce tutD and tutH message. These results were not expected, as in-frame deletion of a gene on a plasmid would not be expected to affect other genes located on the plasmid, and indicated the possibility that toluene was insufficient to fully induce the tutE tutFDGH gene cluster (22). RNA and protein analysis of the wild-type strains, induced with either toluene or benzylsuccinate revealed the presence of the tutE, tutD and tutH messages and TutD and TutH proteins, thereby suggesting that both toluene and benzylsuccinate are capable of inducing the tutE tutFDGH operon. Further, Northern analysis of the tutF, tutD and tutG chromosomal deletion mutants (induced by benzylsuccinate) demonstrated the presence of the tutE message (not evident in the corresponding toluene induced chromosomal deletion mutants), indicating that benzylsuccinate is required for full induction of the tut genes

(22). Benzylsuccinate is an intermediate product of anaerobic toluene degradation (see

Figure 2), produced by the enzyme benzylsuccinate synthase, which is comprised of subunits encoded by the tutF, tutD and tutG genes (see Figure 5) (22, 23, 25, 26). It is hypothesized that toluene (or other aromatic compounds) can induce the tutE tutFDGH gene cluster at low levels. The benzylsuccinate synthase produced will convert toluene

(but not other aromatics) to benzylsuccinate which in turn carries out full induction of the tut genes. However, benzylsuccinate cannot serve as a carbon source for T. aromatica strain T1 and is believed to be incapable of entering the cells (22). One of the possible reason might be potential toxicity of the benzylsuccinate concentrations added to the media as compared to that produced by the cells (after induction of tut operon) (22).

Although, the exact mechanism of benzylsuccinate mediated induction of the tut operon

is not clear, it might be a possible that benzylsuccinate from the medium binds to the cell

(surface) receptors and induces the tut genes via a signal transduction mechanism. A

similar mechanism of induction by benzylsuccinate has not been reported in any other

known toluene degrading bacteria.

1.11 β-oxidation pathway

Toluene is metabolized to benzylsuccinate in the first step of the anaerobic

toluene degradation pathway (11, 17, 59, 60). Benzylsuccinate is converted to an

intermediate benzoyl CoA, via a modified β-oxidation pathway (59) (see Figure 7).

Genes involved in the β-oxidation of benzyl succinate to benzoyl-CoA (bbs) have been identified in T. aromatica strain K172, T. aromatica strain T1 and Azoarcus sp strain

EbN1 (46, 55, 59). In T. aromatica strain K172 nine genes (bbsA-I) involved in β- oxidation pathway have been identified. These genes are found to be organized in an 8.5 kb operon, with a single transcriptional start site located upstream of bbsA. (see Figure

8A). The bbs operon is toluene regulated (59). bbsE and bbsF are proposed to code for subunits of succinyl- CoA: benzylsuccinate CoA transferase while bbsG is proposed to code for benzylsuccinyl CoA dehydrogenase. Other enzymes in the pathway are proposed to be encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl CoA dehydrogenase) and bbsB (3-oxyacy-CoA thiolase). The functions of the bbsA and bbsI

gene products have not yet been determined (59). In T. aromatica strain T1, five

individual genes involved in the β-oxidation pathway, bbsA, bbsB, bbsC, bbsE and bbsH

have been amplified and cloned (46). These genes are toluene regulated and exhibit 97%

similarity with genes of T. aromatica K172 at the nucleotide level (46, 59). In T.

aromatica strain T1, the bbs genes are found to be organized on several transcriptional

units, but the exact transcriptional organization of these genes has not yet been

determined. The absence of the homologous genes bbsD, bbsF, bbsG and bbsI in T. aromatica strain T1 suggests that these genes might not have a crucial role in toluene degradation or other genes might have functionally replaced these genes (46). In

Azoarcus sp. strain EbN1, the bbs operon consists of nine genes (bbs A-H) and it has

89.1% similarity with the operon from T. aromatica strain K172 (55) (see Figure 8B).

However, the bbs operon from Azoarcus sp. strain EbN1 does not contain gene bbsI,

which is present in T. aromatica strain K172. Further, the bbs operon of Azoarcus sp.

strain EbN1 contains an additional gene bbsJ between bbsF and bbsG, coding for a 9.8

kDa protein of unknown function. The absence of bbsJ in the corresponding operon from

T. aromatica strain K172 indicates that this gene might have been deleted from the bbs

operon in T. aromatica strain K172 during the course of evolution (55, 59).

Figure 7: Proposed pathway of anaerobic toluene oxidation of toluene to benzoyl-CoA in T. aromatica K172

(59) (1) benzylsuccinate synthase (2) succinyl-CoA:benzylsuccinate CoA-transferase (3) benzylsuccinyl-CoA dehydrogenase (4) phenylitaconyl-CoA hydratase (5) 3-hydroxyacyl-CoA dehydrogenase (6) benzoylsuccinyl-CoA thiolase, BbsB (7) succinate dehydrogenase.

(A)

bbsA bbsB bbsC bbsD bbsE bbsF bbsG bbsH bbsI

(B)

bbsA bbsB bbsC bbsD bbsE bbsF bbsJ bbsG bbsH

Figure 8: Organization of bbs genes involved in β-oxidation pathway

(A) T. aromatica strain K172 (59) (B) Azoarcus sp. strain EbN1 (55)

1.12 Benzoyl-CoA pathway

1.12.1 Biochemistry of Benzoyl-CoA pathway

Most aromatic compounds are anaerobically degraded by substrate specific peripheral pathways to the central intermediate benzoyl CoA, which is then further degraded to non-aromatic end products via a central benzoyl-CoA pathway (21).

In T. aromatica strain K172, the benzoyl-CoA pathway is initiated by the reduction of benzoyl-CoA to cyclohex-1,5-diene-1-caroxyl-CoA. This reaction is ATP dependent and uses ferridoxin as an electron donor. Benzoyl-CoA reductase is involved in this initial step of benzoyl-CoA metabolism. In T. aromatica strain K172, dienoyl-CoA hydratase catalyzes the conversion of cyclohex-1,5-diene-1-caroxyl-CoA to 6- hydroxycyclohex-1-ene-carboxyl-CoA, which is then dehydrogenated to yield 6- ketocyclohex-1-ene-1-carboxyl-CoA. Further transformation of 6-ketocyclohex-1-ene-1- carboxyl-CoA to 2,3-unsaturated pimelyl-CoA is catalyzed by β-oxoacyl-CoA hydrolase.

The active enoyl-CoA hydratase present in bacteria can then catalyze conversion of 2,3- unsaturated pimelyl-CoA to 3-hyroxylpimeyl-CoA, which can be further oxidized to yield three molecules of acetyl CoA and one molecule of CO2 (21). Alternatively addition of a water molecule can precede hydrolytic ring cleavage (see Figure 9).

The latter steps of benzoyl-CoA metabolism pathway are found to diverge between T. aromatica strain K172 and Rhodopseudomonas palustris (R. palustris). The initial step of both pathways involves the reduction of benzoyl-CoA to form a cyclohex-

1,5-diene-1-caroxyl-CoA. The two organisms have highly similar benzoyl-CoA

reductases (45). Later steps of the pathway in R. palustris involve the reduction of cyclic diene to form 6-hydroxycyclohex-1-ene-1-carboxyl-CoA (catalyzed by a dehydrogenase).

6 hydroxycyclohex -1-ene-1-carboxyl-CoA is then hydrated by enoyl CoA hydratase to form 2-hydroxy-cyclohexane-1-caroxyl-CoA, which is then further reduced to 2- ketocyclohexane-1- caroxyl-CoA, followed by hydrolysis to pimelyl-CoA (see Figure 9)

(45).

BadJ

Cyclohex-1,5-diene-1-carboxyl-CoA

Cyclohex-1-ene-1-carboxyl-CoA 6-Hydroxycyclohex-1-ene-carboxyl-CoA

2-Hydroxy-6-keto-1- 2-Hydroxy-cyclohexane-1- caroxyl-CoA 6-Ketocyclohex-1-ene-1-carboxyl-CoA cyclohexanecarboxyl-CoA

2-Ketocyclohexane-1- caroxyl-CoA 2,3-unsaturated pimelyl-CoA 3-Hydroxypimelyl-CoA

Figure 9: Comparison of benzoyl-CoA metabolism by R. palustris and T. aromatica

strain K172

X: 3-enoyl-CoA hydratase (21, 45)

1.12.2 Genes involved in the benzoyl-CoA pathway

Genes involved in the degradation of benzoyl-CoA have been cloned and sequenced from the phototropic α-proteobacterium Rhodopseudomonas palustris (see

Figure 10 A) and the denitrifying β-proteobacteria T. aromatica strain K172 (see Figure

10 B) and Azoarcus evansii (32, 33, 45). In Azoarcus evansii, not much is known about the precise roles of most of the genes involved benzoyl-CoA pathway. However, similarities at the nucleotide level with the genes of T. aromatica strain K172 indicate that its benzoyl-CoA degradation pathway may be similar (45).

In T. aromatica strain K172, eight genes involved in the benzoyl-CoA pathway have been identified (see Figure 10B) (21). These genes are organized in a single transcriptional unit. had is proposed to code for 6-hydroxycyclohex-1-ene-1-carboxyl-

CoA dehydrogenase (hydroxyacyl -CoA dehydrogenase). oah is proposed to code for a ring hydrolyzing enzyme (3-oxoacyl-CoA hydrolase). Other genes in the pathway are dch

(for dienoyl-CoA hydratase), bcrCBAD (for the four subunits of benzoyl-CoA reductase) and fdx (for ferredoxin) (21).

badH (R. palustris) and the corresponding gene had (T. aromatica strain K172) catalyze dehydrogenation reactions for different substrates. oah (T.aromatica) and its counterpart badI (R. palustris) exhibit only 30% similarities at amino acid level. dch (T. aromatica strain K172) codes for dienoyl-CoA hydratase while badK (R. plaustris) codes

48 for enoyl CoA hydratase. These hydratases are specific to the respective organisms and act on different substrates (32, 33, 45).

At present no work has been done on the benzoyl-CoA pathway in T. aromatica strain T1. However, based on the high level of the nucleotide similarity between T. aromatica strain T1 and T. aromatica strain K172 (87), it can be predicted that the benzoyl-CoA pathway in T. aromatica strain T1 might follow the same or similar route as that in T. aromatica strain K172.

Figure 10: Genes involved in benzoyl-CoA metabolism.

(45) (A) R. plaustris (B) T. aromatica strain K172

2 MATERIALS AND METHODS

2.1 Plasmids and Strains

Isolation, classification and characterization of T. aromatica strain T1 has been previously reported (36, 87). The construction and characterization of the insertion/deletion tutF tutD and tutG mutants have also been reported (25). Bacterial

plasmids and strains used for this study are listed in Table 3 and Table 4 respectively.

Escherichia coli HB101, XL-1 Kan Blue (Stratagene, La Jolla, CA), and XL-1 Blue

(Stratagene) were used to propagate and transfer DNA and were purchased as competent cells. Strain HB101 (pRK2013) (Kanr) contains a helper plasmid that allows for

mobilization of the donor plasmid into the T. aromatica strain T1 background (39). The

pBluescript vector (Stratagene) and the modified broad host range plasmid pRK415 were used for the construction of subclones and their mating into the T. aromatica strain T1 background (23, 25).

Plasmid pPWC-314 carries the wild type tutE tutFDGH gene cluster. Plasmids

pPWC314-∆tutF, pPWC314-∆tutD, pPWC314-∆tutG and pPWC314-∆tutH carry the same wild-type DNA, only with an in-frame deletion in tutF, tutD, tutG and tutH genes,

respectively (25). The plasmids pPWC314-C9YA10S and pPWC314-C29S carry site-

directed mutations in the tutF and tutG genes, respectively (16). Plasmids pPWC314-

G52AK53RS54 (16), pPWC314-HL158SN159A, pPWC314-HG161A and pPWC314-

HR177AF178S carry site-directed mutations in the tutH gene (see section 2.6). All the plasmids are stable in the T. aromatica strain T1 background and confer tetracycline resistance (see Figure 13).

Table 3: Bacterial plasmids used for study

Plasmid Description Reference pRK2013 Plasmid carrying mobilization gene, essential for (39) triparental mating pRK415 A broad host range vector (52) pPWC-314 A plasmid carrying the tutE tutFDGH gene cluster (23) pPWC-314ΔtutF Plasmid pPWC-314 with an in-frame tutF deletion (25) pPWC-314ΔtutD Plasmid pPWC-314 with an in-frame tutD deletion (25) pPWC-314ΔtutG Plasmid pPWC-314 with an in-frame tutG deletion (25) pPWC314-ΔtutH Plasmid pPWC-314 with an inframe tutH deletion (16) pPWC314- Plasmid pPWC-314 carrying the C9YA10S mutations (16) C9YA10S in TutF pPWC314-C29S Plasmid pPWC-314 carrying the C29S mutation in (16) TutG pPWC314- PlasmidpPWC-314carrying the G52AK53RS54A (16) G52AK53RS54A mutations in TutH pPWC314- Plasmid pPWC-314 carrying the L158SN159A This work HL158SN159A mutations in TutH pPWC314- PlasmidpPWC-314 carrying the G161A mutation in This work HG161A TutH pPWC314- Plasmid pPWC-314 carrying the R177AF178S This work HR177AF178S mutations in TutH

Table 4: Bacterial strains used for study

Srain Description Reference pBluescript Escherichia coli (Stratagene La Jolla CA) HB101 E. coli (19) XL-1 Kan Blue E.coli derived (Stratagene) XL-1 Blue E.coli derived (Stratagene) T1 tutF::kan T. aromatica strain T1 carrying the tutF::kan (25) chromosomal insertion/deletion mutation T1 tutD ::kan T. aromatica strain T1 carrying the tutD::kan (25) chromosomal insertion/deletion mutation T1 tutG ::kan T. aromatica strain T1 carrying the tutG::kan (25) chromosomal insertion/deletion mutation

2.2 Media

T. aromatica strain T1 and all the strains derived from this strain were grown on

either brain heart infusion (BHI; Difco Laboratories, Detroit, MI) medium or a mineral

salts medium (35). Either toluene (0.3–0.5 mM) or pyruvic acid (1 mM) was used as the

carbon source to supplement the minimal medium. Pyruvic acid was directly added to the

liquid media, while toluene was directly added only to serum bottles. Toluene was

provided in vapor phase (for growth of cells in small tubes or on plates) by adding a

mixture of toluene and hexadecane (1:10 v/v) to an open tube and incubating. Nitrate was

supplied at a concentration of 10–20 mM. Plates contained 2% Noble agar (Difco

Laboratories). Liquid medium was prepared and placed in serum bottles as described

previously (35). E. coli was grown in Luria-Bertani agar or broth (LB) (15) or on BHI

agar plates. The antibiotics kanamycin (used at 50 mg/ml) and tetracycline (used at 25 mg/ml) were supplied where necessary. A 12.5 mg/ml stock of tetracycline was made in

50% ethanol (23, 25).

2.3 Anaerobic cultivation

T. aromatica strain T1 cells were cultivated under anaerobic conditions. Cells were grown for 3 days at 300C, in tubes containing mineral media (supplemented with

1mM pyruvic acid). Tubes were placed in an anaerobic jar, (Model no PT798, W.R.

Brown company, IL) with a palladium catalyst. The jar was filled with hydrogen for the removal of oxygen. In a reaction catalyzed by palladium, hydrogen reacts with oxygen from the jar to form water. A BBL™ Gaspak ™ indicator (Becton Dickson & Company,

MD) was used to confirm anaerobic conditions in the jar. Toluene was supplied in vapor phase (see section 2.2) (35).

Liquid media was placed into 50 ml serum bottles and sealed with teflon coated butyl-rubber stoppers and aluminum crimps. Oxygen was removed from the bottles and replaced with argon to create anaerobic conditions as described previously (35, 46). 0.5 ml of cells (from the tube growth) were inoculated into the 50 ml serum bottles and grown for 5 days at 300C.

2.4 DNA preparation and subcloning

DNA plasmid mini-preps were performed by either the boiling method of Holmes and Quigley (49) or by using QIAprep spin mini-prep kit (Qiagen, Santa Clarita, CA).

Qiagen midi-preps were used whenever larger DNA samples were required. DNA manipulations were carried out as described by Ausubel, et al. (7). All the enzymes were

56 obtained from New England Biolabs (Beverly, MA). The tutE tutFDGH gene cluster (or mutated gene cluster) was assembled in the pBluescript vector (2.8 kb), containing Pac I restriction sites. Since the pBluescript vector is not stable in T. aromatica strain T1, the mutated tutE tutFDGH gene cluster was subcloned, using PacI restriction sites, into pRK415 (10.5 kb) for mobilization into the T. aromatica strain T1 background via triparental mating as described previously (23, 24).

2.5 Selection of amino acids for site-directed mutagenesis

In the TutF and TutG proteins, cysteines were chosen for site-directed mutagenesis since cysteines are often involved in metal binding and might be important for protein structure (16). NCBI blastP (5) was used to compare the TutH protein sequence with protein sequences from the non-redundant protein sequence (nr) database.

Default blastP parameters were used for the search. The first ten best hits were selected for sequence comparison. In addition, protein sequences from other closely related denitrifying bacteria (NorQ from Paracoccus denitrificans (28) and Rhodobacterium sphaeroides (8) and NirQ from Pseudomonas stutzeri (51)), which also displayed a conserved, putative ATP/GTP binding domain (similar to the TutH protein) were also used for sequence comparison (26). Selected sequences were aligned using the

CLUSTAL X (1.83) Multiple Sequence Alignment Tool (89). Default parameters were used for alignment. Computer analysis has indicated the presence of a putative ATP binding domain in the TutH protein (26, 47, 55, 67). In the TutH protein, a putative ATP

57 domain, which was also conserved in the other closely related bacteria (see Figure 11), was disrupted by substituting the glycine at position 52, lysine at position 53 and serine at position 54. Further, in TutH, other amino acids from the aligned sequences (other than the putative ATP binding domain), that were highly conserved and contiguously located with amino acids from closely related bacteria (leucine and asparagine at positions 158 and 159, glycine at position 161 and arginine and phenylalanine at positions 177 and 178 respectively) were selected for mutagenesis (see Figure 12).

58 Putative ATP binding domain G52, K53, S54

Figure 11: A. Multiple sequence alignment using CLUSTALIX (1.83)

Amino acids selected for mutagenesis, '*' positions which have a single, fully conserved residue, ':' fully conserved strong group

(strong score >0.5 using Gonnet Pam250 matrix), '.' fully conserved weak groups (weak score =<0.5 using Gonnet Pam250 matrix)

R177, N159 G161 F178

Figure 12: B. Multiple sequence alignment using CLUSTALIX (1.83)

Amino acids selected for mutagenesis, '*' positions which have a single, fully conserved residue, ':' fully conserved strong group

(strong score >0.5 using Gonnet Pam250 matrix), '.' fully conserved weak groups (weak score =<0.5 using Gonnet Pam250 matrix)

2.6 Site-directed mutagenesis

The pBluescript vector containing fragments of the tutE tutFDGH gene cluster was used as the starting material for site-directed mutagenesis. Mutations in tutF, tutG and tutH genes were constructed using the QuickChange site-directed mutagenesis kit

(Stratagene), as described previously (23). Mutagenic primer pairs were designed to obtain the desired amino acid(s) change(s) (see Table 5). Specific base changes were also designed to generate restriction sites for the rapid identification of desired mutations (see

Table 6). Candidates were screened by restriction analysis and confirmed by sequencing.

Subcloning was then carried out to transfer the altered tutE tutFDGH gene cluster into the modified pRK415 vector as described previously (16). The resultant plasmids were subsequently mobilized into the desired T. aromatica strain T1 background.

Plasmid carrying the tutE tutFDGH gene cluster with the site-directed mutations in the tutH

pRK415

tutE tutF tutD tutG tutH

Chromosomal insertion/deltion mutant(ΔtutG) of T. aromatica strain T1

Figure 13: Mobilization of the plasmid carrying tutH site-directed mutations into

ΔtutG chromosomal mutant background

Table 5: Primer sequences used for site-directed mutagenesis

Gene Mutation Primer Primer Sequence (5’-3’) tutF TutFC9YA10S F CCA CCA CAT GCA AGC AGT ACT CAA ACT TCT TTC CCG TCC C R GGG ACG GGA AAG AAG TTT GAG TAC TGC TTG CAT GTG GTG G tutG TutGC29S F GGC CGT GCC GGA GTT CGA AAT GGC AAA CCC CCG ACC CCA C R GTG GGG TCG GGG GTT TGC CAT TTC GAA CTC CGG CAC GGC C tutH TutHG52AK53RS54A F CAA GGC TGC GCC CGG GCT TCC CTA GTG CGG CAA TAC G R CGT ATT GCC GCA CTA GGG AAG CCC GGG CGC AGC CTT G TutHL158SN159A F GTT TTC TTC GCA ACG CTA GCC GAA GGG TCC GAA TTC GTC R GAC GAA TTC GGA CCC TTC GGC TAG CGT TGC GAA GAA AAC TutHG161A F CGC AAC GCT CAA CGA AGC TTC CGA ATT CGT CGG TAC R GTA CCG ACG AAT TCG GAA GCT TCG TTG AGG GTT GCG TutHR177AF178S F CCC GGC CCT GCG CGA CGC TAG CTA TGT CAC TAC C R GGT AGT GAC ATA GCT AGC GTC GCG CAG GGC CGG G

F- Forward primer, R-Reverse primer

Table 6: Generation of desired amino acid changes using site-directed mutagenesis with simultaneous engineering of restriction sites in the mutant constructs.

Mutation Changes in the DNA sequence Desired amino acid change/Simultaneous engineering of restriction sites TutFC9YA10S CAGTGCGCA Changing cysteine at CAGTaCtCA position 9 to tyrosine, alanine at position 10 to serine and generating ScaI restriction site TutGC29S ATGTGCAAA Changing cysteine at the AGTTcgAAA position 29 to serine and generating BstBI restriction site. TutHG52AK53RS54A GGCAAGTCT Changing glycine at the CcCcgGgCT position 52 to alanine, lysine at the position 53 to arginine, serine at the position 54 to alanine and generating SmaI restriction site TutHL158SN159A ACGCTCAACGAA Changing leucine at ACGCTagcCGAA position 158 to serine, asparagine at position 159 to alanine and engineering NheI restriction site TutHG161A AACGAAGGGTCC Changing glycine at AACGAAGctTCC position 161 to alanine and engineering HindIII restriction site TutHR177AF178S GACCGTTTTTAT Changing arginine at GACgcTagcTAT position 177 to alanine, phenylalanine at position 178 to serine, and engineering NheI restriction site

2.7 Triparental Mating

Triparental mating was carried out as described previously (23, 24). The tutF, tutG and tutH chromosomal deletion mutants of T. aromatica strain T1 were grown for 3 days in minimal medium containing nitrate and pyruvic acid. E. coli carrying the donor plasmid pRK415 (TetR), was grown overnight in LB-tetracycline. E. coli carrying the

helper plasmid pRK2013 (KanR), was grown overnight in LB-kanamycin. Sample

cultures were centrifuged and the pellets were resuspended in potassium phosphate buffer

(pH 7.0). 10 µl of the donor, helper and recipient were spotted on a BHI agar plate (same

spot) and allowed to air dry. The helper plasmid (pRK2013) has transfer and mobilization

genes, which facilitate its self-transfer into the donor strain containing pRK415. This is

followed by the transfer of the donor plasmid into the recipient strain (T. aromatica strain

T1). The plates were incubated anaerobically at 300C for three days and the resulting cell

growth was then transferred to minimal plates containing pyruvate, ethanol, nitrate, and

tetracycline to select for the transconjugant strains carrying the donor plasmid. Only T.

aromatica strain T1 cells carrying the donor plasmid pRK415 (TetR) should be able to

grow. After an additional anaerobic incubation at 300C for three days, cells were streaked

onto the same medium and again incubated without oxygen. The growth obtained from

these plates was used to inoculate 1 ml of liquid medium containing nitrate, pyruvate, ethanol and tetracycline, which was incubated under anaerobic conditions at 300C for three days and used to test for complementation (23, 25).

2.8 Testing for complementation

2.8.1 Cell growth and sample preparation

The transconjugant strains generated above were examined to determine if the construct complemented the chromosomal mutation. The transconjugants were streaked onto a minimal-plus-nitrate agar plate and toluene was supplied in the vapor phase (25).

After 5 to 7 days of anaerobic incubation (300C) (see section 2.3), the strains were

examined for the ability to grow on toluene. The transconjugants were also inoculated

from a 1 ml culture into sealed 50 ml serum bottles of minimal liquid medium plus nitrate

(10 mM), pyruvic acid (1 mM), and toluene (0.4 mM) with an argon headspace (see

section 2.3). Samples were withdrawn for analysis immediately following inoculation and

after 3 to 4 days of incubation (300C). The strains were tested for toluene utilization and

production of phenylitaconic acid by High Performance Liquid Chromatography (HPLC).

Phenylitaconic acid (intermediate product of anaerobic toluene degradation) was detetced

to attribute the loss of toluene from media to the anerobic toluene degradation (and not

due to escape from bottles because of volatile nature of toluene). If the transconjugant

could grow on toluene, utilize toluene in liquid culture, and produce wild-type levels of phenylitaconic acid then the subclone was considered to complement the mutation (23,

25).

2.8.2 HPLC analysis for detection of toluene and phenylitaconic acid

Samples were analyzed for toluene utilization and phenylitaconic acid production as described previously (23, 25). Phenylitaconic acid (an intermediate product of anaerobic toluene degradation) was detected inorder to confirm that the loss of toluene from media was due to anaerobic toluene degradation (but not due to escape from botttles). About 1 ml of each culture was withdrawn anaerobically with a sterile syringe flushed with argon. The samples were centrifuged (5 min in a micro centrifuge), and the supernatant was filtered through a 0.45-mm-pore-size filter (Millipore Corp., Bedford,

MA) into a sample vial. Samples were analyzed by HPLC with a System Gold High-

Performance Liquid chromatograph (Beckman, Fullerton, CA) equipped with a C18 column (250 by 4.6 mm; particle size, 5 mm [Beckman]) with UV detection at 254 nm for toluene and 260 nm for phenylitaconic acid. The mobile phase used for toluene analysis was 55:45 vol/vol acetonitrile-water, while the mobile phase used for analysis of phenylitaconic acid was methanol-water-acetic acid (30:68:2, vol/vol/vol) (23, 35, 40) at a flow rate of 1 ml/min. Peaks for the toluene analysis were identified by using toluene as an external standard, while the peaks for phenylitaconic acid production were identified by comparison to the external standard benzylmaleic acid (23, 35, 40).

2.9 Cell growth and induction

T. aromatica strain T1 chromosomal deletion strains carrying various plasmids

(see Table 3 and Table 4) were grown under anaerobic conditions, in 50 ml cultures of mineral salt medium as described previously (22, 35). After 3 days cells were fed nitrate

(5mM) and toluene (0.5mM) or benzylsuccinate (0.5mM). The following morning cells were refed with toluene (0.5mM) or benzylsuccinate and allowed to grow for 2 hr before the cells were harvested (22).

2.9.1 RNA isolation

50 ml of cultures of anaerobically grown cells were used for RNA isolation and

Western blot. Total RNA was extracted from 30 ml of culture using the RNeasy mini kit from Qiagen (Valencia, CA) as described previously (26). Additionally, on column DNA digestion was performed to remove any residual DNA contamination, using RNase-Free

DNase Set (Qiagen). The remaining 20 ml were centrifuged at 28,000 x g for 30 min, the cells were resuspended in 0.5 ml of 100 mM potassium phosphate buffer (pH 7.0) and used for Western analysis (see section 2.9.7) (22).

2.9.2 RT-PCR

Gene specific primers (see Table 7) were used for RT-PCR analysis. Two μg of

RNA was reverse transcribed using a gene specific reverse primer (see Table 7) and

Omniscript RT® Kit (Qiagen) according to the manufacturer’s instructions. Samples were also processed with 16 R reverse primer (for 16S rRNA) as a control (see Table 7).

Control reaction mixtures without reverse transcriptase were also performed, to check for any possible DNA contamination. The reaction mixtures were incubated at 370C for 1 hr.

PCR reactions were performed in a PCT-100 programmable thermal controller (MJ

Research Inc. Waltham, MA), using forward and reverse primers (see Table 7) and Taq

DNA polymerase (New England Biolabs), at the following conditions. Melting at 950C,

annealing at 52 0C and extension at 720C (37 cycles). The PCR product was run on an

agarose gel (1% aquapore, 1% metaphore) at 100V for 40 min using the Easy-Cast electrophoresis system (Model # B1, Owl Scientific Inc., Portsmouth, NH) to determine the size of the PCR products. A 100 bp DNA ladder (New England Biolabs) was used for size comparison.

Table 7: Primer sequences used for RT-PCR analysis

Mutant Target for Primer Sequence (5’-3’) Product amplification length (bp) TutFC9YA10S tutD gene TR1(F) GCA GAG CAA GTG 541 TCA GCC C BR1(R) GAC GGA GGC CTT GTA GTA CG TutGC29S, tutH gene TL2(F) GTC AGG TAT GGA 541 TutHG52AK53RS54A TGG ACG AG TutHL158SN159A NC1(R) CTC GCC GGG AAT TutHG161A GAC GAA AC TutHR177AF178S TutFC9YA10S 16 S 16F(F) AGC CAT GCC GCG 432 TutGC29S, Ribosomal TGA GTG AAG AAG TutHG52AK53RS54A RNA TutHL158SN159A 16R(R) TCG ACA TCG TTT TutHG161A AGG GCG TGG ACT TutHR177AF178S

F- Forward primer, R-Reverse primer

2.9.3 Protein extraction

Crude protein was extracted from T. aromatica strain T1 cell cultures using the

BugBuster® Protein Extraction Reagent (Novagen, Madison, WI). Cells were harvested from a 30 ml liquid culture by centrifugation at 28,000 x g for 30 min. The cell pellet was resuspended in 2 ml of BugBuster® Protein Extraction Reagent (Novagen) supplemented with lysozyme (in TE buffer, pH 8.0) to a final concentration of 100 μg/ml. The cell suspension was incubated on a shaking platform for 50 min at room temperature. The suspension was centrifuged at 28,000 x g for 20 min. The supernatant was used for ATP binding analysis as per the manufacturer’s instructions (see section 2.9.6).

2.9.4 Protein quantitation

Before being used for analysis, the protein concentration in all the samples was determined by the Bicinchonic acid method (85), using the BCA protein assay kit (Pierce,

Rockford, IL.), before using it for analysis (see section 2.9.6 and 2.9.7).

2.9.5 Coomassie staining

In addition to the protein quantitation by the Bicinchonic acid method (85) (see section 2.9.4), Coomassie staining of gels of the crude cell lysate (see section 2.9.3) was

71 carried out for visual comparison of the proteins. Crude cell extracts (see sections 2.9.3 ) were combined with sodium dodecyl sulfate (SDS) loading dye (7) and boiled for 10 min.

The samples were run on a 10% polyacrylamide gel at 15 mA per gel (0.75 mm thick), for 90 min using the Mini–Protean II cell (Bio-Rad, Hercules, CA). The gels were stained for 25 min using Coomassie R250 solution (Bio-Rad) with gentle agitation at room temperature. They were distained two times for 30 min and one time overnight, with gentle agitation at room temperature (using distaining solution (Bio-Rad)).

2.9.6 Analysis for ATP binding

The crude protein lysates were analyzed for the presence of an ATP binding domain using the ProteoEnrich™ ATP-Binders™ Kit (Novagen). The kit is optimized for the enrichment of protein extracts with ATP binding proteins. The lysates were depleted of excess cellular ATP by dialysis against PBS buffer (supplied by Novagen), using

Fisher brand dialysis tubing (Molecular weight cutoff = 3500 Da). Three buffer changes were carried out with sample–to volume ratio of 1:250 and a time interval of 4-6 hr.

Around 300 μg (see section 2.9.4) of dialyzed protein lysates were supplemented with the supplied additives before loading on ATP-resin columns. Binding washing and elution steps were carried out according to the manufacturer’s instructions. The eluates were concentrated according to the manufacturer’s instructions and used for Western blot analysis.

2.9.7 Western blot analysis

All the samples used for Western analysis had the same protein concentration (see

2.9.4). Crude cell extracts (see sections 2.9.1 and 2.9.3) or eluates from the ATP resin column (see section 2.9.6) were combined with sodium dodecyl sulfate (SDS) loading dye (7) and boiled for 10 min. The samples were run on 10% polyacrylamide gel at 15 mA per gel (0.75 mm thick), for 90 min using the Mini –Protean II cell (Bio-Rad).

Prestained protein standards (BioRad) were used for size comparison. Protein was transferred to Hybond enhanced chemiluminescence (ECL) membrane (Amersham

Pharmacia Biotech, Piscataway, NJ). Electroblotting was carried out overnight using the

Bio-Rad Mini protean II Trans blot cell at 14 V in electroblotting buffer (20 mM Tris,

150 mM glycine, pH 8.0) (7). Antibody production against TutH protein has been described previously (22). TutH specific antibody was used as the primary antibody for

Western blot analysis (1:2,500, in Tris-buffered saline Tween buffer), while Anti-rabbit

IgG horseradish peroxidase linked whole antibody from donkey (ECL) (1: 3,000, in Tris- buffered saline Tween buffer), was used as the secondary antibody. The Western blot was developed using the ECL Western blotting system from Amersham Pharmacia Biotech essentially as described by the manufacturer. The proteins were visualized by exposure to

BioMax Light film (Eastman Kodak, Rochester, NY).

3 RESULTS

3.1 Mutations in tutF

3.1.1 Construction and complementation of tutF mutation

In T. aromatica strain T1, the tutF, tutD, and tutG genes are proposed to code for the three subunits of benzylsuccinate synthase, the enzyme which is responsible for the first step of anaerobic toluene metabolism (11, 17, 23, 25, 26, 59, 60). Site-directed mutagenesis of the tutD gene has led to the identification of amino acids in the TutD protein that are essential for enzyme function (23). It was decided to determine if cysteine residues in both the TutF and TutG proteins are essential for enzyme function, because cysteines are often involved in metal binding and might be important for protein structure

(16). Amino acid substitutions were designed to create restriction sites while minimizing the impact on protein structure due to change in charge, polarity or hydrophobicity of the substitute. A mutation in tutF was generated by site-directed mutagenesis such that the cysteine (slightly polar, hydrophobic and neutral) at position 9 was changed to a tyrosine

(polar, hydrophobic, and neutral). Further, the alanine (non-polar, hydrophobic and neutral) at position 10 was changed to serine (polar, hydrophilic, and neutral), to engineer a ScaI restriction site (see Table 6). The plasmid carrying this construct was then tested for its ability to complement the tutF chromosomal insertion/deletion mutation. As can be

74 seen in Table 8 the strain carrying a wild type plasmid pWC314 (positive control) metabolized toluene and produced phenylitaconic acid (intermediate product of anaerobic toluene degradation pathway). The strain carrying the vector pRK415 and the strain carrying a wild-type plasmid with an in-frame tutF deletion (negative controls) were not able to metabolize toluene and produce phenylitaconic acid. The site-directed mutant construct (pPWC314-C9YA10S) failed to grow with toluene serving as the sole carbon source, metabolize toluene (results comparable to the negative controls), and produce phenylitaconic acid at the wild-type levels (also comparable to the negative controls).

Hence, the tutF chromosomal deletion mutation was not complemented by the plasmid carrying the mutated tutF gene. These results indicate that cysteine at positions 9 and/or the alanine at position 10 are essential for toluene metabolism.

Table 8: Test for the ability of site-directed mutations of the tutF gene to complement the tutF::kan chromosomal deletion mutation.

Strain Plasmid carried Percent toluene Percent Growth on left in the Phenylitaconic Toluene media acid producedd

tutF::kan pPWC314 a 0 100 +e tutF::kan pRK415 b 53.7 ±8.4 0 -f tutF::kan pPWC314-∆tutF c 70.3 ±10.9 0 - tutF::kan pPWC314- 82.3 ±11.2 0 - C9YA10S

a The wild type plasmid carrying the tutE tutFDGH gene cluster, serving as a positive control b The vector alone, serving as a negative control c The wild type plasmid with an in-frame tutF deletion d Normalized to 100% for pPWC314, the positive control e+ = Growth with toluene as the sole carbon source f- = No growth with toluene as the sole carbon source

3.1.2 Protein production by tutF mutants

Western blot analysis was used to examine TutH protein (but not TutF protein) production by tutF mutants due to the lack of a TutF specific antibody. It has been demonstrated that a tutF chromosomal insertion/deletion mutation results in the loss of

TutH protein production, due to a polar effect on downstream genes (25). As can be seen from Figure 14, the strain carrying a wild-type plasmid pPWC314 produced TutH protein

(approximately 31.8 kDa) (Lane 1). The strain carrying the vector pRK415 and the strain carrying a wild-type plasmid with an in-frame tutF deletion (negative controls) did not show presence of the TutH protein (Lanes 2 and 3). Another 25 kDa protein band was observed in both the strain carrying a wild-type plasmid pPWC314 and the strain carrying the vector pRK415 (Lane 1 and 2), and was not evident in the samples from other strains (Lane 3 and 4). The 25 kDa protein band could be attributed to non-specific binding of polyclonal TutH antibody. Further, as can be seen from Lane 4, no TutH protein was detected in the site-directed mutant construct pPWC314-C9YA10S (results comparable to negative controls).

37 kDa

TutH 25

M 1 2 3 4

Figure 14: Western blot examining TutH protein production from toluene induced cells with tutF::kan chromosomal mutation.

Lane 1 – Strain carrying the wild type plasmid pPWC314 Lane 2 – Strain carrying the vector alone pRK415 Lane 3 – Strain carrying the plasmid pPWC314-ΔtutF Lane 4 – Strain carrying the plasmid pPWC314-C9YA10S

Note: Equal concentration of protein was loaded in each lane (as determined by the

Bicinchonic acid method (85), using the BCA protein assay kit (Pierce, Rockford, IL)).

3.1.3 RT-PCR analysis of tutF mutants

Since no TutH protein was observed in the strain carrying the tutF site directed mutations (C9YA10S) (see Figure 14), an attempt was made to determine if any message

(RNA) was produced. Hence, RT-PCR analysis was carried out to check for the presence of tutD message in the tutF site-directed mutant. In the tutF deletion mutant, the tutD message should not be produced, due to a polar effect on downstream genes (16, 25). The tutF specific primers used for PCR amplification are listed in Table 7. As can be seen from Figure 15 A, the strain carrying a wild-type plasmid pPWC314 produced a 541 bP tutD message (Lane 1). No tutD message was detected in the strain carrying the vector pRK415 (Lane 2), the strain with an in-frame tutF deletion plasmid pPWC314-∆tutF

(Lane 3), and the strain carrying a plasmid with site-directed mutations in tutF pPWC314-C9YA10S (Lane 4) (results comparable to negative control). No bands were observed in the controls lacking reverse transcriptase (see Figure 15B), indicating the absence of any DNA contamination in the samples. Further, 16S rDNA bands were observed in all the samples (see Figure 15C), indicating the presence of RNA in all the samples. These results indicate that the site-directed mutation interferes with the tutD message production or stability, resulting in the lack of ability of the mutants to produce

TutH protein (see section 4.1).

tutD 16S rRNA

1 2 3 4 1 2 3 4 1 2 3 4 A. B. C.

Figure 15: RT-PCR analysis examining tutD message production from toluene

induced cells with tutF::kan chromosomal mutation.

(A) TR1/BR1 gene specific primers (with reverse transcriptase) (B) TR1/BR1 tutD specific primers (no reverse transcriptase) (C) 16F/16R ribosomal DNA specific primers (with reverse transcriptase) Lane 1 – Strain carrying the wild type plasmid pPWC314 Lane 2 – Strain carrying the vector alone pRK415 Lane 3– Strain carrying the plasmid pPWC314-ΔtutF Lane 4 – Strain carrying the plasmid pPWC314-C9YA10S

3.2 Mutations in tutG and tutH

3.2.1 Construction and complementation of tutG mutation

A site-directed mutation in tutG was generated such that the cysteine (slightly polar hydrophobic and neutral) at position 29 was changed to a serine (polar, hydrophilic and neutral), while engineering a BstBI restriction site (Table 6). The plasmid carrying this construct was tested for its ability to complement the tutG chromosomal insertion/deletion mutation. As can be seen in Table 9, the strain carrying this construct

(pPWC314-C29S) failed to grow on toluene serving as a sole carbon source, failed to completely metabolize toluene (results comparable to the negative controls), and failed to produce wild-type levels of phenylitaconic acid (also comparable to the negative controls). Hence, the plasmid carrying the mutated tutG gene was unable to complement the tutG chromosomal mutation, thereby indicating that the cysteine at position 29 is important for toluene metabolism. Further, no TutH protein or message production was detected in the mutant (see section 3.2.4 and 3.2.5).

Table 9: Test for the ability of site-directed mutation of the tutG gene to complement the tutG::kan chromosomal deletion mutation.

Strain Plasmid carried Percent toluene Percent Growth on left in the media phenylitaconic toluene acid producedd tutG::kan pPWC314 a 0 100 +e tutG::kan pRK415 b 89.3 ±0.4 0 -f tutG::kan pPWC314-∆tutG c 85.9±3.6 1.8± 0.1 - tutG::kan pPWC314-C29S 76.4±11.3 3.7±4.6 -

a The wild type plasmid carrying the tutE tutFDGH gene cluster, serving as a positive control. b The vector alone, serving as a negative control. c The wild type plasmid with in-frame tutG deletion. d Normalized to 100% for pPWC314, the positive control. e + = Growth with toluene as the sole carbon source. f- = No growth with toluene as the sole carbon source.

3.2.2 Role of tutH in toluene metabolism

Chromosomal deletions of the tutF tutD and tutG genes demonstrated that these genes are essential for toluene metabolism (25). Efforts were undertaken to determine if tutH is essential for toluene metabolism. Although numerous attempts were made, a chromosomal deletion of tutH was not successfully generated, so it was decided to take advantage of the polar nature of the tutG chromosomal deletion to determine if the tutH gene product is essential for toluene metabolism. Since tutFDGH are organized in a single operon, a chromosomal deletion of tutG should not result in the production of

TutH protein (due to polar effect on tutH) (16, 25, 26). A plasmid containing the tutE tutFDGH gene cluster with a tutH in-frame deletion was generated, mated into a strain carrying the tutG chromosomal insertion/deletion mutation, and tested for its ability to grow on and metabolize toluene (to complement polar tutG insertion/deletion mutation).

As can be seen in Table 10, the strain carrying a wild-type plasmid (pPWC314) grew on toluene serving as a sole carbon source, fully metabolized toluene, and produced phenylitaconic acid. The strain carrying the vector pRK415 did not grow on toluene as a sole carbon source, metabolize toluene or produce phenylitaconic acid at wild-type levels. Further, the strain carrying a plasmid with an in-frame deletion in tutH

(pPWC314-ΔtutH) also failed to grow on toluene as a sole carbon source, failed to

metabolize toluene, and produce phenylitaconic acid at wild-type levels (results

comparable to the negative control), indicating that the tutH gene product is essential for

83 toluene metabolism in T. aromatica strain T1. Further, no TutH protein or message production was detected in the mutant (see section 3.2.4 and 3.2.5).

3.2.3 Construction and complementation of tutH mutations

An attempt was made to identify amino acids in the TutH protein that are essential for toluene metabolism. A NCBI blastP similarity search was used to obtain protein sequences from closely related bacteria which were highly similar to the TutH protein sequence. Sequences that displayed a high degree of similarities with the TutH protein sequence are the BssE sequence from Thauera sp.DNT1 (83) and Azoarcus sp. T (1), chaperones from Azoarcus sp. EbN1 (75) and T. aromatica K172 (47), putative chaperones from Magnetospirillum (84), “ATPases Associated with a Variety of Cellular

Activities”(AAA) from Geobacter metallireducens GS-15 (GeneBank accession no.

ABB31771), and Geobacter sp. FRC-32 (GeneBank accession no. AASH01000015),

ATPase from Desulfotomaculum reducens MI-1 (GeneBank accession no.

AAOP01000001) and NorQ from Paracoccus halodenitrificans (78). In addition, the

NorQ protein sequence from Paracoccus denitrificans (28) and Rhodobacterium

sphaeroides (8) and the NirQ protein sequence from Pseudomonas stutzeri (51), which

display a conserved NirQ/NorQ binding region with the TutH protein sequence (26),

were also used for sequence comparison. Multiple sequence alignment was used to align

the selected protein sequences that are highly conserved across these bacteria (see section

2.5 ).

Since it has been reported that the TutH protein contains a putative ATP/GTP binding site that might be essential for its function (26), it was decided to use site- directed mutagenesis to alter this site. The glycine, lysine, and serine residues at positions

52–54 (part of the putative ATP/GTP binding domain) of TutH were selected for mutagenesis because they are highly conserved and contiguously positioned in the protein sequences of closely related bacteria (see Figure 11). Amino acid substitutions were designed to engineer a restriction site while minimizing the impact on the protein structure, due to change in charge, polarity or hydrophobicity of the substitute. Glycine

(non-polar and neutral) at position 52 was changed to alanine (non-polar and neutral), lysine (polar, hydrophobic and basic) at position 53 was changed to arginine (polar, hydrophilic and basic) and serine (polar, hydrophilic and neutral) at position 54 was changed to alanine (non-polar and neutral), while engineering a SmaI restriction site. The plasmid carrying this altered tutH gene (with the rest of the tutE tutFDGH gene cluster intact) was mated into the tutG chromosomal insertion/deletion mutant strain. As can be seen from Table 10, the strain carrying a plasmid with an altered ATP/GTP binding site

(pPWC314-G52AK53RS54A) failed to grow on toluene serving as a sole carbon source, metabolize toluene, and produce phenylitaconic acid at the wild-type levels, indicating that the amino acids at positions 52–54 (part of the putative ATP/GTP binding site) are essential for toluene metabolism.

Further, an attempt was made to identify amino acids in the TutH protein (other than the putative ATP binding domain) that are essential for toluene metabolism. Amino acid residues those were identical among the closely related bacteria and also contiguous

85 with neighboring conserved amino acids were selected for mutagenesis (see Figure 12).

The selected amino acid residues were changed using site-directed mutagenesis (see

Table 6). The leucine (non-polar, hydrophobic and neutral) at position 158 was changed to serine (polar, hydrophilic and neutral) and asparagine (polar, hydrophilic and neutral) at positions 159 was changed to alanine (non-polar and neutral), while engineering a

NheI restriction site (HL158SN159A). Glycine (non-polar and neutral) at position 161 was changed to alanine (non-polar and neutral) while engineering a HindIII restriction site (HG161A). The arginine (polar, hydrophilic and basic) at position 177 was changed to alanine (non-polar and neutral) and phenylalanine (non-polar, hydrophobic and neutral) at position 178 was changed to serine (polar, hydrophilic and neutral) while engineering a NheI restriction site (HR177AF178S) (see Table 6). Plasmids containing these site-directed mutations pPWC314-HL158SN159A, pPWC314-HG161A and pPWC314-HR177AF178S were then tested for their ability to complement the tutG chromosomal deletion mutation. As can be seen from Table 11, with toluene serving as a sole carbon source, the strain carrying the wild-type plasmid pPWC314 was able to grow on toluene, completely metabolize toluene and produce phenylitaconic acid. The strain carrying the vector pRK415 and the strain with the in-frame tutH deletion pPWC314-

∆tutH did not show growth on toluene serving as a sole carbon source, were unable to completely metabolize toluene and was unable to produce phenylitaconic acid. Similar results were observed with the tutH site-directed mutants. The strains carrying constructs pPWC314-HL158SN159A, pPWC314-HG161A and pPWC314-HR177AF178S failed to grow with toluene serving as the sole carbon source, were unable to completely

86 metabolize toluene (results comparable to the negative controls), and were unable to produce phenylitaconic acid at the wild-type levels (also comparable to the negative controls). These results indicated that these amino acids are critical for toluene metabolism. Further, no TutH protein or message production was detected in the mutants

(see section 3.2.4 and 3.2.5).

Table 10: Test for the ability of plasmids carrying tutH mutation (in ATP binding domain) to complement a (polar) tutG::kan chromosomal mutation

Strain Plasmid carried Percent toluene Percent Growth Left in the media phenylitaconic on acid producedd toluene tutG::kan pPWC314 a 0 100 +e tutG::kan pRK415 b 89.3 ±0.4 0 -f tutG::kan pPWC314-∆tutH c 72.2 ± 5.2 0 - tutG::kan pPWC314- 73.7± 7.4 2.0 ±0.2 - G52AK53RS54A

a The wild type plasmid carrying the tutE tutFDGH gene cluster, serving a positive control b The vector alone, serving as a negative control c The wild type plasmid with an in-frame tutH deletion d Normalized to 100% for pPWC314, the positive control e + = Growth with toluene as the sole carbon source f - = No growth with toluene as the sole carbon source

Table 11: Test for the ability of plasmids carrying tutH mutations to complement a

(polar) tutG::kan chromosomal mutation.

Strain Plasmid carried Percent toluene Percent Growth Left in the media phenylitaconic on acid producedd toluene tutG::kan pPWC314 a 0 100 +e

tutG::kan pRK415 b 90.7±3.8 0 -f tutG::kan pPWC314-∆tutH c 74.7±4.8 0 - tutG::kan pPWC314- 74.3±0.8 0 - HL158SN159A tutG::kan pPWC314-HG161A 83.0±10.7 0 - tutG::kan pPWC314- 91.0±11.0 0 - HR177AF178S

3.2.4 Protein production by tutG and tutH mutants

Western blot analysis was used to examine TutH protein production in tutG and tutH mutant strains. As can be seen from Figure 16, the strain carrying a wild-type plasmid pPWC314 produced TutH protein (Lane 1). No TutH protein was observed in the strain carrying the vector pRK415 (Lane 2) and the strain carrying a wild-type plasmid with an in-frame tutG deletion (Lane 3). Further, no TutH protein was produced by the strain carrying a wild-type plasmid with an in-frame tutH deletion (Lane 4), the strain carrying a plasmid with the tutG site-directed mutation pPWC314-C29S (Lane 5) and the strain carrying a plasmid with the tutH site-directed mutation pPWC314-

G52AK53RS54A (Lane 6) (results comparable to the negative controls). Similarly, as can be seen from Figure 17, no TutH protein was detected in the strain carrying a wild- type plasmid with an in-frame tutH deletion (Lane 3) and the strains carrying site- directed mutants pPWC314-HL158SN159A (Lane 4), pPWC314-HG161A (Lane 5) and pPWC314-HR177AF178S (Lane 6).

37 KDa

TutH 25

M 1 2 3 4 5 6

Figure 16: Western blot examining TutH protein production from toluene induced cells with tutG::kan chromosomal mutation

Lane 1 – Strain carrying the wild type plasmid pPWC314 Lane 2 – Strain carrying the vector alone pRK415 Lane 3 – Strain carrying the plasmid pPWC314-ΔtutG Lane 4 – Strain carrying the plasmid pPWC314-ΔtutH Lane 5 – Strain carrying the plasmid pPWC314-C29S Lane 6 – Strain carrying the plasmid pPWC314-G52AK53RS54A

Note: Equal concentration of protein was loaded in each lane (as determined by the

Bicinchonic acid method (85), using the BCA protein assay kit (Pierce, Rockford, IL)).

37 KDa

TutH

M 1 2 3 4 5 6

Figure 17: Western blot examining TutH protein production from toluene induced cells with tutG:: kan chromosomal mutation.

Lane 1 – Strain carrying the wild type plasmid pPWC314 Lane 2 – Strain carrying the vector alone pRK415 Lane 3 – Strain carrying the plasmid pPWC314-ΔtutH Lane 4 – Strain carrying the plasmid pPWC314-HL158SN159A Lane 5 – Strain carrying the plasmid pPWC314-HG161A Lane 6 – Strain carrying the plasmid pPWC314-HR177AF178S

Note: Equal concentration of protein was loaded in each lane (as determined by the

Bicinchonic acid method (85), using the BCA protein assay kit (Pierce, Rockford, IL)).

3.2.5 RT-PCR analysis of tutH and tutG mutants

Since no TutH protein production was detected in tutG and tutH site-directed mutants (see Figure 16 and Figure 17), RT-PCR analysis was carried out to check for the presence of tutH message. In tutG deletion mutants, tutH message should not be observed due to a polar effect on downstream genes (16, 25). The tutH specific primers used for

PCR amplification are listed in Table 7. As can be seen from Figure 18A, in the strain carrying a wild-type plasmid pPWC314, a 541 bp DNA band is observed indicating the presence of tutH message (Lane 1). No tutH message was detected in the strain carrying the vector pRK415 (Lane 2), the strain carrying a plasmid with the in-frame tutH deletion pPWC314-∆tutH (Lane 3), or the strains carrying plasmids with the site-directed mutations pPWC314-HL158SN159A (Lane 4), pPWC314-HG161A (Lane 5), pPWC314-HR177AF178S (Lane 6), pPWC314-G52AK53RS54A (Lane 7) or pPWC314-

C29S (Lane 8) (results comparable to negative control). No bands were observed in the controls lacking reverse transcriptase (see Figure 18B), indicating the absence of any

DNA contamination in the samples. Further, 16S rRNA messages were observed in all samples (see Figure 18C), indicating the presence of RNA in all samples. Minor deviations in sizes of the 16S rRNA message in different lanes might be attributed to genetic variations between these strains. These results indicate that the site-directed mutations interfere with tutH message production or stability, resulting in the lack of ability of the mutants to produce TutH protein (see sections 4.1 and 4.3).

tutH

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 A. B.

16S rRNA

1 2 3 4 5 6 7 8 C.

Figure 18: RT-PCR analysis examining tutH message production from toluene

induced cells with tutG::kan chromosomal mutation.

(A) TL2/NC1 gene specific primers (with reverse transcriptase) (B) TL2/NC1 gene specific primers (without reverse transcriptase) (C) 16F/16R ribosomal DNA specific primers (with reverse transcriptase) Lane 1 – Strain carrying the wild type plasmid pPWC314 Lane 2 – Strain carrying the vector alone pRK415 Lane 3 – Strain carrying the plasmid pPWC314-ΔtutH Lane 4 – Strain carrying the plasmid pPWC314-HL158SN159A Lane 5 – Strain carrying the plasmid pPWC314-HG161A Lane 6 – Strain carrying the plasmid pPWC314-HR177AF178S Lane 7 – Strain carrying the plasmid pPWC314-G52AK53RS54A Lane 8 – Strain carrying the plasmid pPWC314-C29S

3.3 Analysis of the TutH putative ATP binding domain

Computer analysis revealed the presence of a putative ATP binding domain in the

TutH protein, similar to the AAA family of ATPases (26, 47, 55, 67), thereby suggesting the possible involvement of TutH in the complex formation of benzylsuccinate synthase.

The ProteoEnrich™ ATP Binders™ Kit (Novagen) was used to confirm the ability of

TutH to bind ATP. Crude cell extracts of toluene induced cells were concentrated and depleted of free endogenous ATP by dialyzing against PBS buffer. Dialyzed extracts were then passed through the ATP columns. Proteins with ATP binding domains bound to the ATP column were eluted using the manufacturer’s provided buffer. The eluate was analyzed using SDS PAGE/Western blot, with a TutH specific antibody. As can be seen from Figure 19, no TutH protein was detected in the protein eluate from the tutG chromosomal deletion strain carrying the plasmid pPWC314-ΔtutH with an in-frame tutH deletion (Lane 1). TutH protein was detected in the protein eluate from the strain carrying a wild type plasmid pPWC314 (Lane 2). Further, it is evident from measuring protein concentration (see section 2.9.4) and Coomassie staining of gels (see section 2.9.5 and

Figure 19B) that protein was initially present in all the samples before loading on the

ATP column. This result demonstrates the ATP binding ability of the wild-type TutH protein which is absent in the TutH mutant protein.

50 KDa

TutH

25 M 1 2 M 1 2 A. B.

Figure 19: Analysis of the TutH protein for its ability to bind ATP

A. Western blot using TutH specific antibody against the protein eluate obtained by using ProteoEnrich™ ATP Binders™ Kit (Novagen), from toluene induced cells, with tutG::kan chromosomal mutation B. Coomassie staining on crude protein lysates before loading. Lane 1 – Strain carrying the plasmid pPWC314-ΔtutH Lane 1 – Strain carrying the wild type plasmid pPWC314

Note: Equal concentration of protein was loaded on each ProteoEnrich™ ATP Binders™ column during analysis (as determined by the Bicinchonic acid method (85), using the

BCA protein assay kit (Pierce, Rockford, IL.)). Protein concentration of eluates could not be estimated due to limited volume.

3.4 Induction with benzylsuccinate

The tutE tutFDGH gene cluster is reported to be induced by both toluene and benzylsuccinate (22). Further, analysis of tut chromosomal mutants has indicated that benzylsuccinate might be required for full induction of the tut operon (see section 1.10.2 )

(22). It has been observed that tut chromosomal insertion/deletion mutants (carrying plasmids with in-frame deletions of tut genes and site-directed mutations) are unable to produce TutH protein when induced by toluene (see Figure 16 and Figure 17). An attempt was made to detect the expression of TutH protein in benzylsuccinate induced tutH site-directed mutants. As can be seen from Figure 20, a band corresponding to the

TutH protein was observed in the tutG chromosomal deletion strain carrying a wild type plasmid pPWC314 (Lane 1) induced with benzylsuccinate, the strain carrying plasmid pPWC314-ΔtutH (Lane 2) with an in-frame tutH deletion (negative control) as well as the strains carrying plasmids with the site-directed mutations in tutH, pPWC314-

G52AK53RS54A (Lane 3), pPWC314-HL158SN159A (Lane 4), pPWC314-HG161A

(Lane 5) and pPWC314-HR177AF178S (Lane 6). Further, as can be seen from Figure 21, the strain with the tutG::kan chromosomal mutation carrying a wild-type plasmid pPWC314 (Lane 1), the vector pRK415 (Lane 2), plasmid pPWC314-ΔtutG (Lane 3), plasmid pPWC314-ΔtutH (Lane 4) and the strain with the tutF::kan chromosomal mutation carrying the vector pPWC314 (Lane 5) or plasmid pPWC314-ΔtutF (Lane 6) also produced TutH protein. Further, TutH protein was also observed in the strain with the tutD::kan chromosomal mutation carrying the vector pRK415 (Lane 7) or a plasmid

97 pPWC314-ΔtutD (Lane 8). The presence of a TutH protein band in the tutF, tutD and tutG chromosomal insertion/deletion mutants, carrying the vector pRK415 or plasmids carrying in-frame deletions of the corresponding tut genes indicate that TutH might be produced at the chromosomal level (rather than only at the plasmid level). Hence, it is likely that the tutH gene might have its own transcriptional start site subject to induction by benzylsuccinate. Further experimentation is required to confirm these results (see section 5.2).

37 KDa

TutH 25 M 1 2 3 4 5 6

Figure 20: Western blot examining TutH protein production from benzylsuccinate

induced cells with tutG:: kan chromosomal mutation

Lane 1 – Strain carrying the wild type plasmid pPWC314 Lane 2 – Strain carrying the plasmid pPWC314-ΔtutH Lane 3 – Strain carrying the plasmid pPWC314-G52AK53RS54A Lane 4 – Strain carrying the plasmid pPWC314-HL158SN159A Lane 5 – Strain carrying the plasmid pPWC314-HG161A Lane 6 – Strain carrying the plasmid pPWC314-HR177AF178S

Note: Equal concentration of protein was loaded in each lane (as determined by the

Bicinchonic acid method (85), using the BCA protein assay kit (Pierce, Rockford, IL)).

37KDa

TutH

M M 1 2 3 4 5 6 7 8

Figure 21: Western blot examining TutH protein production from benzylsuccinate

induced cells with tutG:: kan, tutF:: kan, and tutD:: kan chromosomal mutations.

Lane 1 – Strain with tutG:: kan chromosomal mutation, carrying the wild type plasmid pPWC314 Lane 2 – Strain with tutG:: kan chromosomal mutation, carrying the vector alone pRK415 Lane 3 – Strain with tutG:: kan chromosomal mutation, carrying the plasmid pPWC314- ΔtutG Lane 4 – Strain with tutG:: kan chromosomal mutation, carrying the plasmid pPWC314- ΔtutH Lane 5– Strain with tutF:: kan chromosomal mutation, carrying the vector alone pRK415 Lane 6 – Strain with tutF:: kan chromosomal mutation, carrying the plasmid pPWC314- ΔtutF Lane 7– Strain with tutD:: kan chromosomal mutation, carrying the vector alone pRK415 Lane 8– Strain with tutD:: kan chromosomal mutation, carrying the plasmid pPWC314- ΔtutD (M –Protein size markers are displayed on the leftmost side of the gel for size comparison)

Note: Equal concentration of protein was loaded in each lane (as determined by the

Bicinchonic acid method (85), using the BCA protein assay kit (Pierce, Rockford, IL)).

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4 DISCUSSION

4.1 Mutations in tutF and tut G

The tutF, tutD and tutG genes code for the three subunits of the enzyme benzylsuccinate synthase and are found to be essential for toluene metabolism (1, 26, 47).

Site-directed mutagenesis was used in an attempt to identify amino acid residues in the

TutF and TutG proteins which are essential for toluene metabolism. Cysteines were selected for mutagenesis since cysteines are often involved in metal binding or play important structural roles.

In TutG, the cysteine at position 29 was changed to a serine, while in TutF, the cysteine at position 9 was changed to a tyrosine and the alanine at position 10 was changed to a serine. The resultant mutant constructs were unable to complement corresponding chromosomal deletions. As can be seen from Table 8 and Table 9, the site- directed mutant strains failed to metabolize toluene and produce wild-type levels of phenylitaconic acid. Further, the mutants also failed to grow on toluene, indicating that the substitution of amino acids might disrupt production of the TutF and TutG proteins

(in tutF and tutG mutants respectively). Since TutF and TutG are subunits of the benzylsuccinate synthase complex, which is required for the anaerobic production of benzylsuccinate from toluene (25, 26, 59), the tut operon might not be completely induced in the site-directed mutants (since benzylsuccinate is required for complete induction of tut operon (22)). Hence, the mutants might not have the ability to produce

101 benzylsuccinate synthase which is required to metabolize toluene, produce tutD and tutH message (Figure 15 and Figure 18) or TutF and TutG proteins (Figure 14 and Figure 16).

Further, since TutF (6.9 kDa) and TutG (9.3 kDa) are smaller in size than TutD (97.6 kDa), they may contain part of an essential site (such as a metal binding site), while the rest of the site may lie with other subunits (TutD) of the benzylsuccinate synthase complex (16, 26). Additionally, it can be hypothesized that, since cysteines are often involved in metal binding (16), substitution of cysteines in TutF or TutG might have disrupted the function of benzylsuccinate synthase. This could occur by disrupting metal binding or structure. Moreover, inactivation or destabilization of the protein subunits can interfere with the complex formation of benzylsuccinate synthase, resulting in the inability of the mutants to metabolize toluene (16). Further, these results are consistent with previous work in T. aromatica strain T1 in which site-directed mutagenesis of TutD disrupted protein function (23).

4.2 Role of tutH gene in toluene metabolism

In T. aromatica strain T1 the tutH gene is co-transcribed with the tutE tutFDG gene cluster when induced with toluene (26). It has been previously reported that the tutE tutFDG genes are essential for toluene metabolism. Strains containing the plasmid with in-frame deletions of the tutF, tutD and tutG genes are unable to complement the corresponding chromosomal insertion/deletion mutations, indicating the importance of these genes in toluene metabolism (25).

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Attempts at deleting the tutH gene from the chromosome of T. aromatica strain T1 have been unsuccessful; however, the tutG chromosomal insertion/deletion strain was used for complementation analysis. The chromosomal deletion of the tutG gene has a polar effect on the downstream tutH gene and therefore does not result in production of

TutH protein. A plasmid with an in-frame tutH deletion was unable to complement the tutG chromosomal insertion/deletion mutation. As can be seen from Table 10, the positive control grows on toluene and metabolizes toluene to produce phenylitaconic acid. The strain carrying a plasmid with an in-frame deletion of tutH does none of these things (results comparable to negative control). These results indicate that the tutH gene product is essential for toluene metabolism. Although the role of TutH in toluene degradation is presently not determined, it is hypothesized that the TutH protein might act as a chaperone and may be involved in complex formation of benzylsuccinate synthase

(encoded by the tutF, tutD and tutG genes) (1, 26, 47, 55, 67). Hence, in the tutH deletion mutant, the absence of TutH protein might interfere with benzylsuccinate synthase complex formation (which is required for anaerobic conversion of toluene to benzylsuccinate (25, 26, 59)). Further, as benzylsuccinate is proposed to be required for complete induction of the tut operon (see section 1.10.2) (22), the resultant mutants were unable to produce tutH message (Figure 18) and hence TutH protein (see Figure 16).

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4.3 Mutations in tutH

It has been proposed that TutH (and the similar protein BssE) has a putative

ATP/GTP binding domain and is similar to a class of chaperone-like ATPases (see page

105) that may play an important role in the assembly/activation or disassembly of benzylsuccinate synthase (1, 26, 47, 55, 67). Since this domain is conserved, it may be important for protein function. Site-directed mutagenesis was used to disrupt the putative

ATP/GTP binding domain (G52AK53RS54A). The resulting construct was not able to complement the tutG polar chromosomal insertion/deletion mutation. As can be seen from Table 10, the positive control could grow on toluene, metabolize toluene and produce phenylitaconic acid, while none of these things were observed in the mutant

(results comparable to negative control). These results indicate that the putative

ATP/GTP binding domain of TutH protein is critical and might play an important role in

ATP binding, apparently required for TutH function (67, 69, 91). Disruption of the putative ATP/GTP binding domain might impede benzylsuccinate synthase complex formation (which is required for the anaerobic conversion of toluene to benzylsuccinate

(25, 26, 59)). Further, benzylsuccinate is proposed to be required for complete induction of the tut operon (see section 1.10.2) (22), which is required for toluene metabolism (see section 4.2) (16), but the resultant mutant was unable to produce tutH message (Figure

18) and hence TutH protein (see Figure 16).

It was decided to carry out site-directed mutagenesis of other amino acids in TutH

(other than the putative ATP-binding domain) to see if they play any role in toluene

104 metabolism. The TutH amino acid sequence exhibits homology with chaperones,

ATPases and NorQ from various denitrifying bacteria (see section 3.2.3). Amino acids that are highly conserved between the TutH protein and these homologous proteins were chosen for site-directed mutagenesis. Since the substituted amino acids were found to be conserved across closely related bacteria, they might have important structural or functional roles in these bacteria. Plasmids carrying the site-directed mutations in tutH gene (HL158SN159A, HG161A and HR177AF178S) were tested for their abilities to complement a tutG chromosomal insertion/deletion mutation. The mutant constructs failed to grow on toluene, metabolize toluene or produce phenylitaconic acid at the wild- type levels (see Table 11). Further, these mutants did not produce tutH message (see

Figure 18) and hence TutH protein (see Figure 17), which is required for benzylsuccinate synthase complex formation. Since, benzylsuccinate synthase catalyzes anaerobic conversion of toluene to benzylsuccinate and benzylsuccinate is required for the complete induction of the tut genes (see section 4.2) (16), amino acid changes in these mutants might have disrupted benzylsuccinate synthase complex formation and hence toluene metabolism. These results are also consistent with the previous results, where site- directed mutagenesis of the tutF and tutG genes results in failure of the mutants to metabolize toluene (see section 4.1) (16).

Computer analysis has indicated the presence of a conserved putative ATP binding domain the TutH protein, indicating that it belongs to the AAA family of ATPase

(1, 26, 47, 55, 67). The conserved AAA domain in TutH shows similarity with the conserved AAA domains of other bacteria listed in the NCBI Conserved domain database

105

(CDD). The AAA domain is found to be conserved across the proteins of various denitrifying bacteria such as NirQ of Pseudomonas stutzeri (51), NorQ of Paracoccus halodenitrificans (78) and Rhodobacter sphaeroides (8).

The AAA class of proteins is prevalent among both prokaryotes and eukaryotes, but is considered to have undergone a high degree of functional divergence before the emergence of the major divisions of life (67, 69). Members of the AAA family have a conserved ATPase domain and are involved in various cellular activities such as cell division and differentiation, protein degradation, organelle biogenesis and vesicle mediated protein transport (67, 69). Further, members of the AAA family can act as chaperones and are found to be involved in protein folding, proteolysis and also in the disassembly of stable protein–protein complexes (67, 69, 91). The energy required for such reactions is usually derived from ATP hydrolysis and the ATPase cycle (91). Some of the well known members of AAA class are the Clp proteases, proteins involved in

DNA replication and recombination, the Halobacterium GvpN gas vesicle synthesis

protein, dynein motor proteins, prokaryotic NtrC-related transcription regulators, TorsinA

and Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) activase (67, 69). The most widely studied protein that is involved in the disassembly of protein complexes is

N-ethylmaleimide sensitive factor (NSF). NSF is an ATPase which performs chaperone- like function and is involved in the dissociation of “soluble NSF attachment protein

[SNAP] receptors” (SNAREs), which plays an important role in membrane fusion and

vesicle trafficking (38, 91). Peroxisome biogenesis proteins Pexlp and Pex6p also have conserved AAA domains that are required for their functions (72). A eubacterial AAA

106 metalloprotease FtSH has a conserved ATP domain and a Zn+2 binding region that are important for its function (69, 90). It plays important roles in several cellular processes

such as protein assembly and export. Further, it is also involved in sporulation in Bacillus

subtilis and transcriptional activation of phage k CII (69). Several other members of the

AAA family have been identified but their functions have not yet been determined (69).

A common mechanism of action of the members of the AAA family is not clearly

defined, besides ATP binding/hydrolysis (67, 69, 91). Site-directed mutagenesis has

indicated that the putative AAA domain of TutH is essential for protein function. Further,

using ProteoEnrich™ ATP Binders™ Kit (Novagen), it has been demonstrated that the

TutH protein can bind ATP, consistent with the hypothesis of its role as a chaperone

involved in assembly/disassembly of benzylsuccinate synthase (67, 69, 91).

4.4 Induction by benzylsuccinate

The role of benzylsuccinate in the induction of the tut genes has been previously

reported (22, 25). T. aromatica strain T1 is unable to grow on benzylsuccinate as a sole

carbon source and it is believed that benzylsuccinate is not able to enter the cells (22, 25).

However, it might be able to induce the tut operon by a signal transduction mechanism

(see section 1.10.2 ). Further, it is hypothesized that in response to aromatic compounds,

benzylsuccinate synthase is produced in minute amounts. When toluene is converted to

benzylsuccinate it fully induces the tut genes to allow synthesis of more benzylsuccinate

synthase (22). As can be seen from Figure 16 and Figure 17, no TutH protein production

107 is observed in toluene induced tutH site-directed mutants and negative controls

(chromosomal insertion/deletion mutants and vector alone). However, when induced by benzylsuccinate a band corresponding to the TutH protein is observed in all of the tutH mutants as well as the negative controls (see Figure 20 and Figure 21). Presence of the

TutH band in negative controls (vector pRK415 that lacks a plasmid based copy of the tutE tutFDGH genes) clearly indicates the possibility that TutH protein production might be regulated by benzylsuccinate. Hence there is a possibility that the tutH gene might have its own transcriptional start site which is responsive to benzylsuccinate. However, further analysis including transcriptional analysis needs to be performed to confirm these results (see section 5.2). Further, these results are also consistent with previous results where tut chromosomal deletion mutants that are not capable of producing the wild-type level of tutE message when induced with toluene, produce tutE message when induced with benzylsuccinate (22).

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5 FUTURE PROSPECTS

5.1 Site-directed mutagenesis

Site-directed mutagenesis experiments have lead to the identification of amino acids essential for toluene metabolism. Some other amino acids that may be targeted for mutagenesis are the cysteines at positions 76, 98, 104 and 108 of TutE, the cysteines at positions 6, 29 and 53 of TutF and the cysteines at positions 26, 44 and 68 of TutG.

Cysteines can be primarily chosen for mutagenesis since they are often essential for the structure and function of proteins and might play an important role in metal binding.

Further, mutagenesis of different amino acids may be also carried out to determine their role in toluene degradation. Previous work has lead to identification of a cysteine in TutE that is not essential for toluene metabolism (16). A similar approach could be used to identify amino acids in other tut genes that are not essential for toluene degradation.

5.2 Induction by benzylsuccinate

Preliminary analysis has indicated the possibility that the tutH gene might have a separate transcriptional start site when induced with benzylsuccinate. Transcriptional analysis of tutH (induced by benzylsuccinate) using a nuclease protection assay or primer extension assay is required to confirm these results.

109

Further, an attempt could be made to differentiate between the benzylsuccinate induced, plasmid based TutH from the benzylsuccinate induced chromosomal TutH. This could be done by GST tagging of the tut genes in the plasmid and then distinguishing the

GST-Tut fusion protein from the chromosomal derived Tut protein based on the differences in protein sizes (the GST tagged fusion protein will be larger in size). An alternative approach might be to place the plasmid based copy of the tut genes under the control of an inducible promoter (optimally a T. aromatica strain T1 promoter), followed by a comparison of the expression of the Tut proteins with and without induction of the plasmid based tut genes. Once the technique to differentiate between the plasmid derived and chromosomal derived TutH is established, it might be possible to test the benzylsuccinate induced tutH mutant for ATP-binding analysis. However in the former case there is a possibility that the GST-Tut fusion protein might not be stable and might not bind the ATP-column.

Additionally, it might be also interesting to test the benzylsuccinate-induced tutF and tutG site-directed mutants (in tutF and tutG chromosomal backgrounds respectively) for their ability to produce TutH protein. However, it might not be possible to carry out this experiment with the tutH site-directed mutants because of the possibility of chromosomal production of TutH when induced with benzylsuccinate (see section 4.4).

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7 APPENDIX

A.1 To study protein- protein interactions between TutF, TutD, TutG and TutH proteins of T. aromatica strain T1.

In T. aromatica strain T1, the TutF, TutD and TutG proteins are hypothesized to interact to form a α2ß2γ2 complex, required for the functioning of benzylsuccinate

synthase (11, 17, 23, 25, 26, 59, 60). Based on the existing data, it can be hypothesized

that TutF and TutD should interact. Similarly TutD and TutG are hypothesized to

interact. It is presently not known if TutF can directly interact with TutG. The TutH

protein has an ATP/GTP binding domain and is proposed to be involved in complex

formation of benzylsuccinate synthase. Presently it is not known if TutH can stably

interact with the TutF, TutG or TutD proteins. An effort was made to study protein-

protein interactions between the TutF, TutD , TutG and TutH proteins using The

“Bacterio Match® II Two-Hybrid System kit” (Stratagene).

A.2 Methodology

The “Bacterio Match® II Two-Hybrid System Vector Kit” (Stratagene) was used

to study protein-protein interactions between the TutF, TutD, TutG and TutH proteins.

The BacterioMatch II two-hybrid system makes use of E. coli as the host for protein-

126 protein interaction screening. In the bait plasmid (pBT), the protein under study is fused to the full length λ repressor protein which is located downstream of the reporter promoter. The pBT plasmid contains an IPTG inducible promoter P lac-UV5, a p15A origin

of replication and a chloramphenicol resistance marker. In the target plasmid (pTRG)

another protein under study is fused to the α-subunit of RNA polymerase (N-terminal

domain). The target contains an IPTG inducible promoter P Ipp/lac-UV5, a ColE1 origin of

replication and a tetracycline resistance marker. Interactions between the test proteins

results in the binding of RNA polymerase at the promoter and transcriptional activation

of the HIS3 reporter gene, which encodes a component of the histidine biosynthetic

pathway, enabling the co-transformants (containing the interacting pair of bait and target)

to grow on media lacking histidine. The system also contains a secondary reporter gene

aadA that encodes for streptomycin resistance (see Figure A1).

DNA for cloning was prepared by PCR amplification of the gene of interest from

a plasmid containing tutE tutFDGH gene cluster. The genes under the study were cloned,

in-frame, into the bait plasmid (pBT) or the target plasmid (pTRG). The bait and the

target plasmids were transformed into chemically competent E.coli (XL1-Blue MRF’

Kan). Following transformation, cells were plated onto LB- chloramphenicol and LB-

tetracycline plates respectively and incubated for 24 hr at 300C. The recombinants clones

of interest, containing DNA inserts in the same reading frame as that of the λcI protein in bait and RNAPα protein in target, respectively, were verified using restriction digest,

PCR and DNA sequencing.

127

In addition to carrying out the actual protein-protein interaction analysis, control experiments were carried out to determine if the recombinant bait and target vectors are suitable for the interaction studies. Further, constructs can be tested for protein production by western blot. For protein-protein interaction analysis the BacterioMatch II two-hybrid reporter strain, containing the reporter gene cassette was co-transformed with recombinant bait and target vectors in equal proportion. Positive and negative controls were performed according to the manufacturer’s instructions. The

Figure A1: Schematic representation of Bacterio Match® II Two-Hybrid System

Vector Kit (Stratagene)

128 transformation mixtures were plated on 5mM 3-amino-1, 2,4-triazole (3-AT ) plates

(selective) and non-selective medium without 3-AT. Plates were then incubated at 300C

for 24 hr and further scored to determine the strength of the protein-protein interactions.

3-AT acts as a competitive inhibitor of the product of the HIS-3 gene. After the co-

transformation of the reporter strain with the bait and the target proteins, positive protein-

protein interactions can be detected by growth of the reporter strain on media devoid of

histidine, but supplemented with 3-AT. Transcription of the reporter gene HIS-3 results in

production of sufficient levels of HIS-3 gene product, to overcome the competitive

inhibition by 3-AT, resulting in growth of the co-transformants on selective media plates.

Presence of the colonies on selective plates indicates positive protein-protein interaction.

Presence of the colonies only on non-selective plates indicates successful co-

transformation but no protein-protein interactions. Positive co-transformants from the 3-

AT plates and the positive and negative controls can then be streaked onto dual selective

screening medium (5 mM 3-AT + Strep) plates. The positive clone will express the aadA

gene conferring streptomycin resistance to the cells and thereby allowing their growth on

the dual selective medium.

Fragments of the tutH gene (850 bp) and the tutG gene (200bp) were cloned into

the bait plasmid (pBT). A tutF gene (150bp) fragment was cloned in the target plasmid

(pTRG). The tutD gene, being larger in size, was cut and cloned as two distinct halves,

the tutD-front end (1.2 kb) in bait and the tutD-backend (1.6 kb) in bait and target, with a

200 bp overlap between both ends. Protein-protein interactions were studied using the

construct pairs (see Table A1).

129

Table A1: Construct pairs used for protein-protein interaction study

Bait Target

TutG TutF

TutH TutF

TutG TutD-backend

TutD-frontend TutF

TutD-frontend TutD-backend

A.3 Results

Co-Transformation of the BacterioMatch® II two-hybrid reporter strain was carried out with recombinant bait and target protein pairs. As can be seen from Table A2,

Table A3 and Table A4, negative controls (Row 1 and Row 2) did not show any growth on both selective as well as non-selective media. Positive control (Row 3) showed the

presence of co-transformants on both selective as well as non-selective media. Further,

the protein pairs pBT-TutG and pTRG-TutF (see Table A2 row 4), pBT-TutH and pTRG-

TutF (see Table A3, row 4) and pBT-TutG and pTRG-TutD (backend) (see Table A4,

row 4), pBT-TutD (frontend) and pTRG-TutF (see Table A5) and pBT-TutD (frontend)

and pTRG-TutD (backend) (see Table A6) showed the presence of co-transformation on

130 non-selective plates but the absence of co-transformants on selective plates. Further,

Western blot with TutH antibody did not demonstrate protein production by the pBT-

TutH construct. Western blots could not be carried out using other constructs due to the lack of specific antibodies.

A.4 Discussion

No positive protein-protein interactions were observed using the BacterioMatch®

II two-hybrid system. The positive control worked as expected indicating that that there was no problem with the kit. Western blot analysis of pBT-TutH did not demonstrate protein production by the construct. It is possible that there was no protein production by the constructs. The discrepancies in G+C content between T. aromatica and E.coli might result in differences in codon usage between the two strains. This could lead to less/no protein production from the tut genes when cloned into the E.coli based BacterioMatch®

II two-hybrid system. Further, it is also possible that the protein produced by the constructs is unstable resulting in its degradation, or message production is affected at the transcriptional level.

All of the constructs could not be tested for protein production by Western blot due to the lack of specific antibodies. However, other possible reasons for the lack of protein-protein interactions include improperly folded bait or target proteins and the requirement of certain cofactors/conditions that are not provided in the media. It is also possible that more than two proteins/other proteins are required for interaction. Further,

131 the lack of TuH might also be responsible for the lack of observed protein-protein interactions, consistent with the hypothesis that TutH is required for complex formation of benzylsuccinate synthase.

Table A2: Protein-protein interactions using pBT-TutG and pTRG-TutF

Protein pair Growth on Non- Growth on

selective medium selective medium

pBT-TutG + pTRG (empty vector) - -

pBT (empty vector) + pTRG-TutF - -

P pBT-LGF2 and pTRG-Gal11 + +

pBT-TutG and pTRG-TutF- + -

132

Table A3: Protein-protein interactions using pBT-TutH and pTRG-TutF

Protein pair Growth on Non- Growth on selective

selective medium medium

pBT-TutH + pTRG (empty vector) - -

pBT (empty vector) + pTRG-TutF - -

P pBT-LGF2 and pTRG-Gal11 + +

pBT-TutH and pTRG-TutF + -

Table A4: Protein-protein interactions using pBT-TutG and pTRG-TutD (backend)

Protein pair Growth on Non- Growth on selective

selective medium medium

pBT-TutG + pTRG (empty vector) - -

pBT (empty vector) + pTRG- TutD - -

(backend)

P pBT-LGF2 and pTRG-Gal11 + +

pBT-TutG and pTRG-TutD (backend) + -

133

Table A5: Protein-protein interactions using pBT-TutD (frontend) and pTRG-TutF

Protein pair Growth on Non- Growth on selective

selective medium medium

pBT-TutD (frontend) + pTRG (empty - -

vector)

pBT (empty vector) + pTRG- TutF - -

P pBT-LGF2 and pTRG-Gal11 + +

pBT-TutD (frontend) and pTRG-TutF + -

134

Table A6: Protein-protein interactions using pBT-TutD (frontend) and pTRG-TutD

(backend)

Protein pair Growth on Non- Growth on selective

selective medium medium

pBT-TutD (frontend) + pTRG (empty - -

vector)

pBT (empty vector) + pTRG- TutD - -

(backend)

P pBT-LGF2 and pTRG-Gal11 + +

pBT-TutD (frontend) and pTRG-TutD + -

(backend)