Examination of the Sigc Regulon and Cobalamin Biosynthesis In

EXAMINATION OF THE SIGC REGULON AND COBALAMIN BIOSYNTHESIS IN

MYCOBACTERIUM TUBERCULOSIS

BENJAMIN TOBIAS GROSSE-SIESTRUP

(Under the Direction of Russell Karls)

ABSTRACT

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains a leading cause of morbidity and mortality, claiming ~2 million lives annually. The current live-attenuated vaccine protects against meningeal TB in children, but protection against pulmonary TB varies from 0-80% among post-adolescents. Understanding the mechanisms by which M. tuberculosis adapts to successfully reside and cause disease in the host is vital for rational design of therapeutics, vaccines, and diagnostics.

In M. tuberculosis, a transcription initiation protein, sigma factor C (SigC), is a known virulence factor in murine and guinea pig TB infection models. However, no phenotypes in vitro have been reported. How SigC is regulated and how it aids the pathogen in vivo is unknown. In this study, the regulon of genes controlled by SigC was examined. Use of M. tuberculosis strains either lacking or overexpressing the sigC gene has revealed a role for SigC in trace metal acquisition. Use of medium deficient in trace metals resulted in a growth defect by a sigC mutant and will aid future studies to explore the regulation of SigC. This sigC mutant was confirmed to be attenuated in immune-compromised mice. The genomic location of the monocystronic sigC gene within a cluster of potential cobalamin biosynthesis genes led to our interest in cobalamin production. Cobalamins, such as vitamin B12, function as cofactors in various metabolic processes. While studies over fifty years ago reported vitamin B12 synthesis by mycobacteria, recent studies suggest that it is not made by M. tuberculosis. To investigate this discrepancy, production of cobalamin in mycobacteria was re-examined. Interestingly, B12 was not detected in any of the species that cause tuberculosis in mammals, but was produced in all other species tested. The presence of a full complement of cobalamin biosynthetic genes in M. tuberculosis suggests this pathogen produces the cofactor, but not under the conditions assayed. Differences in acquisition of trace metals for cobalamin synthesis by mammalian versus environmental mycobacteria or limitations of the detection methods utilized may account for the observed differences. Research is underway in the laboratory to test these hypotheses.

INDEX WORDS: Mycobacterium tuberculosis, Vaccine, Sigma factor, Virulence, Gene

regulation, Metals, Metal acquisition, Carbon metabolism, Vitamin B12,

Cobalamin

EXAMINATION OF THE SIGC REGULON AND COBALAMIN BIOSYNTHESIS IN

MYCOBACTERIUM TUBERCULOSIS

BENJAMIN TOBIAS GROSSE-SIESTRUP

FORB, University of Würzburg, Germany, 2006

A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

DOCTOR OF PHILOSOPHY

ATHENS, GEORGIA

2012

Benjamin Tobias Grosse-Siestrup

EXAMINATION OF THE SIGC REGULON AND COBALAMIN BIOSYNTHESIS IN

MYCOBACTERIUM TUBERCULOSIS

BENJAMIN TOBIAS GROSSE-SIESTRUP

Major Professor: Russell Karls

Committee: Frederick Quinn Mary Hondalus Ellen Neidle Eric Stabb

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia May 2012

DEDICATION

Ich widme diese Dissertation meiner Mutter, die während all dieser Jahre immer in meinem

Herzen war.

ACKNOWLEDGEMENTS

I owe thanks to many people who have supported me during my time in graduate school while pursuing my degree. First of all I would like to thank my advisor Dr. Russell Karls, and my committee members Dr. Frederick Quinn, Dr. Mary Hondalus, Dr. Eric Stabb, and Dr. Ellen

Neidle for guiding me through my time as a graduate student. Their input, scientific knowledge and constructive feedback was invaluable for my degree completion.

I also want to thank my family, especially my brother Sebastian and my father Matthias, for supporting me all this time. I owe thanks my wife Ivy who has accompanied me during most of my time at UGA. She shared all the highs and the lows with me and always encouraged me to keep going.

I had the privilege to work with many talented and friendly individuals in the lab. The scientific discussions and personal conversations had a great impact on the completion of my dissertation.

I want to especially mention Shelly Helms who has been there for me since day one. Thank you all.

Lastly I want to thank my friends and the crew of the German radio station “Sunshine Live”.

Your music and energy was part of the everyday lab work and helped me more than once to stay in the lab late at night to finish experiments.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS ...... v

CHAPTER

1 INTRODUCTION ...... 1

2 LITERATURE REVIEW ...... 3

3 MYCOBACTERIUM TUBERCULOSIS SIGC IS A REGULATOR OF A METAL-

SCAVENGING SYSTEM ...... 38

4 EXAMINATION OF COBALAMIN BIOSYNTHESIS IN MYCOBACTERIA ...... 78

5 CONCLUSIONS ...... 97

REFERENCES ...... 103

CHAPTER 1

INTRODUCTION

The bacterium Mycobacterium tuberculosis continues to be a major cause of human pestilence. Annually, 8-10 million people suffer from active tuberculosis (TB) resulting in approximately 2 million deaths. The estimated 2 billion individuals harboring the bacilli as a latent infection carry a 10% lifetime risk of TB reactivation. The existing live-attenuated

Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine protects against meningeal TB in children, however, in adults the protection against pulmonary TB is highly variable, ranging from 0-80%. New vaccines are in phase I trials; each has been imparted with enhanced bacterial survival or antigen presentation, or has been designed as a BCG booster. The problem with such vaccines is that BCG itself can cause serious disease in immune-compromised patients.

Therefore, alternate approaches to vaccine development are needed. One such approach is attenuation of virulent M. tuberculosis strains or further attenuation of BCG by deletion of genes required for growth in the host. Also, for the rational design of vaccines and therapeutics, it is vital to understand the mechanisms by which M. tuberculosis adapts to successfully reside and cause disease in the host.

In M. tuberculosis, the transcription sigma factor C (SigC) is known to be associated with virulence in mouse and guinea pig TB infection models. Thus, we hypothesize that this sigma factor directs transcription of genes required for survival inside the host. The sigC gene is not part of an operon, but is divergently-transcribed from a β-lactamase gene (blaC). A regulating 1

anti-sigma factor has not been identified. BlaC is a β-lactamase and responsible for high-level resistance against β-lactam antibiotics. The sigC gene is also located on the chromosome within a cluster of genes with homology to cobalamin biosynthesis genes from other bacteria.

Cobalamins, such as vitamin B12, serve as cofactors in enzymes that catalyze isomerization and reduction reactions involved in various metabolic processes. At the outset of this research, it was unclear which genes are regulated by SigC, how SigC production or activity is regulated, and if there is a link between SigC and regulation of cobalamin biosynthesis. Publications from the

th middle of the 20 century reported that M. tuberculosis produces vitamin B12; however, at least one recent publication suggests that it does not. Investigations of the SigC regulon and mycobacterial cobalamin biosynthesis are the foci of this dissertation.

CHAPTER 2

LITERATURE REVIEW

1 MYCOBACTERIUM TUBERCULOSIS

1.1 History and Taxonomy

Mycobacterium tuberculosis has been around for a long time. It is thought that the genus

Mycobacterium originated more than 150 million years ago [1] and it is likely that the modern members of the M. tuberculosis complex appeared 20,000-35,000 years ago in Africa [2].

Evidence for actual disease is lacking in that geographical area, however, tuberculosis (TB) disease characteristics and M. tuberculosis DNA sequences were found in Egyptian mummies

[1]. The disease has also been documented in Egyptian writings dating to 5000 years ago.

Mycobacterium tuberculosis has a lipid-rich cell wall and a high G+C DNA content. It is therefore hypothesized, that M. tuberculosis has a more stable genome than many other bacteria

[3]. At the beginning of the 17th century, a TB epidemic, the “Great White Plague”, started in

Europe and continued for the next 200 years, and by 1650, it was the leading cause of mortality

[3]. In 1882, German scientist Robert Koch was the first to obtain pure cultures of M. tuberculosis by growing the bacteria on solid medium. Nine years later, he published a first description of the partially-purified derivative (PPD) of tuberculin [4]. PPD was later developed into a diagnostic TB skin test by Charles Mantoux in 1907.

The members of the M. tuberculosis complex are grouped in the suprageneric rank of actinomycetes that usually have a high GC content (61-72% ), and currently consist of the 3

following species: M. tuberculosis, M. bovis, M. pinnipedii, M. africanum, M. microti, and M. caprae [2]. The obligate human pathogens are M. tuberculosis, and M. africanum, while the other members, M. bovis, M. pinnipedii, M. microti and M. caprae primarily infect animals.

Based on their genetic homogeneity and the lack of significant evidence of genetic exchange among them, it is believed that all species originated 20,000-35,000 years ago from a single common ancestor [5-6].

1.2 Clinical Bacteriology

The M. tuberculosis bacilli are non-motile and non-sporulating rods that have a very slow doubling time averaging 12-24 hr, depending on culture conditions. The genome is approximately 4.5 Mbp. Diagnostic staining relies on acid fastness, rather than on Gram staining.

This distinction is based on the cell wall which has a high content of lipids such as mycolic acids. The composition of these lipids varies throughout the life cycle of the bacteria depending on culture density and nutrient availability. Surface lipids are thought to be the primary cause for various phenotypes including: extreme hydrophobicity and resistance to injury, many antibiotics, and assaults by the host immune system [7-10].

1.2.1 Cell wall structure

The cell envelope of M. tuberculosis is probably the most outstanding feature of mycobacterial physiology and has been a primary focus of many researchers. It consists of the cytoplasmic membrane, a peptidoglycan/arabinogalactan matrix, and an outer membrane [10]. The cytoplasmic membrane contains various proteins including those that function in sensing, energy generation, and transport. Is also contains some lipopolysaccharides typical for actinomycetales.

Surrounding this membrane is the cell wall which is responsible for protection and mechanical support of the bacterium. Unlike the plasma membrane, the cell wall of M. tuberculosis is unique among prokaryotes. It consists of an inner layer of peptidoglycan covalently-attached to membrane proteins. Compared to other bacteria, the M. tuberculosis peptidoglycan exhibits a high degree of cross linking [3]. A branched polysaccharide (arabinogalactan) matrix is covalently-linked to the exterior of the peptidoglycan matrix. The arabinogalactan matrix is esterified to long fatty acids (60-90 carbon units in length), known as mycolic acids, which form the inner leaflet of the outer membrane [7, 10]. The outer leaflet consists of several lipids such as phthiocerol dimycocerosates (PDIM), trehalose-containing glycolipids, and phenolic glycolipids

(PGL). Glycolipids such as lipomannan (LM) and lipoarabinomannan (LAM) are anchored to the cytoplasmic membrane and extend beyond the cell wall. The outer membrane also contains porins, which allow the transport of small hydrophilic molecules through the hydrophobic layer.

Because of its unusual cell envelope, the bacteria stain red with the Ziehl-Neelsen acid-fast staining method [3]. The reason for this is that the bacteria resist decolorization with acid-alcohol after staining with carbol fuchsin.

1.2.2 Growth requirements

Mycobacterium tuberculosis can grow prototrophically or heterotrophically. That means it can either build all its components from basic carbon and nitrogen sources or consume already- synthesized partially-reduced carbon compounds as a source of energy and carbon. Nutritional limitations as well as physical factors, including oxygen availability, temperature, salinity, and pH, influence the growth of M. tuberculosis. However, the bacteria can adapt to survive in many harsh environments. This pathogen has been reported to persist in adipose host tissues, where

fatty acids are the major carbon source [11]. Robert Koch showed that the tubercle bacilli can grow on coagulated blood serum. It is also able to grow in solutions containing glycerol as carbon source and ammonium ions and asparagine as nitrogen sources [3]. M. tuberculosis is able to metabolize glycerol into pyruvate, which is further dissimilated into acetyl-CoA for entry into the TCA cycle and used for energy production [3]. Since the bacillus respires aerobically, it utilizes oxygen as the terminal electron acceptor and reduces it to water. It cannot respire anaerobically [12]. Therefore, it is not surprising, that M. tuberculosis is frequently found in tissues such as the lungs, with high oxygen tension. However, lipid anabolism has been reported as an electron sink that aids survival of the pathogen in low-oxygen conditions [13]. This obligate human pathogen has a growth optimum of 37°C and neutral pH.

1.2.3 Physical resistance

Because of its cell wall, M. tuberculosis can withstand harsh environment conditions, even though it does not form spores. The pathogen can survive within nonactivated macrophages in vitro, inside human granulomas, and is naturally resistant to very low temperatures. Upon freezing without cryoprotectant, bacterial viability, metabolic and pathogenic properties remain largely intact. However, sunlight and ultraviolet (UV) radiation are detrimental to the bacilli, killing them within a few minutes [14-15].

1.3 Immunology

In 90% of the cases, the immune system of a host is able to control the infection and symptoms of active TB never develop [3]. However, 10% of those infected develop active TB disease. The risk of developing disease is considerably higher in persons co-infected with an immune-

suppressing pathogen such as the AIDS virus [16] . Hence, the immunological status of the host plays a fundamental role in the outcome of the disease.

Macrophages are considered the main target for M. tuberculosis and are among the first cells that come in contact with the pathogen. After binding of M. tuberculosis bacilli to specific receptors on the surface of the macrophage, it is believed that the cholesterol in the cell membrane of the host cell is an anchor point for the bacterium and is involved in the phagocytosis [17]. Inside nonactivated host cells, M. tuberculosis bacilli often reside within the phagosome and block fusion with lysosomes [18-20]. Macrophages that are pre-activated with agonists, such as LPS and IFN-γ or TNF-α, are able to overcome this block and kill the bacilli through maturation of the phagosome [21]. Even though macrophages are considered the main targets for M. tuberculosis, other immune cells have been shown to be targeted as well. One group of targeted cells are neutrophils. It is controversial whether neutrophils are able to kill mycobacteria [22-23]. However, it is thought that neutrophils influence the disease outcome either by killing the bacteria directly through the secretion of chemokines and the induction of granuloma formation, or by transfer of microbicidal molecules to infected macrophages [24-26].

Dendritic cells tend to concentrate in TB lesions [27] and are important in inducing a cellular immune response against M. tuberculosis infections. Dendritic cells bind the bacilli mainly through the C-type lectin dendritic cell-specific intercellular adhesion-molecule-3-grabbing- nonintegrin (DC-SIGN) receptor [28-30]. After uptake of the bacteria, the dendritic cells mature and migrate to regional lymph nodes [31], where they present the antigens and release cytokines to induce maturation of T-cells. The T-cells then migrate to the infection site where they release

IFN-γ, thus activating macrophages. This activation however, can also be blocked by M. tuberculosis [32]. 7

Since M. tuberculosis is an intracellular pathogen, it is believed that the humoral immune response does not play a large role in the host response to infection. Only during the initial part of the infection or during the extracellular stages might antibodies have some effect to prevent binding of the tubercle bacilli to specific host receptors [33]. However, antibodies do seem to enhance immunity through for example opsonization, complement activation, or promotion of cytokine release [34]. Since the bacteria are located inside compartments within macrophages, their antigens are being presented to the cells of the cellular immune response. This antigen presentation results in the activation of CD4+ T-cells which produce IFN-γ. That in turn activates macrophages which promotes destruction of the intracellular tubercle bacilli.

Additionally, CD4+ cells have been shown to support the development of CD8+ cells [35], which are thought to be important in controlling M. tuberculosis infections [36]. While the innate immune system plays a part in preventing M. tuberculosis infections, the adapted immune system has a major influence on the outcome of the disease progression.

1.4 Pathogenesis

Following the initial infection in the lungs, there are two types of infections caused by M. tuberculosis: progressive/active TB or latent TB. It is estimated that 90% of immune-competent individuals are able to prevent initial M. tuberculosis infection from progressing to active TB disease, but are unable to clear the infection entirely. In active TB in humans, a characteristic lesion, the caseating granuloma, may result. These lesions normally form in the lungs and consist of a central area of caseous necrosis surrounded by epitheloid macrophages and lymphocytes. In healthy hosts, M. tuberculosis bacilli are constrained within the center of these granulomas but are not necessarily killed [37]. Inside the granulomas, the bacilli face low oxygen, acidic 8

conditions, reactive nitrogen and oxygen species, and other caustic agents [38]. Some bacteria survive this hostile environment and remain in a dormant or slowly-replicating state for decades.

This latent TB infection is clinically asymptomatic and the host is not contagious. The centers of some granulomas heal and become fibrotic and calcified; however, scarring often remains and is detected via chest autoradiograms. If the immune system of the infected individual becomes compromised, the granulomas can break open and the bacteria can return to an actively-growing state, causing pulmonary TB. If the disease is not controlled, it rapidly destroys the pulmonary parenchyma leading to a disseminated infection. Factors such as alcoholism, malnutrition, HIV infection, or immune-suppressive drugs promote the development of active TB disease. The most-recognized symptoms of active TB include lack of appetite, low-grade fever, and night sweats. Additionally, symptoms such as coughing up blood, weight loss, or chest pain can also occur. Most active TB cases result from reactivation of dormant bacteria [39]. In later stages of the disease, the bacteria can spread and tuberculomas can form in lymph nodes, bone, brain, kidney, and other organs [40-43]. However, pulmonary TB occurs much more frequently than systemic infections. Using latent infection models and tissue samples from human patients with latent TB, mycobacterial DNA was found in a variety of cell types, including non-professional phagocytes [44]. Since these cell types are not necessarily able to kill ingested bacteria, it seems to be an efficient strategy for M. tuberculosis to avoid elimination inside the host. As mentioned earlier, M. tuberculosis can infect organs other than the lungs. Adipose tissue is postulated by some to be an important location for the bacilli during dormancy, since mycobacterial DNA has been frequently found in adipose tissue of individuals that died from causes other than TB [11].

1.5 Diagnosis

The gold standard for diagnosing active TB today is acid fast bacilli smear microscopy in combination with culturing of the bacteria. Those diagnostic techniques are used to detect tubercle bacteria in the respiratory tract (pulmonary TB) or in other host sites (extrapulmonary

TB). The bacilli smear is fast and inexpensive and therefore especially important for third world countries. However, culturing the bacteria takes up to 6 weeks in which the physician has to decide about treatment of the patient. More problematic is the detection of latent TB that arises when the adaptive immune system is able to control but not eliminated the pathogen. This results in asymptomatic infection without detectable bacteria. The most commonly used test for detecting M. tuberculosis exposure or latent TB in nonvaccinated individuals is the tuberculin skin test (TST): the delayed hypersensitivity reaction to PPD [45] . This test takes advantage of specific cellular immune responses of the host to the mycobacterial disease. It works by injecting

PPD intradermally into the forearm. The test is read 48-72 hours after the injection. A positive diagnosis is indicated by indurations of >10 mm in diameter. The active ingredients that are being used in the TST are several proteins that also exist in M. bovis Bacillus Calmette-Guérin

BCG, the vaccine strain, and other mycobacteria that commonly exist in the environment.

Therefore, people who have been vaccinated are expected to show a false-positive reaction.

Additionally, people tested frequently by this test (e.g. healthcare workers) may eventually develop a response from essentially being immunized with PPD. Because of these potential complications, if people have been BCG vaccinated, or have shown a positive result previously, an alternate test such as chest X-ray is used [46]. A newer diagnostic test that has been developed in the past 10 years is the IFN-γ release assay (IGRA) [47]. This test measures the

IFN- γ released from T-cells specific to M. tuberculosis antigens. Whole blood from a 10

potentially-infected individual is mixed with protein antigens that are present in M. tuberculosis but not in the BCG vaccine or in the majority of nontuberculous mycobacteria. The IGRA test is therefore, considered more specific for M. tuberculosis exposure than is the skin test [48].

1.6 Treatment

Treatment of TB tries to achieve two goals: First, fast killing of the dividing bacteria in the extracellular lung cavities to prevent further transmission of the disease. Second, complete elimination of less-active bacteria within lesions and intracellular semi-dormant bacteria inside of other host tissues [49]. Latent TB infections with a high risk of developing active TB are normally treated for six to nine month with isoniazid (INH), which is the first-line antituberculosis medication [50]. However, since resistance can develop quickly if only a single antibiotic is administered, INH is almost never used by itself for an active TB infection. Drugs frequently combined with INH are ethambutol (EMB), rifampicin (RIF), and pyrazinamide

(PZA), which are also considered first-line drugs. Second-line antibiotics like kanamycin and amikacin are generally less effective than first-line drugs and are associated with greater side effects. Second-line drugs are used once resistance to first-line drugs is detected or suspected. In the past decade, poly-, multiple-, and extensively drug resistant strains have emerged and no new antituberculosis drug has been developed since 1970. In 2008, the World Health Organization

(WHO) reported 11.1% of cases in the European region as being multi drug resistant [51].

Several strategies are currently being used by academic and industrial institutions, trying to develop new effective drugs against TB. An important strategy is the development of new molecules derived from already-known drugs that have been proven safe and successful in the past, for example ethambutol or isoniazid analogues. Two new molecules in the 11

chemotherapeutic group of fluoroquinolones have been developed recently: moxifloxacin and gatifloxacin. There are also several completely new drugs in clinical trials right now. The most promising of them is TMC-207, a diarylquinoline. It inhibits the mycobacterial ATP synthase and has been shown to be successful in small trials [52]. However, large-scale tests have to be conducted before these drugs gain FDA approval.

1.7 TB and HIV

TB re-emerged in the late 80s in the developed world due to the appearance of the human immunodeficiency virus (HIV). The pandemic with HIV resulted in an increase in TB cases worldwide, especially in the sub-Saharan Africa. Diagnosis of TB in HIV patients is more challenging because of a lower rate of positive results through a conventional sputum microscopy smear or a greater proportion of extrapulmonary disease [53]. Even though some progress has been made in diagnostics, for example fluorescence microscopy increases sensitivity [54-55], there is still need for more specific and more sensitive diagnostics tools that are cheap and simple to use in developing countries. Culturing the bacteria on selective media is still the most sensitive method for M. tuberculosis detection in clinical samples. Mycobacterium tuberculosis and HIV increase the negative effects of both pathologies. Weakening of the immune system caused by HIV, results in activation of a latent TB infection. In turn, a TB infection increases HIV replication [56]. It has also been shown that HIV impairs the IFN-γ production associated with M. tuberculosis infection and that antiviral treatment does not reverse this effect [57]. TB can occur anytime during a HIV infection, but the clinical symptoms are dependent on the severity of the immune deficiency. AIDS patients are more likely to develop disseminated forms of TB, especially TB meningitis, and have a higher risk of death [58]. The 12

treatment in HIV-positive patients is similar to the treatment in HIV-negative patients. Similar antimycobacterial drugs are being used, but the Centers for Disease Control and Prevention recommends extending the treatment beyond 6 month [59]. Treating the patients at the same time with highly active anti-retroviral therapy substantially improves the prognosis. However, some antibiotics interact with certain classes of anti-retroviral medication and it needs to be carefully determined which treatment the patient will undergo.

2 MYCOBACTERIUM TUBERCULOSIS SIGMA FACTORS

2.1 Basics of Sigma Factors

Sigma factors (σ factor) are prokaryotic transcription initiation factors. They transiently bind to core RNA polymerase (RNAP) to form an RNAP holoenzyme. The core RNAP is a large ~400 kDa protein and often contains five subunits: α2, β, β', and ω. The RNAP holoenzyme is able to bind gene promoters recognized by the specific sigma factor. Although it enables transcription initiation, the σ factor dissociates from the transcription complex when a 9-12 nt transcript is produced. Core RNAP continues to elongate the transcript until it reaches a terminator. The

RNAP then releases the transcript and DNA. The core RNAP is then able to bind a free sigma factor. The relative abundance and binding affinity of individual sigma factors for core RNAP influences which holoenzymes are formed and which genes are transcribed.

Sigma factors are divided into several groups. These groups contain those related to E. coli

σ factors which are sometimes labeled based on their molecular weight (e.g. σ32, σ54, or σ70). The group containing the σ factors related to the E.coli σ70 is the only group that is represented in mycobacteria [60]. The σ70 family members contain up to four conserved regions connected by flexible linkers [61]. The four regions can be further divided into subunits [62]. Region 1 contains the subregions 1.1 and 1.2 and is located at the amino terminus of the σ factor. This region is responsible for preventing DNA binding of σ factors that are not bound by an RNAP

[63]. Region 2 contains four subregions. Region 2.3 is required for melting the transcription bubble while region 2.4 binds the -10 promoter element [64-65]. Region 3 contains three subregions. It is thought that subregion 3.0 is involved in recognition of the extended -10 promoter region [66]. The last region is region 4, which consists of subregions 4.1 and 4.2. The

latter subregion is required for the recognition of the -35 promoter element. It is also responsible for the interaction with several transcription activators [67-68].

The σ70 group can be further divided into four different groups: principal or primary σ factors (group 1), non-essential primary-like σ factors (group 2), alternative σ factors (group 3) and extracytoplasmic function (ECF) σ factors (group 4). Group 1, the primary σ factors are also known as housekeeping σ factors and are essential for the bacterial growth in vitro. They contain all four regions. The primary-like σ factors from group 2 are structurally related to the primary σ factors but are not essential under laboratory conditions and lack most of region 1. They are often involved in transcription of stress-response and stationary-phase survival genes. Group 3, the alternative σ factors, show less sequence similarity with those of group 1 and include proteins required for the heat-shock response and motility [69-70]. The ECF σ factors, group 4, are the largest and most heterogeneous group [62], and contain only the conserved regions 2 and 4.

Sigma factors of this type transcribe genes with products involved in cell envelope synthesis, secretory or transport functions, or those located within or outside the cytoplasmic membrane

[71-72]. They often transcribe genes associated with virulence [73-76].

2.2 Anti-Sigma Factors

A common mechanism of post-translational regulation of σ factors happens through antagonistic proteins called anti-σ factors. These proteins bind to the σ factor, hence preventing its interaction with the core RNAP. Certain external stimuli can be sensed by anti-σ factors, which react by releasing the σ factor under stress conditions facilitating the transcription of the σ factor’s regulon. Anti-σ factors can be negatively regulated by molecules called anti-anti-σ factors [77].

To date, five anti-σ factors have been identified in M. tuberculosis: RseA, RshA, RslA, RskA, 15

and UsfX. RseA inhibits the transcriptional activity of σE in a dose-dependent or temperature- dependent manner [78]. RshA is part of the sigH operon and negatively regulates σH [79]. RshA is a reductive-stress sensor and inhibits σH only under reductive stress conditions. The inhibition is reversed under oxidative stress or elevated temperature conditions. Phosphorylation of RshA by PknB decreases its interaction with σH [80].The rslA gene encodes a transmembrane protein that is co-transcribed with sigL. It was shown that RslA specifically interacts with σL and causes negative regulation the σ factor [81-82]. The rskA gene, is located downstream of, and co- transcribed with sigK. Like rslA, it encodes a transmembrane protein. Its intracellular N-terminal domain is involved in interaction with σK. The stimulus that results in activation of RskA remains unknown. UsfX is the anti-σ factor specific to σF. It directly interacts with σF and inhibits its transcriptional activity. UsfX has two antagonists; the anti-anti-σ factor RsfA and

RsfB [83]. RsfA has two cysteine residues that function as redox sensor. Under reductive conditions, UsfX is inhibited by RsfA.

2.3 Mycobacterium tuberculosis Sigma Factors

According to DNA sequence analyses, M. tuberculosis strains H37Rv and CDC1551 have 13 σ factors from all four groups of the σ70 family [84]. They include the house keeping σ factor SigA from group 1[85], a primary-like σ factor, SigB from group 3 [86], and an alternate σ factor,

SigF, which has homology to stationary phase σ factors in Bacillus subtilis [87] and which is part of group 3. The other ten belong to group 4 and are classified as extracytoplasmic function

(ECF) σ factors [71-72].

Generally, the number of σ factors increases with the size of the bacterial genome. This is measured by the alternative sigma factor (ASF)/genome size (megabase pairs, Mb) ratio. It has 16

also been observed, that the number of σ factor genes usually depends on the diversity of lifestyles encountered by a bacterium. The more diverse the lifestyle is, the more σ factor genes tend to be present in the genome [62]. Examples include the two mycobacterial strains M. smegmatis strain MC2 155 and M. leprae. While the genome of the saprophyte M. smegmatis contains 28 σ factors, the genome of the obligate pathogen M. leprae contains only four functional σ factors. Mycobacteria have a relatively high ASF/Mb ratio compared to other bacteria. M. tuberculosis and M. avium subspecies have a ratio of 2.7 and M. smegmatis has a ratio of 3.6. The only exception is M. leprae, which has a very low ASF/Mb ratio of only 0.9

[60].

It is generally believed that every σ factor has unique promoter specificity, allowing the transcription of defined subsets of genes. The expression and activation of each σ factor is also theoretically tailored to respond to distinct environmental conditions. Under non-stressed conditions, transcription is dominated by the primary sigma factor SigA. Expression of a σ factor gene may change in response to an environmental stress such as denaturants, heat shock, nutrient depletion or oxidizing agents [88-89]. Levels of several M. tuberculosis σ factors (sigF, sigE, and sigH) were elevated during M. tuberculosis infection of macrophages [90], and several have been reported to be associated with virulence in murine and/or guinea pig infection models [81,

91-95].

2.3.1 Sigma Factor A

In M. tuberculosis, the primary sigma factor, σA is a member of the group 1 σ factors and is essential for mycobacterial viability [85]. The gene sigA is often used as internal standard in quantitative RT-PCR experiments, as it is theoretically constitutively-expressed under various in

vitro growth conditions [88]. However, it has recently been shown that after phagocytosis, the expression of sigA increases in certain clinical strains, but not in H37Rv [96]. Subregion 4.2, located at the C-terminus of σA, is known to interact with transcriptional activators in some bacteria [68]. In M. tuberculosis, it was shown that this σA domain interacts with WhiB3, a putative transcription regulator [97]. This interaction was prevented by substituting an arginine for a histidine at residue 515 (R515H). This mutation was also shown to cause attenuation of M. bovis ATCC35723 in a guinea pig model but not in an opossum animal model [98]. A whiB3 mutant in M. bovis caused the same phenotype as the sigA R515H mutant. However, in M. tuberculosis it was shown that inactivation of whiB3 resulted only in partial attenuation, which suggests that the different genetic backgrounds of M. tuberculosis and M. bovis play a role in the sigA-whiB3 interaction.

2.3.2 Sigma Factor B

The σ factor group 2 is solely comprised of σB. This σ factor is not required for growth of M. tuberculosis or M. smegmatis. It contains regions 2, 3 and 4 but lacks most of region 1. The gene sigB is located approximately 3 kb downstream of sigA. Even though it is not required for growth, it has been shown that a M. tuberculosis sigB mutant is more sensitive to environmental stresses, including SDS-induced surface stress, heat shock, oxidative stress and vancomycin exposure [60]. However, σB is not required for growth in human macrophages. The regulation of sigB is rather complex. When exposed to surface stress, its expression depends on σE, while under oxidative stress and heat shock, its expression depends on σH [88-89, 99]. Also, promoters recognized by σL or σF-containing RNA polymerase have been found upstream of sigB [82].

Sigma factors frequently initiate their own transcription. This is not the case for σB. It is thought

that the response regulator MprA has a small effect on transcription of sigB as MprA has been shown to bind upstream of sigB during growth under physiological conditions [100]. Overall one can assume that σB is involved in general stress response, adaption to stationary phase and carbon starvation.

2.3.3 Sigma Factor F

SigF is a member of the group 3 σ factors and was originally thought to only appear in some slow-growing mycobacteria [87]. Later however, it was found that all sequenced mycobacterial genomes contain a sigF homolog. Expression of σF is post-translationally hindered by the anti-σ factor UsfX and induced after exposure to several antibiotics (rifampin, ethambutol, cycloserine and streptomycin), hypoxia, cold shock, oxidative stress, and entry into stationary phase [87,

101]. However, this induction was only observed when the M. tuberculosis sigF gene was introduced into M. smegmatis and M. bovis BCG. SigF induction was not detected after exposing

M. tuberculosis to the same stresses, except nutrient depletion [88, 102]. This indicates, that σF is differentially regulated in M. tuberculosis compared to M. smegmatis and M. bovis BCG. A sigF mutant in M. tuberculosis was shown to grow to higher densities in stationary phase than its parent strain [103]. The same sensitivity to various stresses, including heat shock, cold shock, hypoxia, and long-term stationary phase was observed between mutant and parent strain and no difference was observed when grown in human monocytes. However, it was observed that the sigF mutant was more permeable to a hydrophobic solute and that it was attenuated in a mouse model. In that model, the mutant strain was able to persist in the lungs of the mice but the bacterial load in the organs was lower compared to the parent strain [104]. Microarray data of cDNA comparing sigF mutant versus parent suggests, that σF plays a major role in the adaption

to stationary phase. It was also shown that sigC was downregulated in the sigF mutant [104], and a σF promoter was identified upstream of sigC [105].

2.3.4 Sigma Factor D

The sigD gene, encoding ECF sigma factor D, is thought to be part of the RelM. tuberculosis regulon

[106] and is induced under starvation [102]. This regulon produces hyperphosphorylated guanidine which is used as a signaling molecule that effects bacterial gene expression for long- term survival and under starving conditions [107]. A sigD mutant strain has been constructed in both M. tuberculosis strains H37Rv and CDC1551 [93, 108]. In a mouse model, both strains were able to grow at the same level as the wildtype in the organs. However, the virulence of the mutants was severely attenuated as determined by increased mouse survival times and by histopathology.

2.3.5 Sigma Factor E

The sigma factor E is also part of the group 4 ECF sigma factors and one of the most-studied sigma factors of M. tuberculosis. The sigE gene has been shown to be induced after exposure to various stress conditions, including heat shock, oxidative stress, and cell surface stress [88-89,

99]. The sigE gene is also induced after M. tuberculosis infection of human macrophages [109].

A sigE mutant was more sensitive to all of the mentioned stress factors and was shown to be attenuated in immune-competent BALB/c mice and severe-combined immune-deficient (SCID) mice [94, 110]. Similar phenotypes of sigE mutants of M. tuberculosis strains H37Rv and

CDC1551 have been reported [111]. SigE does not directly regulate transcription of its own gene. Instead, it is regulated by two known promoters that are located upstream of sigE. One

promoter is regulated by the two-component system MprA/MprB [100] and the other one has been shown to be responsible for σH-dependent induction of sigE after exposure to certain stress conditions such as heat shock or exposure to diamide [99]. Using DNA microarray analysis, several genes have been identified that are regulated by σE following surface stress [110]. The σE regulon genes encode proteins involved in fatty acid degradation, such as isocitrate lyase (icl1), fadE23 and fadE24. These last two genes have been reported to be induced by isoniazid or ethionamide treatment [112] and downregulated by starvation [102]. Additionally, σE also regulates expression of σB, MprA/MprB, heatshock and surface exposed proteins.

2.3.6 Sigma Factor H

Mutants of the ECF σH have been derived from M. tuberculosis strains H37Rv and CDC1551

[89, 95, 99]. A sigH knockout mutant was shown to be sensitive to high temperatures and oxidative environments, and sigH was induced after heat shock, after exposure to the oxidizing agent diamide, and during infection of macrophages [90]. It was also shown that animals infected with the mutant presented reduced pathology even though the mutant bacilli grew to the same levels in the mouse organs as did the parent strain [89, 95]. The animals also survived longer when infected with the mutant and produced a delayed pulmonary inflammatory response.

However, the sigH mutant was not impaired for growth in THP-1derived macrophages and was similarly sensitive as the parent to killing by activated murine macrophages. While regulating sigE and sigB, σH also regulates its own structural gene sigH. Additionally, genes encoding stress response proteins, DNA repair proteins, thiol metabolism enzymes, and cysteine and molybdopterin synthesis enzymes are also present within the σH regulon [89].

2.3.7 Sigma Factor J

While sigJ, encoding the ECF σJ, is strongly induced in stationary-phase cultures [113], the survival in stationary phase by M. tuberculosis bacilli is greatly reduced in a sigJ knockout mutant [114]. Transcription of sigJ is also upregulated in human macrophages; however, mice infected with the mutant were as equally virulent as the parent strain [115]. The only stressor identified that affects a sigJ mutant is exposure to H2O2, which suggest a role in oxidative stress response [114]. Recently, a σJ-dependent promoter has been identified upstream of sigI [116]

2.3.8 Sigma Factor K

SigK, another ECF sigma factor, was shown to be regulated by the anti-σ factor RskA [117].

SigK was shown to control expression of the antigen-encoding genes mpb70 and mpb83 [118].

This was determined by the identification of a mutation in the translation start codon of sigK in some strains of M. bovis BCG. Strains with this mutation had a low expression of mpb70 and mpb83, while other BCG strains without this mutation and M. tuberculosis expressed mpb70 and mpb83 on high levels. SigK also regulates its own operon, which also encodes a putative amine oxidase, a cyclopropane-fatty-acyl-phospholipid synthase, and four other proteins of unknown function [118].

2.3.9 Sigma Factor L

A sigL mutant has been constructed in M. tuberculosis strain H37Rv and tested in a mouse model [81-82]. It was shown that the mutant grew in the organs at the same rate as the parent strain, but the lethality of the mutant was strongly reduced. While exposure to SDS and oxidative stress did not increase transcription of sigL, sensitivity to these stresses was increased in the

mutant compared to the parent strain. RslA is the anti-σ factor regulating σL activity. To examine the σL regulon, a strain was constructed containing sigL but lacking rslA. Twenty-eight genes were positively-regulated by σL. Among them sigL, sigB, several genes encoding proteins involved in cell envelope processes, and two genes encoding proteins that are possibly involved in controlling the redox state of exported proteins. These results were confirmed using an acetamide-inducible promoter to overexpress the sigma factor.

2.3.10 Sigma Factors M, G, and I

Little information is available on the three ECF sigma factors σM, σG and σI. SigM is expressed at low levels in vitro and was not found to be involved in virulence. This sigma factor positively regulates four esat-6 homologues (esxE, esxF, esxT, and esxU) [119], and genes encoding two members of the proline-proline-glutamate (PPE) family. It also negatively regulates genes involved in the synthesis of surface lipids, such as phthiocerol dimycocerosates (PDIM), and the kasA-kasB operon which is required for mycolic acid synthesis [120]. A mutant lacking the sigG gene has recently been constructed. It was reported that the mutant is attenuated in mouse macrophages and more resistant to mitomycin C treatment than the parent strain [121]. A σJ- dependent promoter was identified upstream of sigI [116].

2.3.11 Sigma Factor C

SigC belongs to the group 4 (ECF) σ factors. It is suggested that σC and σE play an especially important role in mycobacterial physiology and virulence since they are the only two from that group that have homologs in the M. leprae genome [122]. Transcript levels of sigC remain elevated or decline during several stress conditions, including heat shock, cold shock, oxidative 23

stress and denaturing stress suggesting that SigC does not respond to these stressors [88].

Mutants lacking a functional sigC gene have been created in M. tuberculosis strains H37Rv and

CDC1551 [91-92]. The laboratory-passaged strain H37Rv was the first M. tuberculosis strain sequenced. Strain CDC1551 is a more recent clinical isolate subsequently sequenced. A sigC mutant in the strain CDC1551 was not altered for survival in activated mouse macrophages.

However, sigC is required for virulence in mice and guinea pigs. DBA/2 mice infected with a sigC mutant of M. tuberculosis strain CDC1551 showed a much lower death rate than mice infected with strain CDC1551 [92]. The sigC mutant strain reached the same bacterial load as the parental strain in the mouse lung. It was also reported that the mutant was more sensitive than the parent to mechanical killing caused by nebulization in a Madison aerosol chamber [94]. It was suggested that differences in the cell envelopes of the two strains may exist. Guinea pigs infected with a sigC mutant of M. tuberculosis strain H37Rv showed fewer and smaller granulomas compared to the parental strain, with most of the granulomas resolving [91]. SCID mice were infected with the sigC mutant in strain CDC1551, the parent strain and a complemented mutant. It was shown, that the mutant grew slower than the parent or the complement. Also, mice infected with the sigC mutant survived 60 days longer and haematoxylin-eosin staining of the lungs showed that the mutant caused less internal damage than the parent strain or the complemented mutant [123]. In the same study, bronchoalveolar lavage fluid analysis showed lower numbers of neutrophils in the lungs of DBA/2 mice infected with the mutant compared to lungs of mice infected with the parent strain or the complemented mutant. Additionally, reduced levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IFN-γ) were measured in the lungs of DBA/2 mice infected with the sigC mutant [123].

2.3.12 The SigC Regulon

Two studies have been published attempting to define the SigC regulon. Each takes a different approach and identifies unique genes theoretically controlled by SigC. Both are detailed below because they provide the foundation for our efforts to define the SigC regulon.

In the first published report, gene expression of a CDC1551 sigC mutant was compared to the parental strain during exponential phase (A600 = 0.5), early stationary phase (A600 = 2.0), and late stationary phase (3 days after the early stationary phase) using DNA microarrays [74].

Approximately 200 genes were reported down-regulated at least two-fold in the sigC mutant under these conditions, but error bars for several of the most highly-affected genes approached or exceeded 100%. The genes included some virulence-associated genes, a two-component sensor kinase and a two-component response regulator. Although RT-PCR was performed on the RNA samples to confirm the same general trends in gene expression of some of these genes, faint in vitro transcription results with SigC-containing RNAP holoenzyme was only shown for one of the genes. A consensus promoter (SSSAAT -20 bp- CGTSSS, where S = G or C), compiled by examining sequence upstream of these genes, was reported.

The second article employed chromatin immunoprecipitation (ChIP) assays to identify in vivo chromosome binding sites by artificially-induced levels of SigC in M. bovis BCG [124]. In this study, after induction of myc-tagged σC, a cross-linker was added. Cells were then sheered and DNA fragments bound presumably by RNAP:SigC holoenzyme were immunoprecipitated using an antibody to the myc tag. After removal of the protein, the DNA was denatured and reverse-transcribed to make labeled cDNA. The cDNA was hybridized to microarrays containing the intergenic regions of the M. tuberculosis H37Rv genome. The intergenic regions bound by

SigC-RNAP were as follows (listed in order of decreasing abundance): Rv0095c-ppe1, ctpB- 25

Rv0104, Rv0311-Rv0312, Rv0330c-Rv0331, ansP2-Rv0347, grcC1-htpX, Rv0575c-Rv0574c, secE1-nusG, rplJ-rplL, Rv0680c-Rv0681, rplW-rplB, rpsQ-atsA, rplO-sppA, mbtI-Rv2387,

Rv3483c-Rv3482c. Binding to the first two sites was far greater than binding of to other sites and was approximately 15-fold over background. For these hotspots, qRT-PCR was performed to determine which of each divergently-transcribed gene pair was controlled by SigC. It was reported that Rv0095c and ctpB were transcribed by SigC. The DNA sequences upstream of the transcripts were reported to have the same -35 and -10 promoter elements (GGGAAC – 17 bp –

CGACT). However, examination of the promoter region of the first gene (PPE1/Rv0096) in the operon, revealed that it contains the SigC promoter sequence reported for the RACE 5′ transcript end mapping of Rv0095c [124]. As the sequence preceding Rv0095c is different than that reported, this suggests an erratum is present in the Rodrigue et al. 2007 article. Neither of these genes appeared among the 200 reported to be differentially regulated in the sigC mutant relative to the CDC1551 parent [74].

2.3.13 Regulation of sigC

In M. tuberculosis, sigC is unique among the sigma factors in that it is not located within an operon. No anti-σ factor has been identified for SigC, and it is possible that SigC is not regulated by an anti-σ factor. The gene blaC might potentially influence sigC expression. Both genes are divergently- transcribed and share a 134 bp intergenic region. This leads to a potential overlap of regulatory elements of these genes. Active transcription of blaC might sterically block transcription of sigC. Secondary sigma factor synthesis or activity is generally induced by certain stress conditions. No stressors to date have been reported to upregulate sigC transcription. A

SigF-dependent promoter within blaC, upstream of sigC, has been reported [105]. Knock-in

expression of SigF was reported to upregulate sigC after induction of sigF. Transcription of sigF is induced in M. smegmatis and M. bovis BCG after exposure to different antibiotics, hypoxia, cold shock, oxidative stress, and entry info stationary phase. However, in M. tuberculosis, sigF was only induced after nutrient depletion [101].

3 M. TUBERCULOSIS CARBON METABOLISM

Intracellular pathogens must adapt their metabolism to survive and replicate inside their hosts.

The central carbon metabolism (CCM) of M. tuberculosis includes glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the glyoxylate shunt, and the pentose phosphate shunt. It does not appear to contain the Entner–Doudoroff pathway, which catabolizes glucose to pyruvate using enzymes different from those used during glycolysis. This pathway is present in other intracellular bacteria such as Salmonella Typhimurium and Shigella flexneri [125]. The glyoxylate cycle is a variation of the TCA cycle and converts acetyl-CoA to succinate. The isocitrate lyase, encoded in M. tuberculosis by icl1 and icl2, plays an important role in that cycle by converting isocitrate to glyoxylate and succinate. The glyoxylate is converted to malate by the enzyme malate synthase. The glyoxylate cycle allows bacteria to utilize simple carbon sources when complex carbon sources are absent. The pentose phosphate pathway is an alternative to glycolysis and generates NADPH and 5-carbon sugars. The genome of M. tuberculosis also contains numerous fad homologs encoding putative enzymes involved in lipid metabolism or utilization of cholesterol [126]. It has been hypothesized that in bone-marrow-derived macrophages the preferred carbon sources for M. tuberculosis are fatty acids and possibly glycerol [125]. This theory is based on the upregulation of many fad genes, and ugp and glpD which are responsible for glycerol-3-phosphate uptake and metabolism, when the bacteria are grown in resting and activated bone-marrow-derived macrophages. It has been shown that mutants of M. tuberculosis lacking genes responsible for β-oxidation of fatty acids were strongly attenuated [127]. Together this suggests that M. tuberculosis uses host cell lipids as carbon sources in the host. However, carbohydrates might also play a role during infection, as

disaccharide transporters also have been reported to be required for mycobacterial replication in mice [128].

Based on the analysis of the genetic requirements of M. tuberculosis during growth and infection, it is thought that the CCM plays an important role in pathogenicity of this bacterium

[128-129]. This is emphasized by the finding that M. tuberculosis preferentially metabolizes fatty acids over carbohydrates when recovered from the lungs of infected mice [130]. Profiling studies also revealed that the gene expression of M. tuberculosis CCM genes is undergoing vast remodeling during the infection of a host [109, 131]. Mutants lacking CCM enzymes showed strong attenuation in animal models. One example is the enzyme isocitrate lyase which converts isocitrate into glyoxylate and succinate. Mutants lacking this enzyme showed impairment of intracellular replication and rapid elimination from the lungs [132]. Another example is the methylcitrate cycle which also was shown to be required for intracellular growth and virulence.

Mutants lacking the genes prpC and prpD, both playing an important role in the methylcitrate cycle, could not grow in murine bone marrow-derived macrophages [133]. Unlike other bacteria that consume individual carbon sources in a preferred sequence, M. tuberculosis is able to co- metabolize multiple carbon sources simultaneously [134]. This allows enhanced growth of the bacterium when infecting its natural host.

4 COBALAMIN

4.1 Structure and Function

Vitamin B12 is the prototypical cobalamin. It is a molecule consisting of a corrin ring with a central cobalt atom, a di-methylbenzimidazole ribonucleotide loop which binds to the lower axis of the cobalt atom, and a variable upper axial cobalt ligand (Figure 2.1). Known upper ligands include hydroxyl, methyl, adenosyl, or cyano moieties. The predominant commercial form of vitamin B12 is cyanocobalamin in which the cyano group replaces the upper ligand as a result of the extraction process. Cobalamins function as cofactors for enzymes involved in protein, DNA, and carbohydrate synthesis. This includes the following enzymes:

Methionine synthetase transfers a methyl group from methyltetrahydrofolate to homocysteine as the final step in the synthesis of methionine [135]. In Salmonella and E. coli, this methyl transfer reaction is performed by either the B12-dependent MetH enzyme or by the

B12-independent enzyme MetE. The M. tuberculosis genome contains both metE and metH homologues.

Epoxyqueuosine reductase performs the last step in formation of the hypermodified tRNA base queuosine. This base is found in tRNAtyr, tRNAhis, tRNAasn, and tRNAasp [136]. In

E.coli, the modified base is not essential for bacterial growth under laboratory conditions [137].

A homolog has not been identified in M. tuberculosis.

In acetyl-CoA synthesis, methy-corrinoids are often involved; a methyl group is transferred from methyltetrahydrofolate via a methyl-corrinoid/iron-sulphur protein to carbon- monoxide-dehydrogenase [138]. This methyl-corrinoid/iron-sulphur enzyme synthesizes acetyl-

CoA from this methyl group, carbon monoxide, and the coenzyme A. No homolog to the methyl- corrinoid/iron-sulphur protein was found in M. tuberculosis.

Ribonucleotide reductases generate the deoxyribonucleotides needed for DNA synthesis by reducing the ribonucleotide sugar. There are four different classes of this reductase; Class II enzymes are cobalamin-dependent and are mainly found in bacteria [139-140]. In M. tuberculosis there are two ribonucleotide reductases, one of which is class II and vitamin B12- dependent. The other one is a class I reductase that is vitamin B12-independent.

Methylmalonyl-CoA mutase interconverts (R)-methylmalonyl-CoA and succinyl-CoA.

This enzyme plays an important role in the fat and carbohydrate metabolism in animals and humans. In certain bacterial fermentations, this enzyme converts succinate to propionate. There is one known homolog of methylmalonyl-CoA mutase in M. tuberculosis.

It is thought that abundance of fatty acids in the human host dictate mycobacterial metabolism in M. tuberculosis [84]. To use and break down fatty acids, the bacteria use β- oxidation and the anaplerotic glyoxylate cycle, which enables the assimilation two-carbon acetyl- coenzyme A (CoA) subunits [133]. However, when odd-carbon-length fatty acids are present, the C3 adduct propionyl-CoA is produced. The accumulation of propionate is toxic to the cell, possibly due to a buildup of 2-methylcitrate. Buildup of this product was found to inhibit aconitase and citrate synthase activities [141]. Alternatively, it was speculated that if the ratio of propionyl-CoA to other acyl-CoA intermediates was increased too much, the cell may find itself with insufficient levels of acetyl CoA and succinyl CoA to function properly [142]. As such, efficient mechanisms are required for the dissimilation of propionyl CoA [143]. Two pathways for utilization of propionyl-CoA in M. tuberculosis have been described. One pathway utilizes

the methyl citrate and glyoxylate cycles. The other is the methylmalonyl pathway which utilizes the B12-dependent enzyme methylmalonyl-CoA mutase (MCM) [144].

4.2 Cobalamin Biosynthesis

In bacteria, two possible pathways for cobalamin synthesis have been identified. One is oxygen- dependent and typified by Pseudomonas denitrificans. The other is oxygen-independent and present in Salmonella enterica subspecies typhimurium. M. tuberculosis is considered to utilize the oxygen-dependent route, since it has homologs to cobalamin genes found in P. denitrificans but lacking in S. typhimurium. In M tuberculosis, 17 genes have been identified [145] with potential roles in cobalamin synthesis (Fig. 2.2). The complete pathway of the oxygen-dependent cobalamin synthesis including the involved genes, starting with precorrin-2 (a common precursor in tetrapyrrole biosynthesis), has been identified in P. denitrificans [146-148]. The precorrins are biosynthetic intermediates of corrin synthesis and the given numbers indicate the number of methyl groups that have been added to the tetrapyrrole framework.

The major differences between the oxygen-dependent and the oxygen-independent routes occur at the timing of cobalt insertion. In the oxygen-dependent pathway, cobalt is inserted by

CobNST at the level of hydrogencobyrinic acid a,c-diamide which is a comparatively late stage step in the oxygen-dependent route, whereas cobalt is inserted into precorrin-2 under the anaerobic pathway. Finally, the two pathways rejoin at the point of adenosylcobyric acid synthesis. The last three steps leading to synthesis of the end product adenosylcobalamin are conserved in the oxygen-dependent and the oxygen-independent pathway.

The different genetic requirements for cobalamin synthesis can often be used to identify which pathway an organism uses for corrin synthesis [149]. The presence of cobG, which

encodes the mono-oxygenase, and cobN, which is part of the heterotrimeric cobalt chelatase complex, is an indicator that M. tuberculosis uses the oxygen-dependent pathway.

Cobalamin plays an important role in enteric bacteria where many known B12-dependent reactions support anaerobic fermentation of small molecules. It was hypothesized that cobalamin supports fermentation by catalyzing molecular rearrangements that generate both an oxidizable compound and an electron sink for use in balancing redox [150]. Examples for this are the B12- dependent degradation of ethanolamine, propanediol, and glycerol. Since M. tuberculosis is not an enteric bacterium, those degradation reactions may not be employed by this pathogen.

4.3 Cobalamin regulation in M. tuberculosis

Regulation of cobalamin gene expression often occurs through a conserved RNA element named the B12 riboswitch [151]. A B12 element (an RNA motif) bound by B12 forms a structure that obscures the ribosome binding site, thereby inhibiting translation. Approximately 200 such B12- elements were identified in cobalamin-related genes in 67 bacterial genomes, including M tuberculosis and M. leprae [151]. Two B12-riboswitch motifs have been identified in the M. tuberculosis genome. Riboswitch regulation of the methionine synthase has been recently described for M. tuberculosis strain CDC1551 [152]. The vitamin B12-independent methionine synthase gene, metE, is controlled by a B12 riboswitch. A mutation in the metH gene, encoding the vitamin B12-dependent enzyme, methionine synthase, prevents functional MetH synthesis.

When 10 µg/ml cobalamin was present, the bacteria were unable to grow because of metE translation inhibition [152]. The second riboswitch is located immediately upstream of PPE2

[153]. The function of this gene is unknown, but this gene appears to be the first gene in an

operon that includes the cobalamin genes cobQ, and cobU, both involved in the late stages of cobalamin synthesis.

In S. typhimurium, translation of the cob operon is reduced in the presence of the end product adenosylcobalamin due to binding to a B12 element [154]. Inhibition by adenosylcobalamin is seen even when transcription is initiated from foreign promoters [155].

Genetic analysis has not revealed any proteins that might mediate the repressive effects of adenosylcobalamin. All mutations leading to elimination of translational control altered the mRNA leader region [156-157]. Hence, a mechanism similar to that of the B12 riboswitch was suggested in which a direct interaction occurs between the effector (adenosylcobalamin) and the mRNA leader. In this model, the interaction may induce mRNA folding that stabilizes the hairpin, thereby blocking the ribosome binding site. The message termination at a site within the structural gene might be provided by the untranslated coding sequence [156]. However, it remains uncertain how an mRNA might specifically recognize adenosylcobalamin or how that binding might affect mRNA folding.

4.4 The Cobalamin Biosynthetic Cluster

The biosynthetic cluster of M. tuberculosis that we are targeting contains several homologs of cobalamin synthesis genes, genes of unknown function, blaC, and sigC (Figure 2.2), and Region of Difference 9 (RD9). The gene blaC encodes beta-lactamase C. The sigC gene encodes the transcription factor SigC. The RD9 region is present in Mycobacterium species that principally infect primates (M. tuberculosis, M. canettii, and M. africanum), but is absent in species that primarily infect lower animals (M. microti, M. bovis, and the various M. bovis BCG vaccine strains). The RD9 region includes the genes: Rv2073c, Rv2074, and portions of cobL and 34

Rv2075c. It remains to be determined if loss of this region is partially responsible for the attenuation of M. bovis BCG.

Figure 2.1: Chemical structure of vitamin B12 (cobalamin). The central cobalt atom is covalently bound to four nitrogen atoms in a planar tetrapyrrole ring. The lower axial ligand of cobalt forms a loop with the tetrapyrole ring through a dimethylbenzimidazole ribonucleotide moiety. Various upper ligands have been identified (R).

[ ]

Figure 2.2: The cobalamin biosynthetic cluster in M. tuberculosis strain H37Rv. Open reading frames (ORFs) and orientations are shown (arrows). Genes in red are putative cobalamin biosynthetic genes. Predicted functions of ORFs in green have not been described. The β- lactamase gene blaC (blue arrow) and the sigma factor C gene, sigC (black arrow) are also shown. Brackets indicate the cobalamin biosynthetic cluster. Figure was created using information obtained from the Tuberculist webserver (http://genolist.pasteur.fr/TubercuList/) and drawn using Vector NTI 10.

CHAPTER 3

MYCOBACTERIUM TUBERCULOSIS SIGC IS A REGULATOR OF A METAL-

SCAVENGING SYSTEM 1

1 Benjamin T. Grosse-Siestrup, Tuhina Gupta, Shelly Helms, Martin I. Voskuil, Frederick D.

Quinn, Russell K. Karls. To be submitted to Science or Molecular Microbiology. 38

ABSTRACT

Mycobacterium tuberculosis reigns as the leading cause of human death by a single infectious bacterium. Success of this pathogen lies in its ability to acquire host nutrients and circumvent immune defenses. Sigma factor C is a known M. tuberculosis virulence determinant in murine and guinea pig infection models. However, the lack of in vitro phenotypes associated with mutations in the sigC gene encoding this transcription factor has hindered efforts to define its regulon. Here we report that growth of a sigC mutant of M. tuberculosis strain Erdman is affected by carbon source availability. Growth rate is reduced in chemically-defined, glycerol- based Sauton medium, but not in Middlebrook 7H9 medium. Supplementation of Sauton medium with glucose or Tween 80 erases the growth defect of the mutant, while addition of valerate improves growth of the mutant but retards growth of the parent or complemented mutant. Global gene expression studies of the mutant and parent strain cultured in Sauton medium revealed that the nrp operon was most-highly expressed in the parent, while genes including ald, icl and the cyd operon were elevated in the mutant. This initially suggested a role in central carbon metabolism. However, artificial overexpression of sigC in M. tuberculosis in complex medium, Middlebrook 7H9, resulted in early upregulation of the nrp operon and ctpB, with subsequent elevation of several metal stress response genes. Comparison of medium components supports a model in which the nrp operon encodes a trace-metal scavenging system akin to the iron-chelating mycobactin. In metal-restricted Sauton medium, the nrp operon is induced in strains carrying wildtype sigC resulting in sufficient trace metals to be taken up and trace-metal-requiring enzymes such as Zn-dependent glycerol kinase to function. In metal-rich

7H9 medium, artificial induction of sigC causes expression of the putative nrp-encoded metal scavenger, which results in excess metal uptake and subsequent expression of stress responses 39

against toxic concentrations of metals such as copper. The predicted polyketide, synthesized by enzymes encoded by the nrp operon, represents a novel target for both drug and vaccine development. Studies to characterize the metal scavenging system are in progress.

Introduction

The etiological agent of tuberculosis (TB), Mycobacterium tuberculosis, remains a leading cause of morbidity and mortality in the world. In 2010, an estimated 8.8 million people developed active TB disease and 1.45 million deaths resulted [158]. Additionally, one third of the world population is estimated to harbor this pathogen as a latent infection [159]. The Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine provides only variable protection against pulmonary TB in post-adolescents [160]. Importantly, multi-drug resistant M. tuberculosis strains are common and totally drug-resistant strains have been reported, highlighting the importance of novel vaccines and treatments [161].

The success of M. tuberculosis lies in part in its ability to sense stress cues and mount effective responses against assaults by the host immune system. A primary mechanism by which bacteria direct stress responses is through the use of sigma factors, dissociable components of

RNA polymerase that dictate the DNA sequences (promoters) at which an RNA polymerase holoenzyme initiates transcription. Each sigma factor is believed to recognize a unique promoter, enabling a coordinated response tailored to a specific stressful condition. In addition to the primary sigma factor SigA, M. tuberculosis has a dozen secondary sigma factors. We and others have reported that mutants defective for production of SigC are attenuated in animal models [74,

123, 162]. Such mutants are not impaired at replicating in macrophages or for growth in vitro, nor have in vitro stress stimuli been identified that upregulate sigC transcription in M. tuberculosis [74, 88, 162].

Acquisition of nutrients from the host is vital to M. tuberculosis bacilli. Over the years, in vitro culture of tuberculous bacilli was optimized to accelerate growth. A commonly-used medium is Middlebrook 7H9, a complex medium that is supplemented with bovine albumin and 41

which is rich in minerals and contains glucose as one of its carbon sources [163]. The observation that an M. tuberculosis mutant defective in production of isocitrate lyase failed to persist in murine infections was pivotal in demonstrating the importance of intermediary metabolism, the glyoxylate shunt, and a dietary shift from carbohydrates to lipids as principal carbon source for persistence in the host [164].

To define what conditions require expression of SigC and to determine which genes are regulated by SigC, a sigC mutant in the virulent strain Erdman was created. We employed different culture conditions and performed SCID (severe combined immunodeficiency) mouse infections to define a SigC phenotype. Additionally, we utilized microarray technology to determine global gene expression profiles of the sigC mutant compared to the wildtype under different culture conditions.

MATERIAL AND METHODS

Bacterial strains and culture conditions

Mycobacterium tuberculosis strain Erdman and its mutants were cultured in Middlebrook 7H9 medium supplemented with 0.5% glycerol, 0.05% Tween 80 and 10% ADS (albumin, dextrose,

NaCl) (7H9tgADS) [163]. When required for plasmid maintenance, kanamycin was added to a final concentration of 25 µg ml-1 or hygromycin was added to a final concentration of 50 µg ml-1.

For RNA isolation, cells were grown in Middlebrook 7H9 medium supplemented with 0.5% glycerol, 0.05% Tween 80 and 10% ADS or Sauton medium (0.05% KH2PO4, 0.05% MgSO4 ·

7H2O, 0.2% Citric acid, 0.005% Ferric ammonium citrate, 6% glycerol, 0.4% asparagine, pH adjusted to 7.4) supplemented with 0.025% Tylaxopol (Sigma). For carbon utilization growth studies, bacteria were cultured in Sauton medium supplemented with 0.025% Tylaxopol (SMT) or in SMT supplemented to 0.2% with one of the following: Tween 80, glucose or valeric acid.

The pH of the Sauton-based media was adjusted to 7.4 with NaOH prior to use. Liquid cultures were grown in 250 ml polypropylene bottles (VWR) at 37°C on a rotary platform (75 rpm). For culture of mycobacteria on plates, Middlebrook 7H10 agar was supplemented with 0.5% glycerol, 0.05% Tween 80 and 10% ADS. Escherichia coli strains were cultured on Luria base medium supplemented when necessary with 50 µg ml-1 kanamycin or 200 µg ml-1 hygromycin.

Growth was also examined on Löwenstein-Jensen medium (BD/Difco).

Construction and complementation of a sigC mutant in M. tuberculosis Erdman

An internal-deletion mutant of the sigC gene (Rv2069) was generated in M. tuberculosis strain

Erdman using homologous recombination with plasmid pLJ∆sigC as previously described [162].

The sigC gene was disrupted by allelic exchange without inserting an antibiotic resistance gene. 43

The mutant was identified by isolating genomic DNA from several candidates and screened by

PCR using primers 5′-ACC GAC CAA CGG GAA GC-3′ and 5′-TTA CCA CGA TGA GTT

CGC AC-3′. The deletion mutant was confirmed by Southern analysis (Figure 3.1).

The complementation plasmid pGSsigC was constructed as follows: the M. tuberculosis sigC region with ~600 bp of upstream regulatory sequences was amplified with primers 5′-GCT

AAG CTT GCT CGT CCG TAG TCA C-3′ and 5′-CGC AAG CTT CGG TGG TCA TGA TAG

C-3′, digested with HindIII and inserted into the multiple cloning site of the mycobacterial integrating vector pMV306 [165]. The resulting plasmid, pMV306SigC, was transformed into the sigC deletion mutant selecting for kanamycin resistance. This plasmid should permit single- copy sigC complementation by integrating into the M. tuberculosis chromosome at the mycobacteriophage L5 attachment site.

Southern immunoblotting

Genomic DNA from strains Erdman and ΔsigC was digested with BamHI and separated on a 1% agarose gel. After transferring the digested DNA to a nylon membrane, it was hybridized with a sigC-specific DNA probe. The probed was synthesized by PCR using the primers 5′- GGT GTC

ACC CAA GCT GCG -3′ and 5′- TAG ATC CCG CCG CCG AG -3′, and labeled using a the

North2South biotin random prime labeling kit (Thermo Scientific) according to manufacturers instructions.

Animal infections

SCID mice were infected in parallel with strains Erdman, ∆sigC, and the complemented ∆sigC strain (comp). Prior to infection, each strain was cultured to an OD600 = 1 in 7H9 medium. Mice 44

were infected transorally with 104 CFU/strain or mock-infected with phosphate-buffered saline

(PBS). Twelve mice were infected with each M. tuberculosis strain. The animals were housed under institutionally-approved animal care and use protocols. Time-to-death was determined by monitoring the animals for signs of morbidity at which point animals were humanely euthanized.

The Kaplan-Meier Log Rank test was used to determine statistical significance.

Protein lysates and western immunoblotting

For detection of myc-SigC in M. bovis BCG/pSR173, bacteria were cultured in 7H9 medium. At

OD600 =0.2, 50 ng/ml aTc was added to one culture (+aTc), but not to the other (-aTc) and both cultures were incubated 48 hr prior to harvesting the cells and preparation of whole cell lysates.

For protein isolation, cultures were centrifuged at 3500 rpm and 4°C for 5 min. The supernatant was filtered through a 0.22 µM PVDF filter and stored at 4°C. The pellets were washed with

PBS and resuspended in PBS containing 2mM EDTA and a proteinase inhibitor (Roche). The resuspended pellets were then shaken three times with 0.1-mm diameter zirconia/silica beads in a bead beater (BioSpec Products) with 40 second pulses at 4800rpm each round. Between each round, the lysates were chilled on ice for 2 min. In the final step, the lysates were filtered through a 0.22 µM PVDF filter and stored at -20°C. Equivalent protein loads (~10 µg) from each condition were loaded on NuPAGE 4-12% Bis/Tris gels (Invitrogen) and transfered to a PVDF membrane. Western immunoblotting was performed with a monoclonal antibody to Myc

(Sigma).

RNA isolation

Bacterial RNA was obtained from M. tuberculosis strains cultured to the indicated optical densities in the indicated media. To isolate RNA, pellets were thawed on ice in the presence of

Trizol (Gibco BRL) and cells disrupted with 0.1 mm zirconium beads in a BeadBeater (BioSpec

Products, 4800 rpm, 40 sec per cycle, 3 cycles, with chilling tube for 1 min on ice between cycles). To each tube 200 µl chloroform was added. Samples were mixed, incubated at RT for 3 minutes, and phases separated by centrifugation (12,000 g, 15 min). The aqueous phase was transferred into a new tube and extracted with an equal volume of phenol/chloroform. Following centrifugation (12,000 g, 10 min), the aqueous phase was transferred to a new tube and RNA precipitated with 500 µl isopropanol and incubation (RT, 10 min). After centrifugation (12,000 g, 10 min) RNA was washed twice with 75% ethanol, and suspended in DEPC-treated water.

Two rounds of DNAse treatment with Turbo RNAse-free DNAse (Ambion) followed by RNeasy

(Qiagen) purification, were performed following the manufacturers’ instructions. After elution,

25 units RNaseOUT (Invitrogen) were added to each sample. To test for genomic DNA contamination, RNA was subjected to 40 cycles of PCR with primers 5′-AAC AGA TCG GCA

AGG TAG-3′ and 5′AAC TTG TAC CCC TTG GTG-3′ specific to sigA.

Microarray studies

Microarray slides consisting of 70mer oligonucleotides were obtained through I) the TB Vaccine

Testing and Research Materials Contract administered by Colorado State University (Fort

Collins, CO) (Tables 3.4 and 3.5), II) the Pathogen Functional Genomics Resource Center

(PFGRC) at the J. Craig Venter Institute (Table 3.3), and III) the M.I. Voskuil laboratory,

University of Colorado-Denver (Tables 3.1 and 3.2). The slides from sources I and III consisted 46

of probes to 4,269 M. tuberculosis genes from strain H37Rv plus 26 controls. The slides from source II consisted of 4,750 M. tuberculosis genes from strains H37Rv and CDC1551 plus 500 controls. Each gene was printed four times on the slides available from source II. Unfortunately, these slides are no longer available through PFGRC. Complementary DNA (cDNA) was prepared using a protocol from the PFGRC. Briefly, 2 µg RNA was incubated overnight at 42°C in the presence of 6 µg random hexamers (Invitrogen), 0.5 mM dNTP/aminoallyl-dUTPs, 2 µl

SuperScript III Reverse Transcriptase, and 10 mM DTT. The cDNAs were purified by MinElute

PCR purification (Qiagen) using manufacturer’s instructions with the following exceptions: the wash and elution buffers provided in the kit were replaced with a phosphate wash buffer (5 mM

KH2PO4, pH 8.0, 80% EtOH) and a phosphate elution buffer (4 mM KH2PO4, pH 8.5). The aminoallyl-labeled cDNAs were coupled to Cy3 or Cy5 dyes (Amersham Biosciences).

Uncoupled dye was removed by MinElute PCR purification with the kit’s buffers and instructions. The Cy3- and Cy5-labeled cDNAs were combined in pairs, dried to completion, and suspended in hybridization buffer. The microarray slides were prehybridized by incubation in prehybridization buffer (5x SSC, 0.1% SDS, 1% BSA) for at least one hour at 42°C, washed with water and isopropanol, dried, and hybridized with the labeled cDNA mixture overnight at

42°C. The next day, slides were washed with high (0.1x SSC), medium (0.1x SSC, 0.1% SDS) and low (2x SSC, 0.1% SDS) stringency buffer, and scanned on a ProScanArray microarray scanner (Perkin Elmer, Waltham, MA). The resulting images were analyzed using

ScanArrayExpress software and exported into Microsoft Excel. For each experiment, four biological replicates were prepared with at least two hybridizations performed per replicate.

Dyes were swapped for the two hybridizations of each biological replicate. A minimum of eight intensity values were obtained for each gene. All data was normalized using the LOWESS 47

algorithm [166]. To identify the significant differentially-expressed open reading frames (ORFs), the Significance Analysis of Microarrays (SAM) test procedure was conducted using the

Microsoft Excel plug-in with a stringent false discovery rate of 0 [17]. To maintain stringency, only genes differentially-regulated 2-fold or more were examined. Microarray data will be uploaded to the NCBI Gene Expression Omnibus website (http://www.ncbi.nlm.nih.gov/geo/) upon acceptance of the manuscript for publication.

Quantitative RT-PCR assays

For reverse transcription, 500 ng RNA of each biological replicate was used with ImProm-II

Reverse Transcriptase (Promega, Inc.) according to the manufacturer’s instructions. The expression of each target gene was determined with 0.5 µl of cDNA reaction using the Platinum

SYBR Green kit (Invitrogen) with a BioRad iCycler and the ∆∆Ct method [167], normalized to sigA. Each qRT-PCR reaction was performed with three biological replicates.

RESULTS

A M. tuberculosis sigC mutant of strain Erdman exhibits no significant growth or gene expression differences in log-phase Middlebrook 7H9 cultures

In previous studies, M. tuberculosis sigC mutants defective for production of sigma factor C were found to be attenuated in murine and guinea pig infections [74, 162]. Recent studies indicate that prolonged laboratory passage of H37Rv strains can result in mutations [168]. As the sigC parent strain H37Rv employed in the guinea pig infection studies was less virulent than expected, to characterize the SigC regulon, a sigC mutant (ΔsigC) in virulent strain Erdman was generated (Figure 3.1). Growth of ΔsigC in Middlebrook 7H9 broth was comparable to strain

Erdman or ΔsigC complemented with a wild type copy of sigC (Figure 3.2). DNA microarray analyses of gene expression differences between ΔsigC and Erdman cultured to OD600 = 1 in 7H9 medium did not reveal any statistically significant differences when results of four biological replicates on ten slides were examined together using the Significance Analysis of Microarrays

(SAM) program (Stanford University) and a 2-fold expression cutoff (data not shown). This was surprising as numerous genes were reported to be differentially expressed by a sigC mutant of M. tuberculosis strain CDC1551 compared the wildtype when grown in 7H9 medium [74]. One explanation for this discrepancy may be a possible mutation in strain Erdman affecting sigC or its regulation. To assess this, virulence of ΔsigC was examined in a murine infection model.

A sigC mutant of M. tuberculosis strain Erdman is attenuated in SCID mice

To determine if loss of sigC from strain Erdman results in attenuation, SCID mice were infected in parallel with 104 CFU ΔsigC, Erdman, and the complemented mutant by trans-oral instillation.

Animals were monitored for signs of morbidity and humanely euthanized when moribund. The 49

mean survival time of mice infected with bacilli of strain Erdman or of the complemented mutant was 68 days or 72 days, respectively (Figure 3.3). The mean survival time of mice infected with

ΔsigC was 131 days. The trends indicate that loss of sigC attenuates M. tuberculosis strain

Erdman in this immune-compromised host. It should be noted that statistical significance was only achieved between the ΔsigC group and the Erdman and complemented groups if two outliers in each of the latter groups were removed. This is attributed to the infection method which employed blind insertion of a tube into the trachea to deliver the bacteria. With this method it is possible that not all bacilli are being delivered successfully into the trachea.

Attenuation of ΔsigC is consistent with that reported for a sigC mutant of strain CDC1551 in which SCID mice infected by aerosol with 100 CFU of the mutant survived an average of 86 days relative to 26 days for those infected with either the parent strain or complemented mutant

[74]. SCID mice infected with CDC1551 by aerosol succumbed more quickly than those infected with a higher infective dose of Erdman by transoral instillation; this could be explained by genetic differences between the strains or by more efficient aerosol delivery of bacilli to the alveoli.

sigC is required for M. tuberculosis growth in Sauton medium

Based on reports suggesting that glucose is not a principal carbon source for M. tuberculosis bacilli in the host, growth of ΔsigC was compared to that of strain Erdman and the complemented mutant using chemically-defined Sauton medium. The growth rate of the sigC mutant was found to be slower than that of the parental strain or complemented mutant when cultured in Sauton medium (Figure 3.4). It is important to note that the growth-defect phenotype required washing Middlebrook 7H9 seed cultures and subculturing several times in Sauton 50

medium. This suggested that nutrients stored in the inoculum must be depleted for the phenotype to manifest.

sigC is required for glycerol utilization

To investigate why the sigC mutant grows more slowly in Sauton medium, global gene expression differences between ΔsigC and parent Erdman were examined from cultures grown to log phase (OD600 = 1) in Sauton medium. Significant differential expression of 2-fold or greater is reported (Tables 3.1, 3.2). Of note, none of these genes correspond to genes previously reported to be SigC-regulated [74, 124]. Only five genes were significantly upregulated at least

2-fold in strain Erdman relative to ΔsigC: Rv0097, Rv0098 (fcoT), Rv0100, Rv0101 (nrp), and

Rv0967 (csoR) (Table 3.1). The first four genes are likely part of a nonribosomal peptide synthase operon. Although the product of the nrp operon is unknown, transposon insertion into the Rv0097 homolog in the homologous 6-gene nrp operon in M. bovis was reported to exhibit polar effects on downstream genes, to alter synthesis of phthiocerol dimycocerosates (PDIM) resulting in a smooth colony morphology, and to attenuate the bacilli in a guinea pig infection model [169]. Rv0098 was reported to encode a long-chain fatty acyl-CoA thioesterase, FcoT

[170]. The only other gene upregulated >2-fold in Erdman versus ΔsigC was Rv0967 encoding a copper-sensitive operon repressor CsoR [171]. In the presence of copper, csoR is derepressed as is the ctpV operon encoding predicted cation transport protein CtpV [172]. Elevated expression of csoR and a representative gene of the nrp operon, fcoT in strain Erdman versus ΔsigC, was confirmed by qRT-PCR (Figure 3.5).

A dozen genes are expressed at significantly higher levels in the sigC mutant than its parent strain in Sauton medium (Table 3.2). The genes affected suggest various stresses may be 51

occurring in the mutant in this medium. The genes include those functioning in denaturing stress

(hsp, encoding a heat-shock protein), oxidative stress (ahpC, encoding alkyl hydroxyl peroxidase), carbon metabolism (icl, encoding isocitrate lyase, and ald, encoding alanine/glycine dehydrogenase), and respiration (cydABDC, encoding a cytochrome b/d ubiquinol oxidase pathway) (Figure 3.6). Elevated transcription of icl and cydB in the mutant relative to strain

Erdman was confirmed by qRT-PCR (Figure 3.5).

The upregulation of genes implicated in PDIM synthesis (the nrp operon) in the parent strain, and the induction of glyoxylate shunt and alternate electron transport genes in ΔsigC, suggested the possibility that the redox state of the cell is affected when the bacteria are cultured on glycerol-based Sauton medium. Production of specific lipids, such as PDIM, has been shown to alter the redox state of the cell in response to growth on reduced carbon sources [13]. To examine the effects of carbon sources, growth of ΔsigC, Erdman, and the complemented sigC mutant were subcultured into Sauton medium supplemented with a sugar (glucose), an even- chain lipid (Tween 80), or an odd-carbon lipid (valerate). Either glucose or Tween 80 reversed the growth defect of the mutant (Figure 3.7 A-B). Improved ΔsigC growth by high redox- potential carbon source glucose (based on the oxidation state of the carbon atoms) supported the reductive stress hypothesis. However, the low redox-potential carbon source Tween 80 also improved growth of the mutant, which indicates that reductive stress does not explain the growth defect on Sauton medium containing only glycerol as the primary carbon source. It remains to be determined if PDIM synthesis is altered in the sigC mutant. Loss of sigC alone does not result in a colony morphology different from strain Erdman when examined on either Middlebrook 7H10 agar or on Löwenstein-Jensen medium. This suggests to us that PDIM synthesis may not be

severely affected in M. tuberculosis upon loss of sigC unlike a transposon insertion into the nrp operon in M. bovis [169].

It was surprising to see that addition of valerate improved growth of the mutant while temporarily retarding growth of the parent or complemented mutant (Figure 3.7 C). Beta- oxidation of valerate results in production of acetyl-CoA and propionyl-CoA [173]. While acetyl-CoA feeds directly into the TCA cycle, propionyl-CoA forms toxic products in mycobacteria and needs to be assimilated via the methylcitrate pathway. The immediate use of valerate by the mutant is likely due to elevated expression of isocitrate lyase, while a lag exists in the parent and complemented strains due to toxic effects of propionyl-CoA metabolites prior to induction of isocitrate lyase. Taken together, the above data indicate that SigC is required for growth on Sauton medium with glycerol as the primary carbon source.

Artificial sigC induction in M. bovis BCG in 7H9 medium

An alternate approach to study the M. tuberculosis SigC regulon is to examine how gene expression changes upon overexpression of the sigma factor. Rodrigue and colleagues utilized a tetracycline-inducible promoter to drive expression of M. tuberculosis SigC with an engineered

N-terminal c-myc epitope in M. bovis BCG to map intergenic regions bound by SigC-RNA polymerase holoenzymes using chromosome-immunoprecipitation/DNA microarray (ChIP-chip) experiments [124]. The authors reported two intergenic chromosomal hotspots for SigC: each between pairs of divergently-oriented genes: Rv0095c-PPE1 and ctpB-Rv0104. Based on qRT-

PCR and 5′RACE mapping of transcript ends, the authors concluded that only Rv0095c and ctpB were transcribed upon sigC induction. This was compelling as identical -35 [GGGAAC] and -10

[CGACT] promoter sequences were shown upstream of the 5′ ends for both genes [124]. The 53

authors confirmed that ctpB, encoding cation transporter B, was transcribed in vitro with E. coli core RNA polymerase reconstituted with myc-SigC, but not with any other M. tuberculosis sigma factor.

To confirm and complement the M. bovis BCG results by using traditional intragenic microarray analyses and to compare the impacts of sigC overexpression in M. tuberculosis, the tetracycline-inducible myc-sigC expression plasmid, pSR173, was obtained from the authors and initially transformed into M. bovis BCG. Prior to microarray studies, induction of myc-SigC production following addition of anhydrotetracycline (aTc) was confirmed by western analysis

(Figure 3.8). A band of the expected size (~21 kD) for myc-SigC was detected only in the lysate from the aTc-induced culture (Figure 3.8). For intragenic expression studies, replicate cultures of

BCG/pSR173 were cultured to OD600=0.2. Half of the cultures were induced with aTc (50 ng/ml) and all were incubated for 48 hours prior to RNA isolation. Results of genes differentially upregulated 2-fold or greater in the presence of inducer are shown (Table 3.3). While no genes were significantly down-regulated upon sigC induction, eleven genes in addition to sigC were up-regulated: four of these were elevated ~2-fold (Rv0847, Rv0848, Rv1183, and MT0196) , seven were elevated >15-fold (Rv0096-Rv0101, and Rv0103c). Based on published BCG/pSR173 chIP-chip studies, it was expected that ctpB and Rv0095c would be upregulated [124]. While ctpB gene was among the highly-expressed genes, Rv0095c did not appear. Instead, all six genes constituting the nrp operon were upregulated >15-fold (Table 3.3). Examination of the promoter region of the first gene (PPE1/Rv0096) in the operon, revealed that it contains the SigC promoter sequence reported for the RACE 5′ transcript end mapping of Rv0095c [124]. As the sequence preceding Rv0095c is different than that reported in the study, this suggests an erratum is present in the Rodrigue et al. 2007 article. The four genes induced approximately 2-fold (Rv0847, 54

Rv0848, Rv1183, and MT0196) were not detected in the ChIP-chip study. Interestingly, all four of these genes have been reported to be upregulated in response to toxic levels of copper [174-

175]. Taken together, these data indicate that the primary SigC targets in M. bovis BCG are ctpB and the PPE1 operon when sigC is induced in 7H9 medium while copper stress response genes are upregulated to a lower level.

SigC-induced gene expression in M. tuberculosis Erdman in 7H9 medium

To determine if SigC-directed gene expression profiles differ between M. bovis BCG and M. tuberculosis Erdman, the latter was transformed with pSR173, and transcription resulting from overexpression of the sigma factor under culture conditions described previously in BCG was examined by microarray analysis. Again employing SAM to identify only genes differentially- expressed significantly, no genes were downregulated 2-fold or more in the presence of inducer, but 13 genes were upregulated >2-fold (Table 3.4). The ctpB and all six PPE1 operon genes were upregulated as seen previously when sigC was aTc-induced in M.bovis BCG/pSR173. Two other loci were upregulated: Rv0190 (ricR), encoding a copper-responsive repressor [175], and genes

Rv0846c-Rv0850 encoding a multi-copper oxidase (Rv0846c), a lipoprotein LpqS (Rv0847), a cysteine synthase CysK2 (Rv0848), and a membrane permease (Rv0849). Only two genes were not significantly upregulated in M. tuberculosis when compared with those elevated when sigC is induced in M. bovis BCG: Rv1183 (mmpL10), encoding a probable conserved transmembrane transport protein and MT0196 (mymT), encoding a copper-protective metallothionein. However, the microarray slides utilized to examine gene expression in M. tuberculosis did not contain the quadruple prints of the array set, which controls better for imperfections on the slides.

Quantitative qRT-PCR of sigC, ctpB, cysK2, and fadD10 confirmed induction of each of these genes in response to artificial sigC induction (Figure 3.9).

To help define the order of genes affected by SigC, expression of Erdman/pSR173 was examined after only 4 hr incubation following aTc addition to 7H9 cultures (Table 3.5). In this time frame, only members of the nrp operon and ctpB were upregulated 2-fold or greater.

Quantitative qRT-PCR of sigC, ctpB and fadD10 confirmed induction (Figure 3.10). Thus, it appears that the primary targets of SigC are the nrp operon and ctpB, while the myriad of toxic metal stress-response genes are induced subsequently.

DISCUSSION

SigC has been known to be an important M. tuberculosis virulence determinant in animal models for several years, but the stress that is mitigated by this sigma factor has remained elusive. The monocistronic nature of the sigC gene offered no co-transcribed genes that might hint at its physiological function. Confusion about the SigC regulon existed because published studies reported different gene sets regulated by SigC [74, 124]. The first report defined a SigC consensus promoter (SSSAAT –16-20 nt– CGTSSS, where S = G or C) based on genes reported down-regulated in a sigC mutant relative to parent M. tuberculosis strain CDC1551 in

Middlebrook 7H9 cultures [74]. The second study identified SigC promoter elements (GGGAAC

-17 nt- CTACT) upstream of two genes (Rv0095 and ctpB) with greatest in vivo binding of plasmid-encoded SigC in M. bovis BCG-Russia after SigC was artificially-induced in 7H9 cultures [124]. Our study did not reveal any genes that were identified as being differentially- regulated when ΔsigC was compared to parent strain Erdman when cultured in 7H9 medium

(data not shown). Applying the same stringency levels to RNA isolated from strains cultured in

Sauton medium resulted in detection of significant upregulation of 4 of 6 genes in the nrp operon in Erdman versus ΔsigC (Table 3.1). Initially, this appeared to conflict with the publications mentioned above. However, sigC overexpression studies in 7H9 medium indicating induction of the nrp operon and the ctpB gene [Tables 3.3-3.5] supported the findings reported in BCG after realizing that the SigC promoter sequence reported for Rv0095c [124] was actually that for

Rv0096, the first gene in the nrp operon.

Examination of the sigC-associated gene expression data and growth phenotypes suggests a correlation of SigC with trace metal acquisition. A model is presented to provide context in which to review the data supporting this link (Figure 3.11). The model is based on the 57

hypotheses, that SigC is the only sigma factor that transcribes the nrp operon and the product of this operon synthesizes a high-affinity scavenging system for essential trace elements. In media with ultra-low levels of such metals, SigC transcribes the operon allowing M. tuberculosis to import sufficient quantities for growth. If SigC production is artificially-induced in a medium with low levels (6 μM) of copper and zinc ions, then the scavenging system imports metals to high levels resulting in induction of metal-efflux and other stress-response mechanisms to prevent toxic effects associated with an excess of transition metals.

Pathogenic bacteria have evolved mechanisms to obtain essential nutrients from the host.

Conversely, the host has evolved systems to maintain tight control of glucose and trace elements to minimize toxic effects to host cells as well as limit availability to microbes that breach epithelial barriers. To acquire iron from the host, M. tuberculosis bacilli produce the siderophore mycobactin from a polyketide synthase pathway encoded by mbtA-I [176-177]. Limited availability of other transition metals within specific host niches supports the concept of the evolution of an additional metal-chelating system by this pathogen. The nonribosomal peptide synthase (nrp) operon is a likely candidate to encode such a biosynthetic pathway. The nature of the product synthesized by the 11-kb nrp gene is unclear. It has been proposed to synthesize a lipid because it is preceded in the operon by genes encoding a fatty acyl-CoA thioesterase

(FcoT/Rv0098) which hydrolyzes C16-C18 fatty acids), and a fatty acid ligase (FadD10/Rv0099) which links the lipids to an acyl-carrier protein (Rv0100) upon which the multi-domained nonribosomal peptide synthase (Nrp/Rv0101) can synthesize its product [169]. No current biochemical data supports that the final product generated is simply a lipid. Mycobactin is also known to have a C17 lipid tail [178]. A role in PDIM synthesis for the conserved nrp operon in

M. bovis was hypothesized based on observed changes in colony morphology and synthesized 58

PDIM lipids for mutants with transposon insertions within the operon [169]. Trace element requirement by an enzyme in the PDIM biosynthetic pathway would be consistent with a role for the operon in both metal acquisition and in PDIM synthesis.

Transition metals are likely imported by the predicted metal-uptake system. As iron is present in both media (Sauton and 7H9) that led to observations supporting a role for SigC in metal acquisition, iron is unlikely to play a role in transcription of the nrp operon or to be imported via the product of the operon. Zinc, copper, and calcium ions are present in 7H9, but not in Sauton medium. Hence, the influence of these metals will be further examined. Retarded growth of ΔsigC on Sauton medium, unless supplemented with a carbon source in addition to glycerol, indicates that glycerol is not utilized efficiently (Figures 3.4, 3.7). The M. tuberculosis glycerol kinase has 68% homology to E. coli glycerol kinase, which requires a zinc cofactor

[179]. This supports that zinc may be a ligand for the proposed chelator. Copper is another likely ligand. Induction of the ubiquinol oxidase operon by the mutant (Table 3.2) provides an alternate respiratory pathway to compensate for reduced function of the copper-dependent cytochrome a/a3 oxidase [180]. Induction of the alkyl hydroperoxidase C gene in Sauton-cultured ΔsigC

(Table 3.2) provides an alternate enzyme to respond to reactive oxygen radicals to replace

Cu/Zn-dependent superoxide dismutase SodC [181]. Up-regulation of several copper detoxification genes when sigC is artificially-induced for a prolonged period in 7H9 supports that this metal is imported (Tables 3.3-3.5, Figure 3.11). The induction of icl in the ΔsigC suggests the possibility of a TCA cycle block between isocitrate and succinate (Table 3.2, Figure

3.6). This pathogen lacks the typical dehydrogenase for conversion of 2-ketoglutarate to succinyl-CoA, but contains alternate enzymes to generate succinate from 2-ketoglutarate [182-

184]. Limitation of metals required for function of these enzymes would explain icl induction to 59

bypass a block in this branch of the TCA cycle. In support of copper and zinc being imported via a SigC-dependent mechanism, preliminary studies indicate that growth rate of Sauton-cultured

ΔsigC increases when supplemented with 6 μM of CuSO4 and ZnSO4 (data not shown).

Abundant transcription of the nrp operon and ctpB in 7H9, but transcription of only the nrp operon in Sauton cultures suggests ctpB (encoding a predicted cation transporting ATPase, CtpB

[172, 185]) is repressed by an unknown metal (Tables 3.1, 3.3-3.5). Rodrigue and colleagues demonstrated that ctpB was transcribed in vitro only by RNA polymerase holoenzymes reconstituted with SigC and that ctpB has identical promoter elements as that preceding the nrp operon [124]. Thus, it appears that, at a minimum, copper and zinc ions are imported by the predicted SigC-controlled metal scavenger.

The importance of the nrp operon to survival of pathogenic mycobacteria is underscored by the conservation of all genes in the nrp operon between members of the M. tuberculosis complex and to distantly-related Mycobacterium leprae [169]. M. tuberculosis mutants with transposon insertion in genes Rv0097 or Rv0101 (nrp) failed to replicate within the lungs of

BALB/c mice [186]. The attenuation of M. tuberculosis sigC mutants in murine and guinea pig models is consistent with its role in transcribing the nrp gene (Figure 3.2) [74, 123, 162].

A variety of testable questions remain regarding the role for SigC in metal acquisition.

Among them is the mechanism by which SigC function is controlled by metal availability.

Studies using a real-time RT-PCR assay with molecular beacons reported sigC transcription is expressed at high levels but not induced in response to various stressors [88]. This is likely explained by the presence of five different transcript 5′ ends recently mapped for sigC [187].

Associated promoters include one for SigA with an overlapping BlaI operator and another for

SigF, suggesting the response regulated by SigC is required under multiple environmental 60

conditions [105, 187]. Our prediction is that SigC is regulated at the post-transcriptional level.

Another major question is the identity of the metal chelator synthesized. Examination for SigC- dependent production of secreted or membrane-associated molecules should enable the identification and purification of this important molecule. Targeting this metal uptake system will provide a new approach to develop drugs or vaccines to aid in the persistent battle to control tuberculosis.

Table 3.1: Genes transcriptionally elevated in M. tuberculosis strain Erdman relative to strain ΔsigC when cultured in Sauton medium Locus Proposed function Fold-upregulation

Rv0097* Oxidoreductase 3.5 Rv0098/fcoT* Fatty acyl-CoA thioesterase 6.7 Rv0100* Acyl-carrier protein 3.0 Rv0101/nrp* Nonribosomal peptide synthase 2.8 Rv0967/csoR Copper-sensitive operon repressor 2.6 DNA microarray results from strain Erdman versus ΔsigC cultured in Sauton medium to OD600=1. Arrays contained probes to ORFs from M. tuberculosis strain H37Rv. Combined data from 4 biological and 2 technical replicates were analyzed using Statistical Analysis for Microarrays software (Stanford University) with false-discovery rate set to zero. Genes statistically upregulated 2-fold or greater in Erdman vs. ΔsigC are shown along with proposed encoded functions. Asterisks indicate genes of the nrp operon.

Table 3.2: Genes transcriptionally elevated > 2-fold in M. tuberculosis strain ΔsigC relative to strain Erdman when cultured in Sauton medium Locus Proposed function Fold-upregulation

Rv0188 Hypothetical membrane protein 3.4 Rv0251c/hsp Heat shock protein 4.8 Rv0467/icl Isocitrate lyase 3.5 Rv1620c/cydC ATP-binding protein ABC transporter 4.0 Rv1621c/cydD ATP-binding protein ABC transporter 7.6 Rv1622c/cydB Cytochrome D ubiquinol oxidase 7.3 Rv1623c/cydA Cytochrome D ubiquinol oxidase 6.5 Rv1624c Probable membrane protein 3.3 Rv1846c/blaI Transcriptional repressor 3.4 Rv2428/ahpC Alkyl hydroperoxide reductase 3.0 Rv2628 Hypothetical protein 2.6 Rv2780/ald Alanine/glycine dehydrogenase 4.9 DNA microarray results from strain Erdman versus ΔsigC cultured in Sauton medium to OD600=1. Arrays contained probes to the ORFs from M. tuberculosis strain H37Rv. Genes statistically upregulated 2-fold or greater in strain ΔsigC versus Erdman are shown along with proposed encoded functions.

Table 3.3: Genes upregulated in M. bovis BCG/pSR173 after 48 hr aTc induction in 7H9 medium Locus Proposed function Fold-upregulation Rv0096/PPE1* PPE family protein 19.5 Rv0097* Oxidoreductase 17.5 Rv0098/fcoT* Fatty acyl-CoA thioesterase 21.3 Rv0099/fadD10* Fatty acid ligase 18.1 Rv0100* Acyl-carrier protein 16.7 Rv0101/nrp* Nonribosomal peptide synthase 15.6 Rv0103c/ctpB Cation-transporting ATPase B 18.0 Rv0847/lpqS Lipoprotein 2.3 Rv0848/cysK2 Cysteine synthase A 2.4 Rv1183/mmpL10 Transmembrane transport protein 2.4 Rv2069/sigC RNA polymerase sigma factor 9.3 MT0196/mymT Metallothionein 2.0

DNA microarray results from strain M. bovis BCG/pSR173 cultured in 7H9 medium to OD600=0.2 and cultured 48 hr +/- aTc induction. Arrays contained probes to ORFs in M. tuberculosis strain H37Rv and additional ORFs present in strain CDC1551. Genes statistically upregulated 2-fold or greater in induced cultures vs uninduced are shown along with proposed encoded function. Asterisks indicate genes of the nrp operon.

Table 3.4: Genes in Erdman/pSR173 up-regulated following 48 hr aTc induction in 7H9 medium

Locus Proposed function Fold-upregulation Rv0096/PPE1* PPE family protein 8.9 Rv0097* Oxidoreductase 5.6 Rv0098/fcoT* Fatty acyl-CoA thioesterase 13.0 Rv0099/fadD10* Fatty acid ligase 11.5 Rv0100* Acyl-carrier protein 8.2 Rv0101/nrp* Nonribosomal peptide synthase 11.2 Rv0103c/ctpB Cation-transporting ATPase B 7.3 Rv0190/ricR Copper-regulated repressor 3.7 Rv0846c Multi-copper oxidase 2.3 Rv0847/lpqS Lipoprotein 13.2 Rv0848/cysK2 Cysteine synthase A 12.7 Rv0849 Membrane permease 16.2 Rv0850 Transposase fragment 4.6 Rv2069/sigC RNA polymerase sigma factor 7.5

DNA microarray results from strain Erdman/pSR173 cultured in 7H9 medium to OD600=0.4 and cultured 48 hr +/- aTc induction. Microarrays used contained probes to ORFs present in M. tuberculosis strain H37Rv. Genes statistically upregulated 2-fold or greater in induced cultures vs uninduced are shown along with proposed encoded function. Asterisks indicate genes of the nrp operon.

Table 3.5: Genes in Erdman/pSR173 up-regulated >2-fold following 4 hr aTc induction in 7H9 medium Locus Proposed function Fold-upregulation

Rv0096/PPE1* PPE family protein 2.6 Rv0098/fcoT* Fatty acyl-CoA thioesterase 2.1 Rv0099/fadD10* Fatty acid ligase 5.4 Rv0100* Acyl-carrier protein 2.5 Rv0101/nrp* Nonribosomal peptide synthase 3.8 Rv0103c/ctpB Cation-transporting ATPase B 3.1 Rv2069/sigC RNA polymerase sigma factor 6.2

DNA microarray results from strain Erdman/pSR173 cultured in 7H9 medium to OD600=0.4 and cultured 4 hr +/- aTc induction. Microarrays used contained probes to ORFs found in M. tuberculosis strain H37Rv. Genes statistically upregulated 2-fold or greater in induced cultures vs. uninduced are shown along with proposed encoded functions. Asterisks indicate genes of the nrp operon.

Figure 3.1: Southern analysis of the sigC genomic regions from strains Erdman and ΔsigC. Consistent with a ~140 bp deletion within sigC, the ΔsigC strain yields a genomic DNA band correspondingly smaller than that observed from strain Erdman.

Figure 3.2: Growth of M. tuberculosis strains in 7H9 broth. The optical density of M. tuberculosis strains Erdman, sigC mutant (ΔsigC) and complemented sigC mutant (comp) in Middlebrook 7H9 medium was monitored over time. Results shown are the average of triplicate experiments performed with three biological replicates each. Growth of the strains was not statistically different based on one-way ANOVA analysis.

Figure 3.3: Impact of ΔsigC on viability following infection of SCID mice. Transoral instillation was used to deliver PBS or 104 CFU of the indicated M. tuberculosis strains into SCID mice. Animal survival was monitored over a 140-day period. By Kaplan-Meier analysis, survival in animals infected with ΔsigC was statistically different from Erdman or comp after the two outliers in the Erdman and comp groups were eliminated.

* * *

Figure 3.4: Growth of ΔsigC is slower in Sauton medium. Growth of M. tuberculosis strains Erdman, sigC mutant (ΔsigC) and complemented sigC mutant (comp) in Sauton medium was monitored by measuring the optical density at 600 nm over time. Results shown are the average of triplicate experiments performed with three biological replicates each. Statistical significance was determined using one-way ANOVA. Asterisks indicate statistical significance between ΔsigC and either Erdman or comp (p-value < 0.05).

A B

Figure 3.5: qRT-PCR confirmation of microarray data for ΔsigC versus Erdman grown in Sauton medium. Expression of the indicated M. tuberculosis genes was measured by qRT-PCR from strain Erdman and ΔsigC cultured in Sauton medium to OD600=1. Data are presented as gene expression ratios from Erdman/ΔsigC (A) or from ΔsigC/Erdman (B). Ratios were normalized to sigA. Experiments were performed in triplicate with three biological replicatesError bars represent the standard deviation.

Figure 3.6: Drawing of M. tuberculosis central carbon metabolism. Shown is a minimalistic depiction of central carbon metabolism in M. tuberculosis. Genes encoding enzymes responsible for specific steps are shown. Genes upregulated in ΔsigC versus Erdman in Sauton medium are indicated (red arrows). The methylcitrate cycle is highlighted (purple box).

A B

C Figure 3.7: Carbon source supplementation * impacts mycobacterial growth on Sauton medium. Growth (OD600nm) of wildtype (Erdman), sigC mutant (ΔsigC) and * complemented mutant (comp) in Sauton medium (SM) supplemented with glucose (A), Tween 80 (B), or valeric acid (C) was monitored over time. Results are the average of triplicate experiments performed with three biological replicates each. Statistical significance between mutant and either control strain was determined using one-way ANOVA. Asterisks indicate statistical significance between ΔsigC and both Erdman and comp (p- value < 0.05).

Figure 3.8: Immunoblot detection of myc-SigC in M. bovis BCG/pSR173. Duplicate cultures of M. bovis BCG carrying aTc-inducible myc-sigC expression plasmid pSR173 were grown in 7H9 medium. At OD600 =0.2, 50 ng/ml aTc was added to one culture (+aTc), but not to the other (- aTc) and both cultures were incubated 48 hr prior to harvesting the cells and preparation of whole cell lysates. Equivalent protein loads from each condition were separated by SDS-PAGE. Following transfer to PVDF membrane, western immunoblotting was performed with a monoclonal antibody to Myc. The migration of protein standards is indicated on the right.

Figure 3.9: qRT-PCR of RNA isolated +/- 48 hr aTc induction of Erdman/pSR173. Cultures of M. tuberculosis strain Erdman harboring plasmid pSR173 were grown to OD600=0.2 in 7H9 medium, split and cultured 48 hr after sigC induction by addition of aTc to half of the cultures. Quantitative RT-PCR was used to examine expression of sigC, ctpB, cysK2 and fadD10. Ratios were normalized to sigA and uninduced samples were used as a reference. Experiments were performed in triplicate with three biological replicates. Error bars represent the standard deviation.

Figure 3.10: qRT-PCR of RNA isolated +/- 4 hr aTc induction of Erdman/pSR173. Cultures of M. tuberculosis strain Erdman harboring plasmid pSR173 were grown to OD600=0.4 in 7H9 medium, split and cultured 4 hr after sigC induction by addition of aTc to half of the cultures. Quantitative RT-PCR was used to examine expression of sigC, ctpB, and fadD10. Ratios were normalized to sigA and uninduced samples were used as a reference. Experiments were performed in triplicate with three biological replicates. Error bars represent the standard deviation.

A. Sauton Medium B. 7H9 Medium

Figure 3.11: A model for SigC function in metal regulation. Diagrammed are genes impacted positively by M. tuberculosis SigC in A) Sauton medium lacking added Zn2+ or Cu2+ or B) 7H9 medium containing each at 6 μM. Genes are not drawn to scale. Promoters (P) are subscripted with known regulators. Upward green arrows denote elevated levels of SigC or SigC-transcribed genes. Metal- mediated promoter derepression is indicated by red arrows. A) In a very low metal environment, SigC transcribes the nrp operon, encoding a predicted trace-metal scavenging system. Imported metals include Zn2+ (activates glycerol kinase to enable growth on glycerol) and Cu2+ (binds copper-sensitive operon repressor, CsoR, and derepresses the csoR operon encoding cation transport protein V, CtpV [185] and other potential metals (M2+). CtpV functions as a copper-exporting ATPase [172]. B) When trace metals are not scarce, induction of sigC from a tetracycline-inducible promoter (TET) by anhydrotetracycline (aTc) results in increased SigC- mediated transcription of the nrp operon which causes an influx of trace metals. Imported Cu2+ binds regulated-in-copper repressor (RicR) [175], resulting in derepression of ricR, mymT (encoding a Cu-binding metallothionein [174]) and the lpqS operon (encoding proteins that function to minimize oxidative damage by excess Cu2+ [175]. Repression of ctpB is relieved by an unknown metal, enabling SigC-mediated transcription. The encoded CtpB protein is predicted to function as ATPase to export the inducing metal. 77

CHAPTER 4

EXAMINATION OF COBALAMIN BIOSYNTHESIS IN MYCOBACTERIA 1

1 Benjamin T. Grosse-Siestrup, and Russell K. Karls. To be submitted to Journal of Bacteriology.

ABSTRACT

The causative agent of tuberculosis, Mycobacterium tuberculosis, remains a leading cause of morbidity and mortality. Understanding the mechanisms by which this pathogen adapts to successfully reside in the host is of importance for rational design of therapeutics, vaccines, and diagnostics. Genomic sequence analysis suggests that this bacterium has a full complement of genes for cobalamin synthesis. However, recent articles report that M. tuberculosis strains do not produce cobalamins under the conditions tested. In the present study, we re-examine cobalamin synthesis in mycobacteria. A cobalamin auxotrophic mutant of Salmonella enterica subspecies enterica, serovar Typhimurium was used to screen for cobalamin production from different

Mycobacterium species. Growth of the auxotroph was detected with extracts from M. smegmatis,

M. marinum, M. fortuitum, M. avium subspecies avium, M. avium subspecies paratuberculosis,

M. shottsii, and M. pseudoshottsii. Growth was not observed using extracts from M. microti, M. bovis BCG, or any of five strains of M. tuberculosis tested. Interestingly, all mycobacteria positive for cobalamin production are not members of the M. tuberculosis complex, while all strains negative for cobalamin production are members of that complex. This suggests that either cobalamin synthesis does not occur in the M. tuberculosis complex species under the conditions tested or the form of cobalamin generated in these species cannot be utilized by the Salmonella auxotroph.

INTRODUCTION

Fueled by HIV co-infections and antibiotic resistant strains, tuberculosis (TB) is re-emerging

[16]. Today, one third of the world population is estimated to be infected by the etiological agent

Mycobacterium tuberculosis resulting in 1-2 million deaths annually [158]. Success of this pathogen lies in its ability to obtain nutrients from the host and thwart immune defenses.

Cobalamins, such as vitamin B12, are molecules consisting of a corrin ring with a central cobalt atom, a dimethylbenzimidazole ribonucleotide loop which binds to the lower axis of the cobalt atom, and a variable upper axial cobalt ligand (Figure 4.1). In its biologically active form, the upper ligand of cobalamin is an adenosyl group. While the lower ligand is most often 5,6- dimethylbenzimidazole, it can show significant variation depending on the nature of the microorganism [188]. Cobalamin synthesis is restricted to bacteria and occurs by either an aerobic or anaerobic pathway, depending on the bacterial species (Figure 4.2). Mycobacterium tuberculosis presumably uses an aerobic biosynthetic process, since it has homologs to cobalamin biosynthetic genes from Pseudomonas denitrificans but not from Salmonella typhimurium (Figure 4.2). Cobalamins, function as cofactors in enzymes that catalyze isomerization and reduction reactions involved in various metabolic processes. These enzymes include: (A) Methionine synthetase which transfers a methyl group from methyltetrahydrofolate to homocysteine as the final step in methionine biosynthesis [135]. In Salmonella and E. coli, this reaction is performed by either the vitamin B12-dependent enzyme MetH or by the vitamin

B12-independent enzyme MetE. The M. tuberculosis genome contains both metE and metH homologues. While both genes are intact in M. tuberculosis strain H37Rv, metH from strain

CDC1551 contains a nonsense mutation which renders it auxotrophic for methionine when bacterial cultures are supplemented with 10 µg/ml vitamin B12 due to the presence of a B12

riboswitch upstream of metE [152]. (B) Epoxyqueuosine reductase performs the last step in formation of the hypermodified tRNA base queuosine [136]. A homolog has not been identified in M. tuberculosis. (C) Ribonucleotide reductase functions in DNA synthesis by reducing ribonucleosides to deoxyribonucleosides [139-140]. In M. tuberculosis, two ribonucleotide reductase genes are present. One encodes an iron-dependent enzyme; the other encodes a vitamin

B12-dependent enzyme [189-190]. (D) Methylmalonyl-CoA mutase interconverts (R)- methylmalonyl-CoA and succinyl-CoA. This enzyme plays an important role in metabolism of odd-chain fatty acids, cholesterol, and some amino acids. The methylmalonyl-CoA mutase homolog in M. tuberculosis has been shown to enable growth of the pathogen on odd-chain fatty acids when isocitrate lyase is blocked by 3-nitropropionate (3NP) [191].

In M. tuberculosis, 17 genes have been identified with potential roles in cobalamin synthesis [145, 153]. Early studies, using feeding assays with Lactobacillus leichmannii, reported vitamin B12 production by various mycobacterial strains including M. smegmatis, M. bovis BCG M. bovis strain BCGp1102, M. tuberculosis strain H37Rv, and a streptomycin-resistant variant H37Rv-SR [192-195]. However, more recent studies using alternate detection methods did not result in vitamin B12 detection from M. tuberculosis strain H37Rv (ATCC 25618) or strain CDC1551 [191]. Interestingly, the gene for the mycobacterial sigma factor C (sigC) and the gene for a β-lactamase C (blaC) are located within this cluster of cobalamin synthesis genes.

Since no anti-sigma factor for sigC has been identified to date, a potential correlation might exist between cobalamin synthesis and SigC regulation. Here we re-examine the cobalamin production and the conservation of cobalamin biosynthetic genes among different mycobacteria.

MATERIAL AND METHODS

Bacterial strains and culture conditions

The M. tuberculosis strains Erdman, H37Rv, H37Ra, and CDC1551, M. microti strain P2, M. smegmatis strain mc2 155, M. avium Serovar #8, and M. avium subspecies paratuberculosis

(ATCC#700535) were obtained from the Tuberculosis/Mycobacteriology Branch of the Centers for Disease Control and Prevention. The strain M. tuberculosis strain 103 was obtained from Dr.

Camille Locht, Institute Pasteur de Lille, France. The strains M. bovis BCG and M. fortuitum were obtained from Dr. Mary Hondalus, Department of Infectious Diseases, University of

Georgia. The strains M. marinum strain M30, M. shottsii strain M175, and M. pseudoshottsii strain L15 were obtained from Dr. David Gauthier, Department of Biological Sciences, Old

Dominion University.

The M. tuberculosis strains were cultured in Middlebrook 7H9 medium (BD/Difco) supplemented with 0.5% glycerol, 0.05% Tween 80 and 10% ADS (albumin, dextrose, NaCl)

(7H9tgADS) [163], in Sauton medium (0.05% KH2PO4, 0.05% MgSO4 · 7H2O, 0.2% Citric acid,

0.005% ferric ammonium citrate, 6% glycerol, 0.4% asparagine, pH adjusted to 7.4) supplemented with 0.025% Tylaxopol (Sigma), or on Löwenstein-Jensen slants (18.75 g/l potato starch, 2.25 g/l L-asparagine, 1.5 g/l KH2PO4, 1.5 g/l MgSO4 · 7H2O, 0.15 g/l magnesium citrate, 0.25 g/l malachite green, 62.5% eggs, 0.0075% glycerol) (BD/Difco). Cultures were incubated at 37°C shaking or static as indicated. Other mycobacteria strains were cultured in one of the following media: Middlebrook 7H9 medium supplemented with 0.5% glycerol, 0.05%

Tween 80 and 10% ADC (albumin, dextrose, catalase) (7H9tgADC), 7H9 medium supplemented with 0.5% glycerol, 0.05% Tween 80 and 10% OADC (oleic acid, albumin, dextrose, catalase)

(7H9tgOADC), 7H9 medium supplemented with 0.5% glycerol, 0.05% Tween 80, 10% ADC,

and 2 mg/L mycobactin (7H9tgADCm), or 7H9 medium supplemented with 0.5% glycerol,

0.05% Tween 80, and 10 mM dextrose (7H9tgD). In studies involving 3NP (Sigma), the chemical was used at a final concentration of 0.1 mM. For 3NP experiments, bacteria were cultured in 7H9 medium supplemented with 0.5% albumin, 0.085% NaCl, 0.05% Tween80 and

0.1% sodium propionate (7H9tASp).

Salmonella typhimurium strain JE212 [genotype: metE205, ara-9, delta299(hisG-cob)] was grown in LB broth. Salmonella typhimurium strain JE7089 [metE205, ara-9, metH::cat] was grown in the same medium supplemented with chloramphenicol (RPI) to a final concentration of

50 µg/ml. The Salmonella typhimurium strains were provided by Dr. Jorge Escalante,

Department of Bacteriology, University of Wisconsin-Madison.

Cobalamin isolation

To isolate cobalamin, 50 ml bacterial cultures were grown in the indicated media to OD600 ≈ 1.

Cells were collected by centrifugation (3000g, 5 min). Cells were washed once with an equal volume 0.1 M sodium phosphate buffer (pH 7.0) and resuspended in 1 ml extraction buffer (0.1

M Na2HPO4 [pH4.5] [acetic acid], 0.005% KCN). Cells were disrupted with 0.1 mm diameter zirconia/silica beads in a Bead Beater (BioSpec Products) with three 40-second pulses at 4800 rpm. The liquid was transferred into glass screw cap culture tubes (Fisher Scientific). With the caps tightened, the lysates were autoclaved for 30 min. Lysates were then transferred into 1.5 ml tubes and clarified by centrifugation (10,000g, 5 min). The supernatants were filtered through

0.22 µM PVDF filter membranes into new 1.5 ml tubes and concentrated to approximately 50 µl in a DNA120 SpeedVac (ThermoSavant). Samples were stored at -20°C until assayed.

Cobalamin bioassay

Salmonella typhimurium strains JE212 and JE7089 were cultured overnight in LB medium at

37°C with shaking (180 rpm). Aliquots of culture (1 ml each) were transferred into 1.5 ml tubes and collected by centrifugation (10,000g, 5 min). Each pellet was washed once with an equal volume 0.9% NaCl solution and resuspended in equal volume 0.9% NaCl solution. For each cobalamin bioassay plate, 200 µl cells were mixed with 3 ml molten (56°C) 0.6% noble agar, vortexed briefly, and quickly poured onto Vogel-Bonner minimal medium agar plates (0.2 g

MgSO4 ∙ 7H2O, 2 g citric acid ∙ H2O, 10 g K2HPO4, 3.5 g NaNH4HPO4 ∙ 4H2O, and 15 g agar per liter) supplemented with a final concentration of 11 mM glucose and 0.1 mM histidine. After solidification of the agar, 5 µl of mycobacterial lysate, 5 µl methionine (5 mg/ml) or 5 µl cyanocobalamin (100 µg/ml) were spotted onto the plates. Plates were incubated at room temperature for approximately 15 min to allow the agar to absorb the liquid. The plates were then incubated inverted overnight at 37°C. Growth of Salmonella typhimurium strain JE212 indicated presence of cobalamin. Growth of Salmonella typhimurium strain JE7089 indicated presence of methionine.

For 3NP growth curves, M. tuberculosis was subcultured to OD600 ≈ 0.05 into 7H9 medium supplemented with 0.5% glycerol, 0.05% Tween 80, 0.1% sodium pyruvate, 0.1% valeric acid, and 1% betaine (Fluka) and incubated shaking (75 rpm) at 37°C. Absorbance readings were taken every 24-48 hours.

RESULTS

Cobalamin production in mycobacteria

To test for the presence or absence of cobalamins in mycobacterial lysates, Salmonella serovar Typhimurium strain JE212, which has a deletion of several cobalamin genes and carries a defective metE gene, was utilized [196]. The lack of the MetE enzyme requires that methionine synthesis is catalyzed by the cobalamin-dependent methionine synthase enzyme MetH. In the absence of cobalamin, strain JE212 is a methionine auxotroph. Since strain JE212 can also grow when methionine but not cobalamin is provided, the metE, metH double mutant Salmonella strain JE7089 was used. This strain will grow only if methionine is provided, regardless of whether or not cobalamin is present. Growth of strain JE212, but not JE7089 indicates the presence of cobalamin but not methionine. Lysates from fourteen different mycobacterial strains were grown to stationary phase in the indicated media (Table 4.1). Several mycobacterial lysates enabled strain JE212 to grow under cobalamin-restricted conditions (Table 4.1, Figure 4.3).

When testing for methionine contamination, none of the lysates enabled strain JE7089 to grow indicating, that the bacterial lysates did not contain sufficient methionine to enable growth

(Figure 4.4). Interestingly, all mycobacterial lysates that supported growth of strain JE212 were not from members of the M. tuberculosis complex (MTC) while all lysates not conferring growth were from members of the MTC (Table 4.1). The MTC bacteria currently consist of the following species: M. tuberculosis, M. bovis, M. pinnipedii, M. africanum, M. microti, and M. caprae [2]. The obligate human pathogens are M. tuberculosis, and M. africanum, while the other members, M. bovis, M. pinnipedii, M. microti and M. caprae primarily infect animals.

To rule out the possibility that components of MTC lysates inhibit growth of the strain

JE212, 500 ng vitamin B12 was added to the purified M. tuberculosis lysates and used as a

control for the feeding assay. Only the M. tuberculosis lysates supplemented with vitamin B12 supported growth of strain JE212 (Figure 4.3).

Karasseva and colleagues reported that M. bovis BCG was positive for cobalamin production when cultured in a different medium and assayed by using a Lactobacillus species as a detection strain [192]. To determine if growth medium differences accounted for the negative results with MTC lysates on strain JE212, efforts to replicate growth conditions used by

Karasseva et al. were undertaken. Specifically, M. tuberculosis strain Erdman was cultured on

Löwenstein-Jensen medium for several weeks, at which time, colonies were transferred into flasks containing Sauton medium without Tylaxopol. The cultures were incubated stationary until turbidity was detected. However, the resulting lysates did not enable growth of strain JE212

(data not shown).

Various hypotheses for the disparity in results remain. Regulation of cobalamin synthesis in

MTC bacteria may be more-tightly controlled than in non-MTC bacteria. Subtle differences in growth conditions or medium components may not have adequately mimicked published studies.

Strain differences might also exist. Additionally, the form of cobalamin produced by the MTC bacteria might be different than that produced by other mycobacteria and may not be utilized by the Salmonella auxotroph.

When cultured with odd carbon-chain fatty acids, β-oxidation of the lipids results in terminal propionyl-coenzyme A (CoA) generation in M. tuberculosis. Accumulation of this molecule will result in formation of toxic propionaldehyde [143]. Mycobacterium tuberculosis can use either an isocitrate lyase-dependent or a cobalamin-dependent methylmalonyl-CoA mutase pathway to dissimilate propionate [133, 191]. By blocking isocitrate lyase function using

3NP, Savvi et al. showed that M. tuberculosis strain H37Rv did not grow on propionate when

3NP was present, but growth was restored upon addition of exogenous vitamin B12 [191]. It was possible that the H37Rv strain used in that study has a mutation in one of the cobalamin biosynthetic genes. Therefore, M. tuberculosis strain Erdman was assayed under similar conditions, but it also failed to grow on propionate in the presence of 3NP until vitamin B12 was added (Figure 4.5). Taken together, these results indicate that M. tuberculosis does not produce sufficient amounts of an appropriate form of cobalamin to support growth of a Salmonella cobalamin autoxtroph or for methylmalonyl-CoA dissimilation of propionyl-CoA.

Comparison of cobalamin biosynthetic genes among mycobacteria

After establishing, that only non-MTC bacteria produce detectable cobalamin under the conditions tested, the extent of conservation of putative cobalamin biosynthetic genes among

Mycobacterium species was examined using the Basic Local Alignment Search Tool (BLAST).

Predicted proteins in the cobalamin biosynthetic pathway were compared among members of the

MTC (M. tuberculosis and M. bovis BCG), to mycobacteria outside the complex (M. marinum and M. smegmatis) and among members outside the complex (M. marinum and M. smegmatis).

Amino acid identity among cob homologs in M. bovis BCG and M. tuberculosis ranges from 98-

100% (Table 4.2). Differences between homologs in M. tuberculosis and those found in either M. marinum or M. smegmatis are more striking, especially with enzymes functioning in the latter part of the pathway (Table 4.2, Figure 4.2). However, the amino acid identity among the cob homologs between M. marinum and M. smegmatis also ranged from 0-85%. Notably, the non-

MTC species M. smegmatis lacks a CobC homolog, while CobU has the least conservation among homologs in M. tuberculosis and among the environmental mycobacteria (Table 4.2).

These enzymes participate in dimethylbenzimidazole addition. Thus it is possible that the MTC

bacilli and the environmental mycobacteria produce cobalamins with different lower axial ligands.

DISCUSSION

Seemingly contradictory studies have either shown the ability [192, 194] or inability [191] of M. tuberculosis to produce cobalamin in vitro. In the present study, we used a cobalamin auxotrophic strain of Salmonella enterica serovar Typhimurium to assay for cobalamin in mycobacterial lysates. Among the fourteen mycobacterial strains examined, only those outside the MTC produced detectable amounts cobalamin. The mycobacteria positive for cobalamin production were the soil saprophyte M. smegmatis; various opportunistic mammalian pathogens:

M. fortuitum, M. avium subspecies avium, and M. avium subspecies paratuberculosis; and the aquatic species M. marinum, M. shottsii, and M. pseudoshottsii. The species that tested negative for cobalamin were the tuberculosis vaccine strains M. bovis BCG, the vole pathogen M. microti, and M. tuberculosis. In an effort to address the possibility that mutations may have occurred in some M. tuberculosis strains due to laboratory passage, M. tuberculosis strains from various sources were obtained and assayed. In addition, various alterations to culture conditions were also examined (Table 4.1). None of the tested conditions resulted in cobalamin production by

MTC bacilli using the Salmonella cobalamin auxotroph assay.

It has been shown that the lower ligand of cobalamin can vary, depending on the nature of the microorganism [188, 197]. However, the most commonly utilized base of the lower ligand is

5,6-dimethylbenzimidazole. This ligand is used by Salmonella enterica and Pseudomonas denitrificans which synthesize the cofactor by distinct pathways. Currently it is unknown what lower ligands are present in cobalamin synthesized in mycobacteria. It is possible that MTC bacilli produce a form that cannot be utilized by the Salmonella auxotroph employed in this study. Bioinformatic comparison of predicted cobalamin biosynthetic genes among MTC and non-MTC mycobacteria suggests the intriguing possibility that the MTC bacilli may produce an

alternate lower ligand. Experiments are underway to utilize a Lactobacillus species to examine this possibility. These experiments will be performed similar to the Salmonella auxotroph feeding assays. However, Lactobacillus will be used instead of Salmonella. Additionally, structural analysis using mass spectrometry will be utilized to determine the nature of the lower ligand. Another alternative is that cobalamin, synthesized by the MTC species, is bound tightly to other cellular components and not solubilized during the extraction process. To address this question, we implemented an alternative test based on the 3NP sensitivity of M. tuberculosis when grown on odd carbon-chain fatty acids [191]. In this assay, M. tuberculosis requires cobalamin to be able to overcome a blockage in the propionate detoxification pathway caused by poisoning of the isocitrate lyase with 3NP. However, this assay failed to result in M. tuberculosis growth unless vitamin B12 was provided exogenously (Figure 4.5). Since genomic sequence analysis suggests that M. tuberculosis has a full complement of genes for cobalamin synthesis, and since cobalamin-dependent enzymes have been reported for this pathogen, it is possible that specific environmental conditions required for induction of cobalamin synthesis have not been adequately replicated. Such conditions might only be present inside a host, where the bacteria are exposed to a more complex and hostile environment.

Table 4.1: Cobalamin production in various mycobacterial strains

Growth Cobalamin Mycobacterial strain Culture medium§ Temperature production*

M. smegmatis strain mc2 155 7H9tgD 37°C yes M. marinum strain M30 7H9tgADS 25°C yes M. shottsii strain M175† 7H9tgADC 25°C yes M. pseudoshottsii strain L15† 7H9tgADC 25°C yes M. avium subspecies avium Serovar #8 7H9tgADC 37°C yes M. avium subspecies paratuberculosis 7H9tgADCm 37°C yes (ATCC#700535) M. fortuitum 7H9tgOADC 37°C yes 7H9tgADS, 7H9tgAS, M. tuberculosis strain Erdman 37°C no Sauton, Sauton† M. tuberculosis strain H37Rv 7H9tgADS 37°C no M. tuberculosis strain H37Ra 7H9tgADS 37°C no M. tuberculosis strain 103 7H9tgADS 37°C no M. tuberculosis strain CDC1551 7H9tgADS 37°C no M. bovis BCG 7H9tgADS, Sauton 37°C no M. microti strain P2 7H9tADS 37°C no

† Cultures were incubated stationary § Bacteria were cultured in Sauton or Middlebrook 7H9 medium with the indicated supplements: t (0.05% Tween 80), g (0.5% glycerol), A (0.5% bovine fraction V albumin), D (0.2% dextrose), S (0.085% NaCl), C (0.0003% catalase), O (0.006% oleic acid), m (mycobactin [2 µg/ml]) * Allowed growth of cobalamin/methionine auxotrophic strain Salmonella typhimurium strain JE212

Table 4.2: Conservation of predicted cobalamin biosynthesis homologs in mycobacteria.

M. tuberculosis M. bovis BCG† M. marinum† M. smegmatis† M. smegmatis* Protein homolog homolog homolog homolog CysG 100% 84% 80% 80% CobIJ 100% 88% 79% 81% CobG 100% 74% 67% 68% CobM 99% 85% 80% 83% CobK 98% 76% 70% 72% CobL (CobLb) 99% 76% 71% 67% CobH 100% 87% 83% 85% CobB 100% 79% 71% 78% CobN 99% 89% 83% 83% CobS 99% 73% 63% 73% CobT 100% 85% 76% 82% CobO 99% 84% 80% 82% CobQ1 100% 86% 50% 51% CobQ2 99% 90% 83% 84% CobD 99% 76% 69% 68% CobC 100% 77% np np CobU 100% 46% 45% 57% np: not present †Percent amino acid identities relative to the M. tuberculosis homologs. *Percent amino acid identities relative to the M. marinum homologs.

Figure 4.1: Chemical structure of cobalamin (vitamin B12). The central cobalt atom is bound to four nitrogen atoms in a planar tetrapyrrole ring. The lower cobalt axial ligand forms a loop with the tetrapyrole ring through a dimethylbenzimidazole ribonucleotide moiety. The upper axial ligand can vary. Heat-stable cyanocobalamin is obtained by addition of a sub-lethal concentration of potassium cyanide to a cobalamin-producing bacterial culture.

Figure 4.2: Bacterial aerobic and anaerobic vitamin B12 biosynthetic pathways. Pseudomonas denitrificans genes responsible for aerobic synthesis steps are italicized, while Salmonella typhimurium genes responsible for anaerobic synthesis are italicized and underlined. Chelatases responsible for insertion of the central cobalt atom are shown in black blocks. Genes with homologs in M. tuberculosis and M. marinum are circled in red. This research was originally published in the Journal of Biological Chemistry [153]. © The American Society for Biochemistry and Molecular Biology.

Figure 4.3: Assaying for the presence of cobalamin in mycobacterial extracts. To assay for cobalamin production in mycobacteria, culture lysate or vitamin B12 was spotted onto Vogel- Bonner minimal medium agar mixed with Salmonella enterica strain JE212 and incubated overnight at 37ᵒC. Strain JE212 has mutations in cobalamin biosynthetic genes and in the metE gene encoding the cobalamin-independent methionine synthase. This methionine auxotroph grows when exogenous cobalamin enables function of the cobalamin-dependent methionine synthase (MetH). Bright spots show growth of strain JE212. Three lysates from independent cultures were made and assayed for each strain. Shown are representative plates for each strain.

Figure 4.4: Assaying for the presence of methionine in mycobacterial extracts. Mycobacterial lysates, methionine, or vitamin B12 was spotted onto Vogel-Bonner minimal medium agar containing Salmonella enterica strain JE7089. Plates were examined for bacterial growth after overnight incubation at 37ᵒC. Strain JE7089 has mutations in cobalamin biosynthetic genes and in both methionine synthase genes rendering the mutant a methionine auxotroph that cannot grow when supplied with vitamin B12. Diffuse bacterial growth was only observed where methionine was spotted. Three lysates from independent cultures were made and assayed for each strain. Shown are representative plates for each strain.

Figure 4.5: Vitamin B12 reverses growth defect. M. tuberculosis strain Erdman was cultured in 7H9tASp with 0.1 mM 3NP (+3NP) or left untreated. Vitamin B12 was added after 300 hr to a final concentration of 10 µg/ml. Growth was monitored by measuring the OD600. Duplicate cultures were assayed under each condition.

Vitamin B12 added

CHAPTER 5

CONCLUSIONS

Mycobacterium tuberculosis is the leading cause of human death by a single infectious bacterial species. Multiple attributes underlie its success as a pathogen. One is the ability to acquire nutrients from the host. For this, tightly-regulated gene expression is required in order to induce and repress genes based on nutritional needs and stresses to minimize wasting energy.

Sigma factors are prokaryotic transcription initiation factors that enable RNA polymerase to direct transcription of genes. Thirteen known sigma factors are present in the M. tuberculosis genome. The only sigma factor known to be essential is the housekeeping sigma factor SigA.

Most other sigma factors are only needed when the bacterium is exposed to certain growth conditions or stresses. While SigC is a known M. tuberculosis virulence determinant in murine and guinea pig infection models, a tractable phenotype associated with mutation of the sigC gene in vitro is lacking and has hindered efforts to define the SigC regulon.

In chapter 3, we reported the identification of a sigC mutant phenotype in vitro. This was the first time an in vitro growth phenotype of a sigC mutant in M. tuberculosis has been described. This phenotype was characterized by the slow growth of a sigC mutant of M. tuberculosis strain Erdman when cultured in chemically-defined Sauton medium which contains glycerol as sole carbon source. Supplementation of this medium with glucose or Tween 80 restored growth to wildtype levels. Additionally, supplementation with valeric acid improved the growth rate of the mutant while causing a delay in growth of strains containing a wildtype sigC

gene. Global gene expression studies of the sigC mutant relative to parent strain Erdman cultured in Sauton medium suggested that while the mutant upregulated various genes including some associated with intermediary metabolism, the parent upregulated an operon thought to be involved in synthesis of surface mycobacterial lipids (PDIM). However, parallel studies in which sigC was artificially induced in the complex medium Middlebrook 7H9 revealed that in addition to the putative lipid biosynthesis operon, transcription of ctpB (encoding a putative cation transporter protein) was also significantly upregulated. This suggested a potential role for SigC in metal transport. After 48 hr sigC induction in the same growth medium, upregulation was also detected for multiple other genes required for detoxification of high copper levels inside the cell.

Comparing the medium components of Sauton and 7H9 media revealed that CuSO4, CaCl2, and

ZnSO4 are lacking in the former. This led to a model in which SigC responds to a deficiency of trace metals, as the zinc requirement for glycerol kinase could explain the growth defect of a sigC mutant with glycerol as the carbon source. Other genes induced in the sigC mutant in

Sauton medium may have been upregulated to replace related enzymes requiring other limiting metals. The presence of an uncharacterized polyketide synthetase within the putative PDIM- associated operon upregulated by SigC suggested that it may synthesize a metal-scavenging system. The intriguing question is what metals this scavenging system is acquiring, and if it might be specific to more than one metal. Mycobacterium tuberculosis has long been known to make another polyketide, mycobactin, an iron siderophore. In support of the metal uptake hypothesis, long-term (48 hr) sigC induction in metal-rich 7H9 medium resulted in upregulation of copper detoxification genes, suggesting that excess amounts of this metal was brought inside the cell. The acquisition of metals is often a battle between the bacteria and the host. Bacteria need small amounts of metals for growth, which the host tries to withhold to prevent growth of

the pathogen. Therefore, mycobacteria utilize molecules such as mycobactin to scavenge the required metals from the host. If SigC regulates the synthesis of a mycobactin-like molecule, this has yet to be shown definitively. Measurements of the copper, zinc, and calcium concentrations inside the bacterial cells under the different growth conditions utilized in chapter 3 (Sauton and

7H9 medium) would help to answer this question. When performing in vitro studies, metals are often present in either the bacterial or mammalian growth media. That could explain why a phenotype has previously not been described when M. tuberculosis was grown in macrophages.

Inside the host however, the availability of metals is much more restricted, which could explain the attenuation of a sigC mutant in mouse and guinea pig infection models. To further address the question if the nrp operon encodes a metal-scavenging system, a mutant in M. tuberculosis, lacking one or more genes of the nrp operon could be created. It would be expected, that the nrp mutant would exhibit a similar growth defect in Sauton medium as the sigC mutant. This predicted metal scavenger, encoded by the nrp operon, represents a novel target for both drug and vaccine development.

Chapter 4 of this dissertation examines cobalamin biosynthesis in mycobacteria. Our interest in this cofactor began because the sigC gene is located in the genome amongst numerous cobalamin biosynthetic genes. The sigC gene is monocistronic and divergently-transcribed from blaC, encoding the major β-lactamase of M. tuberculosis. Cobalamins, such as vitamin B12, play a role as cofactor in enzymes that catalyze isomerization and reduction reactions in protein,

DNA, and carbohydrate metabolism. Cobalamin has a central cobalt atom required for the stability of the molecule. This cation binds to the upper and lower ligand. The metal specificity of the SigC-regulated metal-scavenging system is currently unknown. Because of the genomic location of sigC, it is possible that the nrp operon, regulated by SigC, is involved in cobalt

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acquisition. This scavenging system might have a broader specificity, transporting multiple different cations. As cobalamin biosynthetic genes are conserved in multiple Mycobacterium species, we hypothesized that cobalamins are required for survival in the host. Studies over fifty years ago reported vitamin B12 synthesis by M. tuberculosis and other mycobacteria, but recent studies reported that vitamin B12 is not made by M. tuberculosis. Therefore, we re-examined vitamin B12 synthesis in mycobacteria. For this purpose, we utilized an auxotrophic mutant of

Salmonella enterica subspecies enterica, serovar Typhimurium. This mutant is unable to synthesize methionine, due to mutations in the vitamin B12 independent MetE synthase, and therefore unable to grow, when vitamin B12 is not added exogenously. Multiple mycobacterial species were tested, and growth of the cobalamin auxotroph was detected with extracts from M. smegmatis, M. marinum, M. fortuitum, M. avium subspecies avium, M. avium subspecies paratuberculosis, M. shottsii, and M. pseudoshottsii. Growth was not observed using extracts from M. microti, M. bovis BCG, or any of five strains of M. tuberculosis tested. Interestingly, all mycobacteria positive for cobalamin production are environmental mycobacteria, while all strains negative for cobalamin production are members of the Mycobacterium tuberculosis complex (MTC). This complex comprises closely related mycobacteria that cause tuberculosis in mammalian hosts. Because of the presence of the abundance of cobalamin genes in M. tuberculosis, it seems likely that the pathogen induces these genes only under specific conditions that mimic those present inside the host. In contrast, environmental mycobacteria regulate cobalamin synthesis differently. Alternatively, it is possible that the cobalamins, synthesized by

MTC mycobacteria, are not a form that can be utilized by the Salmonella auxotroph. It has been shown that the lower ligand of cobalamin can vary, depending on the nature of the microorganism. Bioinformatic comparisons of predicted cobalamin biosynthetic genes among

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MTC and environmental mycobacteria showed that genes involved in addition of the lower cobalt ligand were dramatically different between MTC and environmental mycobacteria, supporting the hypothesis that different forms of vitamin B12 are being synthesized. Targeting cobalamin synthesis could be a novel and promising approach for drug or vaccine development.

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