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12-2006

Molecular Screening Of Azoreductase Gene and ItsActivity in Human Intestinal BacteriaSyndia S. Todd

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Recommended CitationTodd, Syndia S., "Molecular Screening Of Azoreductase Gene and Its Activity in Human Intestinal Bacteria" (2006). McCabe ThesisCollection. Paper 37.

The Edwin P. McCabe Honors Program

Senior Thesis

"MOLT,CV£Jl<̂ S(̂ (ENINg 0<F AZO<̂ CDVCMS<E gENEJWD ITS

mCFEQUA*

Syndia S. Todd

December 2006

Langston University Langston, Oklahoma

1

Mo CecuCar Screening Of JLzorecfuctase Cjene J4ncf Its Activity In Human Intestinal (Bacteria

SyncfiaS. rTocfcf Biology Major

School of Arts and Sciences

Submitted in partial fulfillment of the requirements of the E.P. McCabe Honors Program

Fall 2006

iMoCecuCar Screening OfSLzorecCucta.se Cjene J/LncC Its Activity In Human IntestinaC(Bacteria

By

Syndia S. Todd

Thesis approved by:

Thesis ̂ Committee Chairperson

Thesis Committee Member

Director of the Honors Program

Vice President of Academic Affairs

3

Acknowledgments

I would like to start of by thanking my Lord and savior Jesus Christ because

without him nothing is impossible. Second, I want to thank my family and

friends for all the support and love they have given my through all these

years. I would like to give a special thank you to Dr. Sonya Williams without

her help and determination I would probably not be in college receiving all

these great opportunities. Also, special thanks to all the programs and

scholarships that accepted me and helped me with funding my education and

research. Those programs include UBEP (Undergraduate Biomedical

Education Program),LINC (Langston's Integrated Network College), Regents

scholarship, and KU-INBRE. I also want to thank Ms. Irene for always

staying on me to do the best I can.

I definitely want to give special thanks to my mentor Dr. K.J. Abraham for

allowing me to be part of his lab and research. Dr. Abraham went beyond his

duty of a mentor. I also appreciate him for taking on the task of being the

chair person of my committee. Last but not least, I would like to thank

Curtisia Battle for all her help and making my experience in and outside of

Dr. Abrahams lab the best.

4

Table of Contents

Chapter I. Introduction

1. Introduction

2. Background

3. Statement of the problem/topic

4. Purpose of the study

5. Assumption

6. Rationale for Study

7. Definition

Chapter II. Literature Review

1. Organization of Chapter 2

2. Historical background of the topic

3. Summary of existing studies

4. Significant Results

Chapter III. Methodology

1. Organization Chapter III

2. Preparation of media

3. Azoreductase Activity

5

4. Genomic DNA Extraction

5. Agarose gel electrophoresis

6. Ploymersae Chain Reaction PCR

7. Restriction Digestion

Chapter IV. Analysis or Findings

1. Figures

2. Tables

3. Summary

Chapter V. Summary, Conclusions

1. Brief Summary

2. Conclusions

6

Chapter 1: Introduction

Introduction

Xeniobotics are foreign chemicals that are man made or of

natural origin. These chemicals include pesticides, animals, toxins, soil

and water pollutants, food additives and drugs. In many cases,

xeniobotics are metabolized into carcinogens, mutagens, and tumor

promoting agents. Azo dyes and nitrated polycyclic aromatic

hydrocarbons (nitro-PAHs) are two groups of chemicals that are

abundant in our environment.

The species of bacteria capable of reducing azo dyes and nitro-

PAHs from the human intestinal microflora or other sources can be

identified by plating serial dilutions of human feces on brain-heart

infusion agar containing 1-nitropyrene or an azo dye such as Direct

Blue 15. Bacterial colonies with azoreductase and nitroreductase

activities reduce the dye or 1-nitropyreneon on the plate and are

recognized by the appearance of clear zones around the colonies.

Although several azoreductase- and nitroreductase - producing

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bacteria are present in the human intestinal tract, the majority belong

to the genera Clostridium and Eubacterium. Predominant anaerobic

bacteria with azoreductase and nitroreductase activity found in the

human intestinal tract include Clostridium leptum, Eubacterium sp.( C.

dostridiiforme, C. paraputrificum, Clostridium sp., and C. perfringens

(Cerniglia, Rafii, 1995). The aim of this research work was to study the

activity of azoreducatse gene in Citrobacter freundii and isolate the

azroreducatse gene.

Background of the Topic

Azo dye compounds represent a large group of chemicals which

are extensively used in the textile, pharmaceutical, food, and cosmetic

industries. It is one of the largest and most versatile dyes. They are

generally considered to be xenobiotic compounds which are rather

recalcitrant against biodegradative processes in conventional sewage

treatment systems. Nevertheless, during the last few years it has

been demonstrated that several microorganisms are able to transform

azo dyes to noncolored products or even mineralize them completely

8

under certain environmental conditions ( Cerniglia et. al.( 1990).

Statement of the Problem

The intestinal microflora are capable of performing a wide

variety of metabolic transformations. "The intestinal bacteria can

enhance the function of the entire gastrointestinal tract, protect

against pathogenic, maintain the vital chemical balance of the

gastrointestinal system, and produce needed vitamins and hormones"

(http;//www.upwardquest.com/crit2.html). Some of the products of this

metabolism have been associated with carcinogenic processes, such as

cancer, tumor formation, gastrointestinal disease, and infections. The

ability of human intestinal microbes to interact with metabolites

directly or after recirculation may contribute toward different

toxicological disorders and disease.

Purpose of the Study

The purpose of this study is to characterize and isolate the

azoreductase gene in Citrobacter freundii.

Assumption

My hypothesis is that C. freundii possess azoreducatse enzyme

and can influence hepatic P450 enzymes associated with toxicity and

disease.

Rationale for Study

The human intestine is host to food remnants and microbial

organisms, also know as the intestinal microflora, from which the body

derives nourishment and against which the body must be protected.

The first catabolic step in the reduction of azo dyes, which is

accompanied by a decrease in the visible light absorbance of the dye

and the decolonization of the dye, is the reduction of the azo bridge

to produce aromatic amines. These aromatic amines are also known as

human carcinogens. There is a relationship between the intestinal

microflora and metabolic reactions leading to the transformation of

drugs and the production of mutagenic or carcinogenic compounds.

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Definition

The bacterium (Citrobacter freundii) is a gram-negative, rod-

shaped bacterium found in man and other animals including mammals,

birds, reptiles, and amphibians. Its organisms have also been isolated

from soil and water as well as from clinical specimens such as urine,

throat, sputum, blood, and wound swabs as an opportunistic pathogen

(http ;//vm.cfsan.fda.gov/~mow/chap20.html).

Azo dyes are dyes characterized by the presence of one or more

azo groups (Cerniglia, et. al., 1995). More than half of the annually

produced dyes are azo dyes (Blumel et. al., 2002)

Azoreductase is an enzyme capable of transforming azo dyes.

A Gene is a basic unit of heredity.

Genome is the total set of genes carried by an individual or cell.

Chapter 2

Organization Of Chapter 2

This chapter includes a historical background of the work on

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azoreductase enzymes and its presence in human intestinal bacteria,

recent findings, its impact on human health and significant reports.

Historical Background

The history of azo compound goes back to over a hundred years.

William Henry Perkins synthesized the first dye, mauve, in 1856 from

chemicals derived from coal. W.H. Perkins commercialized his

innovation and developed the production process for this new dye.

During manufacturing and usage of azo dyes an estimated amount of

10-15% is released into the environment (Vaidya and Datye, 1982).

Existing Studies

Azoreductase activity of anaerobic bacteria isolated from human

intestinal bacteria was reported by Rafii and Cernigial (1990). Their

results showed azoreductase activity in ten strains of anaerobic

bacteria. Several researchers (Brown, 1981; Chung et. al., 1978, Dubin,

and Wright, 1975) have shown reduction of azo dyes by bacteria.

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Significant results

Azo dye compounds are linked to bladder cancer in humans,

hepatocarcinoma, and to nuclear anomalies in intestinal epithelial cells

in mice (Manning et. al., 1985). A number of azo dyes have been

classified as carcinogenic (Hartman et. al., 1978). All the azo dyes

containing a nitro group were found to be mutagenic (Chung and

Cernigilia, 1992). Furthermore, azo dyes can cause toxic degradation

products.

Chapter 3: Methodology

Preparation of media

The composition of Brain Heart Infusion (BHI) included 425ml of

distilled water, Brain Heart Infusion Broth (18.5g),Yeast Exatract

(5g), Salt Solution 1 (12.5ml), Salt Solution 2 (12.5ml), Calcium Chloride

Solution (12.5ml), Hemin Solution 2ml, Sodium Carbonate (0.2g), and

Cyestine-HCL (0.3g).

13

Composition of Nutrient Agar (NA) included 1 liter distilled water, 8g

of Nutrient broth, and 15g Agar. Colonies of Citrobacter freundii

were maintained on Nutrient Agar medium. BHI was used for liquid

cultures to test for azoreductase activity.

Azoreductase activity

To test the azoreductase activity, different concentrations of azo

dyes (10, 20 and 30^/M of Direct blue 15) were tested for

azoreductase activity and discolorization with C. freundii. Spectronic-

20 was used to measure absorbance at 30 minutes intervals for 3

hours.

Genomic DNA extraction

After testing C. freundii for azoreductase activity , the genomic DNA of

the bacterium was extracted and isolated. During this procedure 100ml

of bacterial cultures, incubated overnight at 37°C was centrifuged for 20

minutes at 6000 rpm. The pellet was resuspended in 9.5 ml of Tris-

Ethylene Diamine Tetra Acetic Acid Buffer (TE buffer). 50|d of

Lysozyme was added, and kept in room temperature for 30 minutes. Next,

14

0.5ml of 10% SDS and 40(al of 20 mg/ml protenase K and 50|J RNAse

(20mg/ml) were added, mixed and incubated at 37° C for 1 hour. 1.8ml of

5M NaCI was then added and mixed thoroughly. After mixing, 1.5ml of

CTAB/NaCI solution was added, mixed, and incubated for 20 minutes at

65° C. After incubation it was extracted with equal volume of

chloroform/isoamyl alcohol (24:1) and centrifuged for 20 minutes, at

6000 rpm, in room temperature. The aqueous phase was transfered to a

fresh tube to precipitate the DNA with 0.6 volume of isopropanol. It was

centrifuged at 6000 rpm for 15 minutes at room temperature. The

precipitate was washed with 70% ethanol, and centrifuged for another 15

minutes at 6000 rpm. The supernatant was removed and the pellet

suspended in 1 ml of TE Buffer. The DNA was stored at -20° C.

DNA measurement

DNA measurement was done to check the purity and the total amount of

the genomic DNA of C. freundii. The measurement was taken at two

different wavelengths (260 and 280nm) and the ratio at 260/280 nm was

calculated. The reading for C. freundii was 1.966 ug/ml.

15

Agarose gel electrophoresis

To ensure that the C. freundii DNA was successfully extracted, gel

electrophoresis was performed. An agarose gel (0.8%) was prepared for

electrophoresis. Genomic DNA (O.l^g) was loaded on to the gel along with

a DNA marker. The gel was stained with ethidium bromide and observed

on a UV Transilluminator

Polymerase Chain Reaction (PCR)

Oligonucleotides were custom synthesized to characterize genes from

Enterococcus faecalis and used as primers. PCR mixture (100|al) for the

amplification of genomic DNA contained 0.1 :g of genomic templates, 0.5|il

of Taq DNA polymerase (5u/jnl), 10 pmol of each primer, lOmM

concentration of each deoxynucleotides triphophate, 25mM MgCI2 and

the corresponding reaction buffer was used for PCR. Electrophoresis

with PCR products showed no fragments of expected size.

Restriction digestion

Restriction digestion of genomic DNA was followed to clone fragments

using a cloning vector. Restriction enzymes, Bam\-\, Hind III, and EcoRT

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were used to perform digestion of genomic DNA at 37° C.

Chapter 4: Analysis and Findings

Figurere 1: Colonies of Citrobacter freundii

Colonies of Citrobacter freundii were maintained onNutrient Agar

medium. BHI was used for liquid culture to test azoreductase activity.

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Figure 2: Gram-negative Citrobacter freundii

C. freundii possess azoreducatse enzyme and can influence hepatic

P450 enzymes associated with toxicity and disease.

Figure 3: Agarose gel showing genomic DNA of Citrobacter freundii

The gel was stained with ethidium bromide and observed on a UV

Transilluminator

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Absorbance

Samples Time (min)

30 60 90 120 150 180

NC (dye) 1.015 0.995 1.020 0.598 0.672 0.672

PC (cit) 0.065 0.072 0.094 0.102 0.161 0.201

Cit and Dye 0.722 0.732 0.766 0.702 0.905 0.990

Cit and Dye 0.734 0.736 0.774 0.700 0.900 0.905

Cit and Dye Table 1

0.672 0.682 0.694 0.658 0.825 0.865

10/vm Direct blue dye added to liquid culture and Citrobacter freundii

Absorbance

Samples

30

NC (dye) 1.045

PC (cit) 0.006

Cit and Dye 1.050

Cit and Dye 0.900

Cit and Dye 0.875 Table 2

20/vm Direct blue dye ac

Time (min)

60 90

1.040 1.005

0.078 0.088

1.100 1.010

0.905 0.865

0.870 0.830

d to liquid culture and

120 150 180

1.005 1.010 1.100

0.105 0.129 0.155

1.045 1.015 1.030

0.865 0.895 0.895

0.825 0.855 0.870

Citrobacter freundii

20

Absorbance

Samples Time (min)

30 60 90 120 150 180

NC (dye) 0.446 0.452 0.448 0.452 0.456 0.682

PC (cit) 0.015 0.018 0.021 0.026 0.046 0.137

Cit and Dye 0.488 0.486 0.480 0.484 0.488 0.744

Cit and Dye 0.468 0.466 0.466 0.468 0.474 0.718

Cit and Dye Table 3

0.506 0.504 0.500 0.504 0.510 0.784

30fjm Direct blue dye added to liquid culture and Citrobacter freundii

Decolonization of Blue 15 dye (lOmM) by C. freundii indicated the

presence of azoreducatse enzyme. The larger the amount of dye the

longer it took for the Citrobacter freundii to decolorize the Blue 15

dye

DNA/ Protein measurement

The concentration of genomic DNA was measured with a UV

21

Spectrophotometer

No Items Results Unit

1 A1 .257 Abs

A2 . 155 Abs

A3 . 051 Abs

C-DNA 9.193 ug/ml

C-Pro 6.647 ug/ml

Ratio 1.966 ug/ml

The ratio for protein measurement should range from 1.5 to 2. Our

protein ratio was 1.966

Chapter 5: Summary and Conclusion

Brief Summary

Genomic DNA was extracted from small bacterial cultures by treating

it with lysozyme or Mutanolysin. The resulting DNA was screened by

22

agarose gel electrophoreses. PCR analysis and restriction endonuclease

digestion was followed for isolating the azoreducatse gene.

Conclusion

• Decolonization of Azo Dye (Direct blue 15)10 - 20/jM

concentrated by Citrobacter freundii.

• Biotransformation of azo dye by Citrobacter freundii indicated

presence of azoreducatse gene.

Isolation of the azoreducatse gene with PCR, restriction

endonucleases, electrophoresis, cloning and transformation are in

progress.

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Work Cited

1. Blumel, S., Knackmuss Hans-Joachim, Stolz Andreas. 2002.

American Society for Microbiology, vol. 68, 3948-3955

2. Brown, J.P. 1981. Reduction of polymeric azo and nitro dyes

intestinal bacteria. Appl. Environ. Microbiol.41:1283-128

3. Cerniglia, C.E., and R. Fatemah. 1995. Environmental Health Issues,

Vol. 103.

4. Cerniglia, C.E., R. Fatemah, Franklin, Wirt. 1990. Azoreductase

Activity of Anaerobic Bacterial Isolated from Human Intestinal

Microflora,_vol. 56, 2146 -2151

5. Chung, K.T, 5.E. Stevens, and Cerniglia. 1992. The reduction of azo

dyes by the intestinal microflora. Crit. Rev. Microbiol.18:175-190

6. Dubin, P., and K.L. Wright. 1975. Reduction of azo food dyes in

cultures of Proteus Vulgaris. Xeniobiotica 5:563-571

7. Hartman, C. P., G. E. Fulk, and A. W. Andrews. 1978. Azo reduction

of intestinal anaerobe. Mutat. Res. 58:125-132

8. Manning, B. W., C. E. Cerniglia, and T. W. Federle. 1985. Metabolismof