Members of the genus Streptomyces are filamentous and spore forming bacteria that live in soil and aqueous environments. Within these environments, Streptomyces as well as other bacteria play a large role in their environment through their ability to interact by producing various chemical signals. To understand what type of chemical signals bacteria produce in these environments and how their signals affects other bacteria, we have established a model system using two soil bacterium: Streptomyces sp. Mg1 (S. Mg1) and Bacillus subtilis. When wild-type S. Mg1 and B. subtilis are cultured together on an agar surface, noticeable lysis and degradation of the B. subtilis colony is observed. We propose a transposon mutagenesis to identify genes in S. Mg1 related to its ability to lyse and degrade B. subtilis colonies

Patel, Roosheel

Texas A&amp;M Repository

IN VIVO TRANSPOSON MUTAGENESIS OF STREPTOMYCES SP. MG1   An Undergraduate Research Scholars Thesis by ROOSHEEL SANDEEP PATEL   Submitted to Honors and Undergraduate Research Texas A&M University In partial fulfillment of the requirements as an   UNDERGRADUATE RESEARCH SCHOLAR   Approved by Research Advisor:        Dr. Paul Straight   May 2014   Major: Biochemistry    TABLE OF CONTENTS                                                  Page ABSTRACT .............................................................................................................................  1 ACKNOWLEDGMENTS ........................................................................................................ 2 CHAPTER      I INTRODUCTION ...................................................................................... 3  II METHODS ................................................................................................. 5   Enzymatic Assembly of pTSR001 .............................................................. 6   Accumulation of pTSR001 ......................................................................... 6   Introduction of pTSR001 to Streptomyces sp. Mg1 .................................... 6  III RESULTS AND DISCUSSION ................................................................. 8  IV CONCLUSION ........................................................................................... 9   Establishment of transposon mutagenesis system ...................................... 9   Screening and identification of non-lytic mutants ...................................... 9   Future endeavours ....................................................................................... 9   Summary ..................................................................................................... 10 REFERENCES .................................................................................................................12          `           1  ABSTRACT  In Vivo Transposon Mutagenesis of Streptomyces sp. Mg1. (May 2014)  Roosheel Sandeep Patel Department of Biochemistry and Biophysics Texas A&M University   Research Advisor: Dr. Paul Straight Department of Biochemistry and Biophysics  Members of the genus Streptomyces are filamentous and spore forming bacteria that live in soil and aqueous environments. Within these environments, Streptomyces as well as other bacteria play a large role in their environment through their ability to interact by producing various chemical signals. To understand what type of chemical signals bacteria produce in these environments and how their signals affects other bacteria, we have established a model system using two soil bacterium: Streptomyces sp. Mg1 (S. Mg1) and Bacillus subtilis. When wild-type S. Mg1 and B. subtilis are cultured together on an agar surface, noticeable lysis and degradation of the B. subtilis colony is observed. We propose a transposon mutagenesis to identify genes in S. Mg1 related to its ability to lyse and degrade B. subtilis colonies.  2 	  ACKNOWLEGMENTS  I would like to thank Dr. Paul Straight for finding me space in his lab and his guidance through my research experience. Furthermore, I would also like to thank Reed Stubbendieck for his continuous guidance through both the research and writing portions of this thesis and my research experience. 3 	  CHAPTER I INTRODUCTION  Bacteria are all around us. As human beings, it is estimated that bacteria outnumber the total number of cells in our bodies by a factor of 10 to 1 (1). Furthermore without these bacteria to carry out vital functions in our bodies, our physiology would be compromised (1). These bacteria survive in an environments where they constantly compete for nutrients and space. To better aid themselves, some bacteria produce secreted products to compete against other bacteria. We know some of these products as secondary metabolites. Additionally, bacteria can also face nutrient poor environments and may undergo transition to dormant states for long term survival, such as sporulation. These spores are dormant cells which upon encountering favorable conditions can germinate and propagate their lineage Streptomyces carries out a large secondary metabolism that allows it to produce typically at least twenty secondary metabolites per strain (2). Within the interaction between Streptomyces sp. Mg1 (S. Mg1) and B. subtilis, we hypothesize that S. Mg1 secretes a molecule that actively lyses cells and degrades the colonies of B. subtilis. The S. Mg1 genome is approximately 8.7 megabases and we estimate it contains around 8700 genes. We hypothesize various gene products working together may be responsible for the ability of S. Mg1 to lyse B. subtilis.  To identify the genes encoding these products, we use transposon mutagenesis to create mutants of S. Mg1 which will be screened in search of non-lytic inducing colonies. A transposon is a piece of DNA that has the ability to change its position within a genome, randomly.  Transposon mutagenesis, when combined with a genetic screen, is a forward genetic approach that we use to identify genes that are responsible for particular phenotypical characteristics. To carry such an 4 	  experiment out, we introduce a non-native transposon to S. Mg1. The introduction of this transposon is facilitated via a plasmid contain the respective transposon. pHTM, shown in Fig. S1 is a plasmid that contains a thiostrepton-inducible Himar1 transposon, which contains two antibiotic markers to test for transfer of the plasmid during transformations and conjugation, as well as identification of mutagenized cells. pHTM has been previously used in successfully mutagenize Streptomyces albus and Streptomyces coelicolor (3). Then through a screen that I have designed, we will be able to screen for non-lytic mutants. As there are many various bacterial interactions in the environment, it is vital to be identifying mutants of the correct interaction as many interactions may result in observation of similar phenotypes. For example, in my project, even though the interaction of growth inhibition and the interaction for cell lysis share a very similar phenotype, the biochemical pathways are highly different. The adjustments to my screen have been appropriated to fit these conditions, such that the interaction that will be visible will be that of cell lysis. Through identification of non-lytic mutants we can identify genes responsible for the lytic activity and gain knowledge that will allow us to identify biological components underlying this interaction.         5 	  CHAPTER II METHODS  The bacterial strains and plasmids that were used in this experiment are shown in Table 1. Table 1. Table of strains and plasmids used 	  To effectively use the transposon mutagenesis as a method to introduce mutations in the S. Mg1 genome, it was vital to test the sensitivity of S. Mg1 to the chemical inducer, thiostrepton. We found that wild type S. Mg1 was extremely sensitive to thiostrepton (Figure 1: Picture of Thiostrepton sensitivity). Therefore to carry out further experiments, it is necessary to introduce thiostrepton resistance to S. Mg1. Strains Relevance         Streptomyces sp. Mg1 Native isolated strain of Streptomyces that shows lytic degradative activity of Bacillus subtilis         E. coli JV36 Used to perform transfer of pTSR001 and pHTM plasmid into S. Mg1 through conjugation         E. coli DH5α Used to obtain greater quantity of pTSR001 plasmid         Bacillus subtilis 3610 Wild type; used to screen for non-lytic mutants of S. Mg1 Plasmids         pDR183 Contains the bla gene; provides ampicillin resistance        pHZ1358 Contains the tsr gene; provides thiostrepton resistance        pSET152 Plasmid used as the backbone for the construction of pTSR001        pHTM Contains the thiostrepton inducible Himar1 transposon with the antibiotic resistant markers of apramycin and hygromycin        pTSR001 Plasmid constructed in this paper; contains pSET152 backbone with antibiotic resistance genes of ampicillin (bla) and thiostrepton (tsr) 6 	  Enzymatic assembly of plasmid pTSR001 conferring Thiostrepton resistance Plasmid maps of pDR183, pHZ1358 and pSET152 were analyzed to design pTSR001, a plasmid that confers resistance to ampicillin and thiostrepton. Primers were designed to amplify gene products of plasmids pDR183 (bla gene), pHZ1358 (tsr gene) and pSET152 (backbone). Primers were also designed to include overhanging regions of homology between the resulting polymerase chain reaction (PCR) products. Resulting fragments were then assembled into pTSR001, using the Gibson assembly protocol. The assembly of pTSR001 was confirmed via gel electrophoresis (Figure 2).  Accumulation of plasmid pTSR001 Assembled plasmid, pTSR001 was transferred into chemically competent cells of a conjugative strain Escherichia coli DH5α.  Transformation of plasmid was confirmed by selection on antibiotic resistance marker encoded by the plasmid on Luria-Bertani agar (LB: 10 g/L tryptone, 5 g/L yeast extract and 5 g/L salt) with the addition of ampicillin at 100 µg/mL. A single surviving colony was picked and grown in an overnight liquid culture of LB with the addition of ampicillin at 100 µg/mL. Resulting culture was concentrated and a plasmid purification procedure (5) was conducted to increase the amount of plasmid pTSR001 available for further experimentation. Introduction of TSR001 to Streptomyces sp. Mg1 The plasmid, pTSR001 was transferred into chemically competent cells of a conjugative strain Escherichia coli JV36. Plasmid transfer was confirmed by selection on antibiotic resistance marker encoded by the plasmid. Single transformed colony was picked and grown in LB liquid culture under selection of ampicillin (100 µg/mL). Resulting culture was diluted to 1:10. 100 µL of 108 spores/mL of S. Mg1 was intergenically conjugated with 2 µL of the diluted overnight 7 	  culture of E. coli JV36 pTSR001 plated on AS-1 agar at 30 °C. Plates were flooded with antibiotics of ampicillin (100 µg/mL) and thiostrepton (50 µg/mL), 15 hours later to select for S. Mg1 exconjugates that contain the plasmid pTSR001. Exconjugants were passaged onto 30 mL agar plates of maltose-yeast extract-malt extract (MYM: 4 g/L maltose, 4 g/L yeast extract, and 4 g/L malt extract) with the addition of the antibiotics, naladixic acid at 30 µg/mL, ampicillin at 100 µg/mL and thiostrepton at 50 µg/mL, to confirm transfer of pTSR001 into S. Mg1 and to inhibit further growth of the donor E. coli. Resulting growth of the S. Mg1 exconjugants containing the plasmid were concentrated and sporulated on MYM plates. The collected spore stock of S. Mg1 pHTM was stored at 4 °C.              8 	  CHAPTER III RESULTS  If successful in randomly mutating the S. Mg1 genome and isolation of non-lytic mutants, inverse PCR experiments would be completed and insertion loci would be identified. Through experiments done by previous members in the lab, the expression of the S. Mg1- pk1 gene cluster constitutes a large role in the production of the LDA conferring metabolite. The genes clusters that might be found through the transposon mutagenesis project could be other genes related in the biosynthesis of LDA or genes encoding transporter proteins aiding in the secretion of LDA metabolite in the surrounding media. These newly discovered genes may or may not be linked to the S. Mg1- pk1 gene cluster, thus identification of these genes may also result in the discovery of profound anabolic pathways that can be pursued as areas of further research in understanding the interactions between S. Mg1 and B. subtilis.  In addition to performing the transposon mutagenesis within the S. Mg1 genome and isolating mutants, the act of transposon mutagenesis itself is an accomplishment. The establishment of a viable transposon mutagenesis system in S. Mg1 in combination with a specific phenotypic screen will allow members of the lab, as well as other labs, to select for mutants of interest in the S. Mg1 genome. By doing so, we will be able to appropriate gene functions to genes scattered through the S. Mg1 genome, further elucidating how S. Mg1 interacts with its environment.    9 	   CHAPTER IV CONCLUSION Bacteria produce small molecules to interact with their environments. In the interaction between Streptomyces sp. Mg1 and Bacillus subtilis, we hypothesize that S. Mg1 produces some molecule that causes the degradation of B. subtilis. To study the lytic ability of S. Mg1, we use a genetic approach of transposon mutagenesis in conjunction with an appropriate screen, to understand this chemically mediated phenotype.  Establishment of transposon mutagenesis system in Streptomyces sp. Mg1 The purpose of the transposon mutagenesis is to randomly introduce mutations in S. Mg1 and effectively generate a library of mutants. There are many advantages to using transposon mutagenesis to generate mutants over other approaches, such as spontaneous mutation. We use a nonreplicative transposon contained on pHTM to conduct the transposon mutagenesis. In theory, the nonreplicative transposon will allow us to introduce a single mutation within one S. Mg1 colony and if the mutagenesis is random, each of these mutants should contain changes within their DNA different from the other. Another advantage is the sequence of the transposable element is known, thus identifying the location loci of the mutation is much easier. Through the establishment of this mutagenesis system, I, as well as future members of the lab, will be able to study any genes of interest in the S. Mg1 genome. Screening and identification of non-lytic inducing mutants The transposon mutagenesis protocol will effectively enable me to generate a library of S. Mg1 mutants. To be able to identify mutants of interest, we make use the genetic tool of a screen. Through a screen that I have designed a screen to identify non-lytic S. Mg1 mutants, I can effectively study genes responsible for the lytic activity of S. Mg1. Two components are needed 10 	  for an effective screen. The first is to be able to isolate single colonies of non-lytic S. Mg1 as lysing colonies produce halos that encompass larger area than the colony itself and will overshadow the non-lytic phenotype. However if a too low density of S. Mg1 mutants are plated, the length of the screening step may be doubled or tripled, therefore an optimal density of mutants must be plated to allow for efficient and accurate screening. By using wild type S. Mg1 and a mutant of S. Mg1 unable to lyse B. subtilis, I have determined the optimal density to be around 50 to 60 mutants a plate. This concentration will allow me clearly to isolate and identify the ability of single S. Mg1 colony to lyse B. subtilis. The second component of the screen is to make sure that the density of B. subtilis in the agar overlay will be able to identify non-lytic/lytic mutants. This component is vital to make the difference between lysis and growth inhibition. The phenotype the lab observes when plating S. Mg1 with B. subtilis on an agar surface, is of lysis. To replicate these conditions within a screen, B. subtilis must be grown to a lawning concentration before interacting with S. Mg1. If a too low density of B. subtilis is plated, the screens may result in false positives, however if a too high density is plated, then the B. subtilis cells might overrun S. Mg1 by greatly outnumbering them. Therefore an optimal density should not only be obtained for S. Mg1 mutants plated, but also for the amount of B. subtilis overlayed on top. I have determined that the optimal concentration of B. subtilis used for an overlay, is at a cell culture OD of .8 to .9. Future endeavours Currently, I have successfully given thiostrepton resistance to S. Mg1 through conjugative experiments. I will perform conjugation experiments to introduce the nonreplicative transposon harbored by pHTM to the S. Mg1 pTSR001 cells. Then I will induce the transposon to mutate    S. Mg1. Next, I will apply the screen I have developed to isolate non-lytic colonies. Then I plan 11 	  to take the non-lytic colonies and perform an inverse PCR protocol to identify the insertion loci of the transposon, thus determining the genes responsible for lytic activity. These genes will be areas of further research to characterize the pathway of the lytic molecule’s synthesis and activity. Conclusion By understanding the function of small molecules in microbial interactions, we may not only better understand our environment, but also learn how to predict and control the outcome of these interactions as well. While in this thesis, I focus on the genetic aspects of the interaction; many other approaches can also be used to study the lytic activity of S. Mg1 such as chemical analysis of the small molecule released, or the molecule’s interaction with B. subtilis. Through the study of this molecule’s production, release and interaction with other organisms, downstream applications may include aiding rationale drug design by discovering new targeting pathways of these therapeutics.          12 	  REFERENCES 1. O’Hara AM, Shanahan F. 2006. The gut flora as a forgetten organ. EMBO. 7(7): 688-693. 2. Procopio RE, Silva IR, Martins MK, Azevedo JL, Araujo JM. 2012. Antibiotics produced by Streptomyces. Science. 16(5): 466-471 3. Bilyk B, Weber S, Myronovskyi M, Bilyk O, Petzke L, Luzhetskyy A. 2013. In vivo random mutagenesis of streptomycetes using mariner-based transposon Himar1. Appl Microbiol Biotechnol. 97:351-359  	  	  	  	  	  	  	  	  	  	  	  	  	  	   

In vivo transposon mutagenesis of Streptomyces sp. Mg1

Texas A&M University

IN VIVO TRANSPOSON MUTAGENESIS OF STREPTOMYCES SP. MG1 
 
 
An Undergraduate Research Scholars Thesis 
by 
ROOSHEEL SANDEEP PATEL 
 
 
Submitted to Honors and Undergraduate Research 
Texas A&M University 
In partial fulfillment of the requirements as an 
 
 
UNDERGRADUATE RESEARCH SCHOLAR 
 
 
Approved by 
Research Advisor:        Dr. Paul Straight 
 
 
May 2014 
 
 
Major: Biochemistry 
 
 
 
TABLE OF CONTENTS 
                                                 Page 
ABSTRACT .............................................................................................................................  1 
ACKNOWLEDGMENTS ........................................................................................................ 2 
CHAPTER     
 I INTRODUCTION ...................................................................................... 3 
 II METHODS ................................................................................................. 5 
  Enzymatic Assembly of pTSR001 .............................................................. 6 
  Accumulation of pTSR001 ......................................................................... 6 
  Introduction of pTSR001 to Streptomyces sp. Mg1 .................................... 6 
 III RESULTS AND DISCUSSION ................................................................. 8 
 IV CONCLUSION ........................................................................................... 9 
  Establishment of transposon mutagenesis system ...................................... 9 
  Screening and identification of non-lytic mutants ...................................... 9 
  Future endeavours ....................................................................................... 9 
  Summary ..................................................................................................... 10 
REFERENCES .................................................................................................................12 
         `     
 
 
 
 
 
 
1 
 
ABSTRACT 
 
In Vivo Transposon Mutagenesis of Streptomyces sp. Mg1. (May 2014) 
 
Roosheel Sandeep Patel 
Department of Biochemistry and Biophysics 
Texas A&M University 
  
Research Advisor: Dr. Paul Straight 
Department of Biochemistry and Biophysics 
 
Members of the genus Streptomyces are filamentous and spore forming bacteria that live in soil 
and aqueous environments. Within these environments, Streptomyces as well as other bacteria 
play a large role in their environment through their ability to interact by producing various 
chemical signals. To understand what type of chemical signals bacteria produce in these 
environments and how their signals affects other bacteria, we have established a model system 
using two soil bacterium: Streptomyces sp. Mg1 (S. Mg1) and Bacillus subtilis. When wild-type 
S. Mg1 and B. subtilis are cultured together on an agar surface, noticeable lysis and degradation 
of the B. subtilis colony is observed. We propose a transposon mutagenesis to identify genes in 
S. Mg1 related to its ability to lyse and degrade B. subtilis colonies. 
 
2 
	  
ACKNOWLEGMENTS 
 
I would like to thank Dr. Paul Straight for finding me space in his lab and his guidance through 
my research experience. Furthermore, I would also like to thank Reed Stubbendieck for his 
continuous guidance through both the research and writing portions of this thesis and my 
research experience. 
3 
	  
CHAPTER I 
INTRODUCTION 
 
Bacteria are all around us. As human beings, it is estimated that bacteria outnumber the total 
number of cells in our bodies by a factor of 10 to 1 (1). Furthermore without these bacteria to 
carry out vital functions in our bodies, our physiology would be compromised (1). These bacteria 
survive in an environments where they constantly compete for nutrients and space. To better aid 
themselves, some bacteria produce secreted products to compete against other bacteria. We know 
some of these products as secondary metabolites. Additionally, bacteria can also face nutrient 
poor environments and may undergo transition to dormant states for long term survival, such as 
sporulation. These spores are dormant cells which upon encountering favorable conditions can 
germinate and propagate their lineage 
Streptomyces carries out a large secondary metabolism that allows it to produce typically at least 
twenty secondary metabolites per strain (2). Within the interaction between Streptomyces sp. 
Mg1 (S. Mg1) and B. subtilis, we hypothesize that S. Mg1 secretes a molecule that actively lyses 
cells and degrades the colonies of B. subtilis. The S. Mg1 genome is approximately 8.7 
megabases and we estimate it contains around 8700 genes. We hypothesize various gene 
products working together may be responsible for the ability of S. Mg1 to lyse B. subtilis.  
To identify the genes encoding these products, we use transposon mutagenesis to create mutants 
of S. Mg1 which will be screened in search of non-lytic inducing colonies. A transposon is a 
piece of DNA that has the ability to change its position within a genome, randomly.  Transposon 
mutagenesis, when combined with a genetic screen, is a forward genetic approach that we use to 
identify genes that are responsible for particular phenotypical characteristics. To carry such an 
4 
	  
experiment out, we introduce a non-native transposon to S. Mg1. The introduction of this 
transposon is facilitated via a plasmid contain the respective transposon. pHTM, shown in Fig. 
S1 is a plasmid that contains a thiostrepton-inducible Himar1 transposon, which contains two 
antibiotic markers to test for transfer of the plasmid during transformations and conjugation, as 
well as identification of mutagenized cells. pHTM has been previously used in successfully 
mutagenize Streptomyces albus and Streptomyces coelicolor (3). Then through a screen that I 
have designed, we will be able to screen for non-lytic mutants. As there are many various 
bacterial interactions in the environment, it is vital to be identifying mutants of the correct 
interaction as many interactions may result in observation of similar phenotypes. For example, in 
my project, even though the interaction of growth inhibition and the interaction for cell lysis 
share a very similar phenotype, the biochemical pathways are highly different. The adjustments 
to my screen have been appropriated to fit these conditions, such that the interaction that will be 
visible will be that of cell lysis. Through identification of non-lytic mutants we can identify 
genes responsible for the lytic activity and gain knowledge that will allow us to identify 
biological components underlying this interaction.   
 
 
 
 
 
 
5 
	  
CHAPTER II 
METHODS 
 
The bacterial strains and plasmids that were used in this experiment are shown in Table 1. 
Table 1. Table of strains and plasmids used 
	  
To effectively use the transposon mutagenesis as a method to introduce mutations in the S. Mg1 
genome, it was vital to test the sensitivity of S. Mg1 to the chemical inducer, thiostrepton. We 
found that wild type S. Mg1 was extremely sensitive to thiostrepton (Figure 1: Picture of 
Thiostrepton sensitivity). Therefore to carry out further experiments, it is necessary to introduce 
thiostrepton resistance to S. Mg1. 
Strains Relevance 
        Streptomyces sp. 
Mg1 
Native isolated strain of Streptomyces that shows lytic degradative activity of Bacillus 
subtilis 
        E. coli JV36 Used to perform transfer of pTSR001 and pHTM plasmid into S. Mg1 through 
conjugation 
        E. coli DH5α Used to obtain greater quantity of pTSR001 plasmid 
        Bacillus subtilis 
3610 
Wild type; used to screen for non-lytic mutants of S. Mg1 
Plasmids  
       pDR183 Contains the bla gene; provides ampicillin resistance 
       pHZ1358 Contains the tsr gene; provides thiostrepton resistance 
       pSET152 Plasmid used as the backbone for the construction of pTSR001 
       pHTM Contains the thiostrepton inducible Himar1 transposon with the antibiotic resistant 
markers of apramycin and hygromycin 
       pTSR001 Plasmid constructed in this paper; contains pSET152 backbone with antibiotic resistance 
genes of ampicillin (bla) and thiostrepton (tsr) 
6 
	  
Enzymatic assembly of plasmid pTSR001 conferring Thiostrepton resistance 
Plasmid maps of pDR183, pHZ1358 and pSET152 were analyzed to design pTSR001, a plasmid 
that confers resistance to ampicillin and thiostrepton. Primers were designed to amplify gene 
products of plasmids pDR183 (bla gene), pHZ1358 (tsr gene) and pSET152 (backbone). Primers 
were also designed to include overhanging regions of homology between the resulting 
polymerase chain reaction (PCR) products. Resulting fragments were then assembled into 
pTSR001, using the Gibson assembly protocol. The assembly of pTSR001 was confirmed via gel 
electrophoresis (Figure 2).  
Accumulation of plasmid pTSR001 
Assembled plasmid, pTSR001 was transferred into chemically competent cells of a conjugative 
strain Escherichia coli DH5α.  Transformation of plasmid was confirmed by selection on 
antibiotic resistance marker encoded by the plasmid on Luria-Bertani agar (LB: 10 g/L tryptone, 
5 g/L yeast extract and 5 g/L salt) with the addition of ampicillin at 100 µg/mL. A single 
surviving colony was picked and grown in an overnight liquid culture of LB with the addition of 
ampicillin at 100 µg/mL. Resulting culture was concentrated and a plasmid purification 
procedure (5) was conducted to increase the amount of plasmid pTSR001 available for further 
experimentation. 
Introduction of TSR001 to Streptomyces sp. Mg1 
The plasmid, pTSR001 was transferred into chemically competent cells of a conjugative strain 
Escherichia coli JV36. Plasmid transfer was confirmed by selection on antibiotic resistance 
marker encoded by the plasmid. Single transformed colony was picked and grown in LB liquid 
culture under selection of ampicillin (100 µg/mL). Resulting culture was diluted to 1:10. 100 µL 
of 108 spores/mL of S. Mg1 was intergenically conjugated with 2 µL of the diluted overnight 
7 
	  
culture of E. coli JV36 pTSR001 plated on AS-1 agar at 30 °C. Plates were flooded with 
antibiotics of ampicillin (100 µg/mL) and thiostrepton (50 µg/mL), 15 hours later to select for S. 
Mg1 exconjugates that contain the plasmid pTSR001. Exconjugants were passaged onto 30 mL 
agar plates of maltose-yeast extract-malt extract (MYM: 4 g/L maltose, 4 g/L yeast extract, and 4 
g/L malt extract) with the addition of the antibiotics, naladixic acid at 30 µg/mL, ampicillin at 
100 µg/mL and thiostrepton at 50 µg/mL, to confirm transfer of pTSR001 into S. Mg1 and to 
inhibit further growth of the donor E. coli. Resulting growth of the S. Mg1 exconjugants 
containing the plasmid were concentrated and sporulated on MYM plates. The collected spore 
stock of S. Mg1 pHTM was stored at 4 °C. 
 
 
 
 
 
 
 
 
 
 
 
 
 
8 
	  
CHAPTER III 
RESULTS 
 
If successful in randomly mutating the S. Mg1 genome and isolation of non-lytic mutants, 
inverse PCR experiments would be completed and insertion loci would be identified. Through 
experiments done by previous members in the lab, the expression of the S. Mg1- pk1 gene cluster 
constitutes a large role in the production of the LDA conferring metabolite. The genes clusters 
that might be found through the transposon mutagenesis project could be other genes related in 
the biosynthesis of LDA or genes encoding transporter proteins aiding in the secretion of LDA 
metabolite in the surrounding media. These newly discovered genes may or may not be linked to 
the S. Mg1- pk1 gene cluster, thus identification of these genes may also result in the discovery 
of profound anabolic pathways that can be pursued as areas of further research in understanding 
the interactions between S. Mg1 and B. subtilis.  
In addition to performing the transposon mutagenesis within the S. Mg1 genome and 
isolating mutants, the act of transposon mutagenesis itself is an accomplishment. The 
establishment of a viable transposon mutagenesis system in S. Mg1 in combination with a 
specific phenotypic screen will allow members of the lab, as well as other labs, to select for 
mutants of interest in the S. Mg1 genome. By doing so, we will be able to appropriate gene 
functions to genes scattered through the S. Mg1 genome, further elucidating how S. Mg1 
interacts with its environment. 
 
 
 
9 
	  
 
CHAPTER IV 
CONCLUSION 
Bacteria produce small molecules to interact with their environments. In the interaction between 
Streptomyces sp. Mg1 and Bacillus subtilis, we hypothesize that S. Mg1 produces some molecule 
that causes the degradation of B. subtilis. To study the lytic ability of S. Mg1, we use a genetic 
approach of transposon mutagenesis in conjunction with an appropriate screen, to understand this 
chemically mediated phenotype.  
Establishment of transposon mutagenesis system in Streptomyces sp. Mg1 
The purpose of the transposon mutagenesis is to randomly introduce mutations in S. Mg1 and 
effectively generate a library of mutants. There are many advantages to using transposon 
mutagenesis to generate mutants over other approaches, such as spontaneous mutation. We use a 
nonreplicative transposon contained on pHTM to conduct the transposon mutagenesis. In theory, 
the nonreplicative transposon will allow us to introduce a single mutation within one S. Mg1 
colony and if the mutagenesis is random, each of these mutants should contain changes within 
their DNA different from the other. Another advantage is the sequence of the transposable 
element is known, thus identifying the location loci of the mutation is much easier. Through the 
establishment of this mutagenesis system, I, as well as future members of the lab, will be able to 
study any genes of interest in the S. Mg1 genome. 
Screening and identification of non-lytic inducing mutants 
The transposon mutagenesis protocol will effectively enable me to generate a library of S. Mg1 
mutants. To be able to identify mutants of interest, we make use the genetic tool of a screen. 
Through a screen that I have designed a screen to identify non-lytic S. Mg1 mutants, I can 
effectively study genes responsible for the lytic activity of S. Mg1. Two components are needed 
10 
	  
for an effective screen. The first is to be able to isolate single colonies of non-lytic S. Mg1 as 
lysing colonies produce halos that encompass larger area than the colony itself and will 
overshadow the non-lytic phenotype. However if a too low density of S. Mg1 mutants are plated, 
the length of the screening step may be doubled or tripled, therefore an optimal density of 
mutants must be plated to allow for efficient and accurate screening. By using wild type S. Mg1 
and a mutant of S. Mg1 unable to lyse B. subtilis, I have determined the optimal density to be 
around 50 to 60 mutants a plate. This concentration will allow me clearly to isolate and identify 
the ability of single S. Mg1 colony to lyse B. subtilis. The second component of the screen is to 
make sure that the density of B. subtilis in the agar overlay will be able to identify non-lytic/lytic 
mutants. This component is vital to make the difference between lysis and growth inhibition. The 
phenotype the lab observes when plating S. Mg1 with B. subtilis on an agar surface, is of lysis. 
To replicate these conditions within a screen, B. subtilis must be grown to a lawning 
concentration before interacting with S. Mg1. If a too low density of B. subtilis is plated, the 
screens may result in false positives, however if a too high density is plated, then the B. subtilis 
cells might overrun S. Mg1 by greatly outnumbering them. Therefore an optimal density should 
not only be obtained for S. Mg1 mutants plated, but also for the amount of B. subtilis overlayed 
on top. I have determined that the optimal concentration of B. subtilis used for an overlay, is at a 
cell culture OD of .8 to .9. 
Future endeavours 
Currently, I have successfully given thiostrepton resistance to S. Mg1 through conjugative 
experiments. I will perform conjugation experiments to introduce the nonreplicative transposon 
harbored by pHTM to the S. Mg1 pTSR001 cells. Then I will induce the transposon to mutate    
S. Mg1. Next, I will apply the screen I have developed to isolate non-lytic colonies. Then I plan 
11 
	  
to take the non-lytic colonies and perform an inverse PCR protocol to identify the insertion loci 
of the transposon, thus determining the genes responsible for lytic activity. These genes will be 
areas of further research to characterize the pathway of the lytic molecule’s synthesis and 
activity. 
Conclusion 
By understanding the function of small molecules in microbial interactions, we may not only 
better understand our environment, but also learn how to predict and control the outcome of 
these interactions as well. While in this thesis, I focus on the genetic aspects of the interaction; 
many other approaches can also be used to study the lytic activity of S. Mg1 such as chemical 
analysis of the small molecule released, or the molecule’s interaction with B. subtilis. Through 
the study of this molecule’s production, release and interaction with other organisms, 
downstream applications may include aiding rationale drug design by discovering new targeting 
pathways of these therapeutics.  
 
 
 
 
 
 
 
 
12 
	  
REFERENCES 
1. O’Hara AM, Shanahan F. 2006. The gut flora as a forgetten organ. EMBO. 7(7): 688-
693. 
2. Procopio RE, Silva IR, Martins MK, Azevedo JL, Araujo JM. 2012. Antibiotics 
produced by Streptomyces. Science. 16(5): 466-471 
3. Bilyk B, Weber S, Myronovskyi M, Bilyk O, Petzke L, Luzhetskyy A. 2013. In vivo 
random mutagenesis of streptomycetes using mariner-based transposon Himar1. Appl 
Microbiol Biotechnol. 97:351-359  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
	  
 


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In vivo transposon mutagenesis of Streptomyces sp. Mg1

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In vivo transposon mutagenesis of Streptomyces sp. Mg1

Abstract

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Texas A&M Repository