X Factor:

Discovering the Influence of a Phosphodiesterase Gene on

Streptomyces scabies.

Cameron Change

Otterbein University

Abstract:

Streptomycetes are members of the bacterial family Streptomycetaceae first classified by

Nobel Prize recipients Selman Waksman and Arthur T. Henrici in 1943. Streptomycetes are soil-

dwelling organisms and are often characterized by the moist, earthy smell that has become more

commonly known to be the scent of dirt. There exists over 900 species of Streptomyces, many of

which are responsible for the vast number of antibiotics that are offered commercially. While the

majority of Streptomyces species have proven to be benign in nature, some of these species are

pathogenic and do have the ability to cause diseases in other organisms; one such species being

Streptomyces scabies. It has been determined that S. scabies is responsible for a disease called

“Common Scab” that produces scab-like formations on the surfaces of tubercle crops (e.g.

potatoes, carrots, etc.). We hypothesize that the presence of cyclic diguanosine monophosphate

(cyclic di-GMP), a common second messenger in bacteria used to regulate various cellular

processes, could be a critical regulator of S. scabies virulence factors. A phosphodiesterase gene

(rmdA) needed for the breakdown of cyclic di-GMP is known to control morphology and

development in Streptomyces coelicolor. Based on a multisequence alignment, we believe that

the functionality of the rmdA orthologue in S.scabies is conserved. The purpose of this study is to

then further determine the gene’s influence on the pathogenic nature of S.scabies via the

inactivation of rmdA.

Table of Contents

Introduction…………………………………………………………….Page 3

Streptomyces Pathogens………………………………………………..Page 4

Common Scab………………………………………………………….Page 5

Cyclic Di-GMP………………………………………………………...Page 6

Purpose…………………………………………………………………Page 7

Materials & Methods…………………………………………………...Page 9

Results………………………………………………………………….Page 17

Discussion……………………………………………………………...Page 21

References……………………………………………………………...Page 23

Introduction

Streptomyces is a member of the bacterial family Streptomycetacecae first classified by

Nobel Prize recipients Selman Waksman and Arthur T. Henrici in 1943 (Kampfer et al 2006).

Since its classification in 1943, the study and

genetic manipulation of the family

Streptomycetacecae has led to many significant

findings in various fields such as agriculture,

microbiology and even medicine.

Streptomyces is a soil-dwelling organism

and is often characterized by its moist, earthy

smell that has become more commonly known to be the scent of dirt. There exists over 900

species of Streptomyces, many of which are responseble for the vast number of antibiotics that

are offered commercially, as well as contributing to the decomposition of organic matter such as

leaves and wood. A large portion of the world’s antibiotics are produced from Actinomycetes,

with a little less than 80% coming from the genus Streptomyces itself (Clermont et al. 2011;

Flärdh et al, 2009).

Streptomycetes have proven themselves to be

a rather unique group of bacteria for a number of

reasons; one of which is their complex life cycle. For

a significant period of time Streptomycetes were

thought to belong to the Fungal family; most likely

due to their strong resemblance. This was once believed so strongly that when it came time to

name this bacterial genus, it was decided that it should be called “Streptomyces” which is loosely

translated to means “chain fungus” (Chater 1984). Streptomyces exhibits a growing method

where it produces vegetative branching hyphae to ultimately form a complex of substrate

mycelium. This mycelium then penetrates organic matter (usually soil) via extracellular

hydrolytic enzymes (Chater 1984). While these mycelia grow indeterminately, Streptomyces

itself is an immobile bacterium and cannot actively transport itself from one place to another.

This presents a problem for Streptomyces reproduction; a problem that was solved via the use of

spores. In addition to the vegetative hyphae that penetrate and gain nutrients from organic matter,

Streptomyces form aerial hyphae that specialize in the formation and distribution of a number of

spores.

Streptomyces Pathogens

While the majority of Streptomyces species have proven to be benign in nature, some of

these species are pathogenic and do have the ability to cause diseases. Of the various

Streptomyces species that have been

discovered, a little over ten of them

have been determined to be

infectious to other organisms

(Labeda 2010). The diseases that

have been found to be pathogenic

seem to be mainly plant pathogens that majorly infect tubercle crops (e.g. potatoes, carrots, etc.).

Some of these diseases, like “Sweet Potato Soft Rot” caused by Streptomyces ipomoea, severely

degrade the crop and cause it to prematurely decompose. Other Streptomyces pathogens seem to

have a less intense effect on the “meat” of the crop itself, but are still a formidable issue in terms

of agricultural yield and distribution. One such pathogen is Streptomyces scabies.

Common Scab

It has been determined that S.scabies is one of the species with the ability to cause a

disease known as “Common Scab” that produces scab-like formations on the surfaces of tubercle

crops (e.g. potatoes, carrots, etc.) (Wharton et al 2007). Even with the formation of these scabs,

S. scabies has yet to produce

any evidence indicating that it

may harm the “meat” of the

tubercle crop itself. While

S.scabies does not harm the

more vital elements of the crop,

the unsightly scab-like

complexes that form on the

outside of the crop cause a serious deficit to the market value of the crop. Due to its source of

nutrients, S.scabies is prominent in environments where most fleshy roots prefer to grow. Prior

research of Streptomyces as a whole has been conducted in order to determine some of the

reasons for select species pathogenic nature. Previous study efforts have focused on the evolution

of Streptomyces pathogenicity in correspondence to horizontal gene transfer as well as further

description of identified virulence factors. Here we present a possible role of the small RNA

dinucleotide cyclic di-GMP in the regulation of S. scabies virulence.

Cyclic di-GMP

Based on previous studies conducted on similar bacterial species, it is hypothesized that

the presence of cyclic

diguanosine monophosphate

(cyclic di-GMP) could be a

critical regulator to S.scabies

virulence factors. Cyclic di-

GMP is a common second

messenger in bacteria used to

regulate various cellular

processes (Holger et al 2012).

It was first discovered to be an enzymatic regulation action activator of cellulose synthase in

Gluconacetobacter xylinus, a bacterium closely associated with grapes (Tamayo et al 2007). In

Streptomyces coelicolor, the presence and abundance of cyclic di-GMP has been linked to

bacterial morphology and developmental regulation (Bennett et al. 2012). Further research was

conducted in order to determine the genes believed to be responsible for the synthesis and the

breaking down of cyclic di-GMP. Further research and purification endeavors were able to

determine the diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes associated with

cyclic di-GMP. Ultimately, it was determined via reverse genetics that the amino acid sequence

of the DGC and PDE genes in G.xylinus had two distinct domains: GGDEF and EAL (Tal et al

1998). Still, it was not until a study conducted on the bacterial pathogen Vibrio cholera that

cyclic di-GMP was indicated as a possible repressor of bacterial virulence (Tischler et al 2005).

Purpose

As it relates to S. ceolicolor, the gene rmdA (originally referred to as SCO0928) is known

to be responsible for phosphodiesterase activity (breaking down) cyclic di-GMP (Hull et al.

2012). It has also been determined that cyclic di-GMP is directly associated with the morphology

and development of S.ceolicolor. Based on the highly conserved nature of rmdA in S.ceolicolor

in comparison to the suspected orthologue in S.scabies, it is suspected that the rmdA homologue

in S. scabies could also be involved in the regulation of morphology and development in

S.scabies. Furthermore, based on previous studies on disease-causing organisms such as

Gluconacetobacter xylinus, it is plausible that the rmdA homologue in S.scabies (SCAB11501) is

particularly influential for S.scabies pathogenesis.

The purpose of this project is to determine if the inactivation of the rmdA homologue will

have an effect on the growth of Streptomyces scabies, as well as its disease-causing nature. Our

efforts to test the validity of this notion were divided into two separate yet equally influential

fields of study. First, our attempts focused on the creation and culture of rmdA inactivated

mutants via the insertion of an S.ceolicolor cosmid containing a disruption cassette with

antibiotic resistant properties into the S.scabies genome. We suspected that due to the seemingly

high homology between the two Streptomyces species, that it should be possible to create an

interspecies hybrid containing the disrupted rmdA gene. It seemed that the most fruitful way to

carry out this experiment would be to conduct two methods of introducing the mutated cosmid

DNA into S. scabies: interspecies conjugation and protoplast transformation. We chose

interspecies conjugation as a means of introducing the DNA from E. coli to S. scabies because a

previous study conducted in our lab where Streptomyces coelicolor was the recipient strain

yielded positive results. As a means of precaution, we also conducted a transformation of the

S.scabies protoplasts with the disruption cosmid. After each attempt to introduce the cosmid into

the S. scabies cells, we then carried out a step designed to help us select for a hybrid

Streptomyces species that had undergone double homologous recombination (gene replacement)

instead of just single homologous recombination (integration of the entire cosmid). While

conducting these experiments, we simultaneously carried out a series of bioinformatics research

on several species of pathogenic Streptomyces and S. ceolicolor in order to compare how similar

they were on a protein level and, in one particular case, nucleotide level. We hypothesize that if

SCAB11501 does control Streptomyces scabies pathogenesis, then the absence of this gene will

show a notable difference in potato scab production from that observed for wild-type S. scabies.

Materials & Methods:

Bioinformatics Data

The bioinformatics research began with the acquisition of the amino acid sequence of the

S.ceolicolor RmdA phosphodiesterase protein. This was done via searching for the gene in the

Streptomyces data base. Once the S. ceolicolor sequence had been procured, we were also able to

attain the S. scabies amino acid sequence by searching S.ceolicolor gene homologues in the same

database. After the two sequences of interest had been attained, we then searched for other

pathogenic Streptomyces species with genes homologous to that of Streptomyces ceolicolor via a

protein BLAST in the NCBI data base. The BLAST search yielded the names and identities of

approximately one hundred Streptomycetes with homologous genes to S.ceolicolor. To narrow

the search down we cross referenced the BLAST search with a list of previously identified

Streptomyces pathogens. We then searched these pathogens in the Streptomyces data base to see

if there was a pre-existing amino acid sequence that could be used to compare with our primary

organisms (S.ceolicolor and S.scabies). The search efforts resulted in the amino acid sequence of

three know Streptomyces pathogens: S.turgidiscabies, S.acidiscabies and S.ipomoeae. Following

this step, an individual domain map was created using the amino acid sequence of each organism

respectively in the SMART database. In addition to this, we compared the amino acid sequences

of the organisms against each other using the CLUSTAL W multi-sequence alignment program

in the “Biology Work Bench” website. In addition to the multi-sequence protein alignment, the

nucleotide sequences of the Streptomyces coelicolor rmdA gene and its Streptomyces scabies

orthologue SCAB11501 were also obtained from the Streptomyces database. Once these

sequences had been obtained, they were entered into the NCBI pair wise nucleotide alignment

program with the intention of discovering the species identity between the Streptomyces

organisms. To conclude the sequence analysis and comparison portion of this study, a pair wise

protein alignment (using the various amino acid sequences) was conducted between S. coelicolor

RmdA versus the suspected protein orthologue from each pathogenic Streptomyces organism.

Following the sequence alignment the study continued onto the creation of three

dimensional images of the projected protein models. We began by entering the entire protein

sequence of each Streptomyces species RmdA orthologue into a threading program known as

Raptor-X. The threading program, over the course of thirteen hours, created a theoretical two

dimensional structure of the gene sequences of each organism respectively. In addition to the two

dimensional structures, the program saved the theoretical protein models in a PDB format. We

took the PDB files and entered them into the NCBI data base so that it could be converted into a

readable file for the CN3D program. Once the files had been successfully converted, we

visualized them in the CN3D program in the hopes of creating three dimensional protein

structures for each Streptomyces organism.

Interspecies Conjugation

Prior to the conjugation attempt, both the wild type Streptomyces scabies (87.22) and the

E. coli containing the cosmid with the antibiotic resistance cassette had to be prepared separately.

The cosmid containing the deletion cassette with antibiotic resistant properties was successfully

synthesized and obtained from another laboratory.

Preparation of E. coli (ET12567/pUZ8002/rmdA::Tn5 cosmid)

Grew an overnight liquid culture of E.coli in LB broth containing the antibiotics

Apramycin and Kanamycin.

150 microliters of the overnight culture was taken and inoculated in 15 milliliters of LB

broth containing the antibiotics Apramycin

and Kanamycin for a period of 7 hours to

reach the optical density of 0.311 at 600

nanometers.

The 15 milliliter culture was then placed

into a centrifuge tube and spun at 4000

rpm for a duration of 5 minutes

Post centrifugation, the supernatant was

discarded and the bacterial pellet

underwent a series of 2 washes in regular

LB broth. (Wash= suspended in broth and centrifuged at 4000 rpm for a duration of 5

minutes.)

Post wash, the bacterial pellet was then suspended in 250 microliters of LB broth.

Preparation of Streptomyces scabies 87.22

Prior to the incubation procedure below, wild type S.scabies (87.22) was grown on Soya

Flour Manitol (SFM) media and was stored at a 30*C incubator for several weeks.

With a sterile plate as the stage, several samples of S. Scabies were taken from the SFM

culture plate with sterile toothpicks and mashed in 50 microliters of 10% Saline solution.

More samples of the S.scabies were collected and mixed with 250 microliters of saline

solution.

The S.scabies/saline solution was place into a 15 milliliter centrifuge tube with 500

microliters of 10% Saline solution.

The procedure would later on be altered to a method of spore harvesting in an attempt to

gain a more fruitful conjugation yield. In order to obtain the spores, a lawn of wild type S.scabies

was streaked on SFM media 72 hours prior to the havest.

3 milliliters of 10% saline solution was added to the S. scabies lawn. Using a sterile

inoculating loop, the lawn was lightly scraped across its surface in order to release the

spores into the overlaying saline solution.

The spore/saline solution was then pipetted into a 15 milliliter centrifuge tube.

The spore/saline solution was then heat shocked at 37°C for a duration of ten minutes

Conjugation Plating

Two SFM plates were distributed and 50 microliters of S.scabies (S.scabies spore)

solution was added to each plate.

50 microliters of prepared E. coli broth was then added to each plate and a sterile

spreader was used to spread the conjugation culture evenly on the plate.

The conjugation plates were then left in the 30°C incubator for aduration of 19 hours.

(The time that two species would be allowed to undergo conjugation would later be

changed to 24 hours.)

After the conjugation period had expired, the Conjugation plates then received an

antibiotic overlay containing 150 micro liters of Apramycin and 250 micro liters of

Naladixic acid.

The conjugation plates were then returned to the 30°C incubator for a period of 72-96

hours.

Transformation of Wild Type S.scabies

Prior to the Transformation attempt, both the wild type Streptomyces scabies (87.22) and

the S. coelicolor mutated cosmid DNA from E. coli containing the antibiotic resistance cassette

underwent separate preparation procedures. The cosmid containing deletion cassette with

antibiotic resistant properties was constructed and acquired from another laboratory.

Preparation of cosmid DNA from ET12567/pUZ8002/rmdA::Tn5 cosmid

Liquid culture of E. coli containing the antibiotic resistance cassette was prepared the

night prior from Dr. Bennett’s private stock. The DNA of said culture was then purified via the

following procedure attained from the QIA Miniprep (QIAGEN) hand book.

Centrifuged liquid culture for 5 minutes at 4000rpm to attain a pellet of bacterial cells.

Then removed the supernatant.

Suspended bacterial pellet in 250 microliters of Buffer P1 (QIA Miniprep kit) then

transferred to a micro-centrifuge tube.

Added 250 microliters of Buffer P2 (QIA Miniprep Kit) and mixed thoroughly by

inverting 4-6 times.

*Did not allow lysing reaction to continue for longer than 5 minutes

Added 250 microliters of Buffer N3 (QIA Miniprep Kit) and mixed thoroughly by

inverting the tube 4-6 times.

Centrifuged the tube for 10 minutes at 13,000 rpm.

Applied supernatants to QIAprep spin column by pipetting. Centrifuged for 30-60

seconds and discarded flow through

Added 500 microliters to Buffer PB to QIAprep spin column. Centrifuged for 30-60

seconds and discarded flow through

Wash- added 750 microliters of Buffer PE to the QIA spin column. Centrifuged for 30-60

seconds

Discarded flow through and centrifuge for an additional minute to remove residual wash

buffer.

Placed QIAprep column in a clean 1.5 milliliter micro centrifuge tube. To elute DNA,

added 50 microliters of Buffer EB (QIA Miniprep Kit) to each QIA spin column. Let

stand for one minute and centrifuge for one minute.

Stored prepared DNA in -80*C freezer.

Preparation of Streptomyces scabies 87.22

Prior to the incubation procedure below, wild type S.scabies (87.22) was grown on Soya

Flour Mannitol media and was stored at a 30°C incubator for several weeks.

Smashed several colonies of S. scabies in 200 microliters of saline.

Added the smashed S. scabies solution to 30 milliliters of YEME in baffled flask.

Placed flask in orbital shaker on 30°C at 250 rpm over the course of 36-48 hours.

Preparation of Protoplasts

On the day of transformation, the following procedure was conducted with the previously

incubated S. scabies (87.22) strain in the YEME solution with the goal of attaining bacterial

protoplasts.

Transferred YEME with pre-germinated spores from baffled flask to 50 milliliter

centrifuge tube.

Added 15 milliliters of sucrose to baffled

flask to rinse the remaining cells and

transferred to the 50 milliliter centrifuge tube

(vigorously shook and inverted tube in order

to suspend spores in YEME/ Sucrose

solution).

Centrifuged the 50 milliliter tube at 4000 rpm

for ten minutes (discarded supernatant).

Added 15 milliliters of 10% sucrose and

shook/ inverted to re-suspend pellet.

Centrifuged at 4000rpm for ten minutes and discard supernatant (the previously stated

steps were completed once more.)

Added 5 milliliters of lysozyme solution and triturated three times to break up any chunks

(Triturate = draw up into a pipette and let roll slowly down the sides)

Incubated at 30*C for 60-90 minutes, triturating (in a fume hood) every 10-15 minutes.

After the incubation period, the entire culture was place in to a syringe with cotton that

had been sterilized (via autoclave) and was allowed to sediment for a minute.

The newly formed protoplast solution was then forced through the cotton syringe in to a

15 milliliter centrifuge tube.

Centrifuged at 4000 rpm for 5 minutes (discard supernatant)

Added 100 microliters to the remaining pellet. Suspended by drawing up and down in a

pipette.

Transformation of Protoplast with DNA

Prior to the protoplast introduction to the DNA, a DNA alkaline denaturation step was

conducted during the final sequences of the protoplasts preparation. The procedure for said

denaturation is as follows:

Placed 24 microliters of the stored E. coli DNA (ET12567/pUZ8002/rmdA::Tn5 cosmid)

into a 1.5 milliliter microfuge tube.

Added 6 microliters of 1M NaOH

Mixed the solution and centrifuged for 3 seconds

Incubated at 37°C for ten minutes.

Post incubation, rapidly chilled on ice

Added 6 microliters of 1M HCL.

After the DNA Alkaline denaturation procedure had concluded, we then preceded with

the transformation steps:

Added 100 microliters of the protoplasts to the tube of alkaline denatured DNA

Immediately added 500 microliters of the 25% polyethylene glycol (PEG) in P-buffer to

the tube.

Mixed the contents of the tube by drawing it up and down repeatedly with a pipette.

Immediately plated 100 microliters of the transformation solution onto each R2YE plate

(6 plates in total).

Incubated at 30°C for a period of 16 hours.

Post incubation, plated each culture with 2 milliliters of antibiotic overlay

Incubated at 30°C for 72-96 hours

Results:

Bioinformatics

The amino acid sequence for each Streptomyces organism had been successfully

obtained. Afterwards, a multi-sequence protein alignment was run via the “Biology work bench”

web site. This program took the individual amino acid sequences and arranged them in a manner

so that they could be compared against one another. The results of this showed that there is

relatively high similarity in the location of amino acids within individual sequences, with

particular emphasis on the conservation of the catalytic regions of sensory and cyclic di-GMP

metabolism. In addition to the multi-sequence alignment results, the domain maps created from

the individual amino acid sequences illustrated the high conservation of the cyclic di-GMP

metabolic pathway within each Streptomyces organism.

In addition to the aligned sequences and domain maps the pair wise protein alignment,

where SCO0928 (rmdA) was compared against each pathogenic organism individually, revealed

that SCO0928 was approximately 86.5% homologous with the rest other Streptomyces organisms

(S.scabies: 88%; S.turgidiscabies: 88%; S.ipomoeae: 87%; S. acidiscabies: 38%). Furthermore,

the pairwise nucleotide BLAST yielded that S.scabies and SCO0928 share an 87% nucleotide

identity.

The efforts to create theoretical three dimensional structures of the various amino acid

sequences were reasonably successful. The RaptorX program, was able to construct the

hypothetical structures of each organism individually, then the structures were visualized in the

CN3D program, provided by the NCBI database. The three dimensional structures were

accompanied by smaller text window with the location of each amino acid within the order of the

organism. By using this text box, we were able to locate and create an annotation for the rmdA

catalytic site in each structure by highlighting the correct EAL region.

Interspecies Conjugation

There were a total of six attempts made to

insert the disruption cassette into S. scabies via

interspecies conjugation. The first tries only yielded a

combination of bacterial contamination and a small

population of indistinguishable red colonies. Latter

attempts were conducted, during which time several

steps in methodology were altered as a means of

improving efficiency. Despite the amount of

attempts, endeavors to create an

S.coelicolor/S.scabies hybrid via interspecies

conjugation with an E.coli vector proved to be unsuccessful.

Protoplast Transformation

There was one effort made to create the

S.ceolicolor/S.scabies hybrid via protoplast transformation.

The entire procedure spanned the course of approximately

five days to complete, during which time all of the reactants

and sterile instruments necessary to carry out the procedure

were made as well. The transformation attempt ended with

the presence of bacterial contamination as well as the

indistinguishable reddish colonies.

Discussion

The study yielded inconclusive results in reference to the phosphodiesterase gene’s

influence on the organism’s ability to cause disease. As the results show, we were unable to

insert the disruption cassette into the S.scabies genome via either interspecies conjugation or

protoplast transformation; the result of this being that we did not create the S.scabies/S.ceolicolor

hybrid and therefore cannot continue with the remaining steps of the study. It is possible that the

inability to create the hybrid could be due in part to some incompatibility between the wild type

S.scabies target and the E.coli vector. It was assumed that positive results from the S.ceolicolor

study, as well as the similarity between S.ceolicolor and S.scabies in homology and nucleotide

identity, provided enough theoretical evidence for the success of the cosmid integration attempts.

However, it is possible that similarities between the two Streptomycetes were not enough for this

study to yield the same results as the previous study with S.ceolicolor. The original goal was to

use S.scabies own homologous recombination system to assimilate the mutated S.coelicolor

gene. It could be, however, that the two Streptomycetes homology was not sufficient enough for

this to occur. Furthermore, the presence of bacterial contamination as well as the interference of

the various antibiotics used in the study could have created conditions too unfavorable for the

desired mutants to grow. It is also important to note that several of the reactants necessary for the

cassette integration attempts were not the same exact reactants that were outlined in the text used

to carry out these procedures, though they differed very minutely in ways such as cloth density

(cotton balls) and molecular weight (polyethylene glycol). Unfortunately, since S.scabies has

never before been studied in this lab and studied very little in other locations, there is not much

information that can be referenced to determine the accuracy of these potential reasons.

Despite the failure to create the Streptomyces hybrid, this study has brought forth many

positive results. We were able to attain the domain maps for each species highlighted within the

study. This is significant because the maps themselves illustrate the highly conserved genes

within the individual species, which could possibly be an indication for similar cyclic di-GMP

use and function in these organisms, respectively. Furthermore, all of the research conducted in

this study provides the framework for which later studies can use as a foundational reference.

Future directions for this study could include efforts to discover the cyclic di-GMP metabolic

pathway function in these other species. However, in the grand scheme of the project, the most

important objective is to create the SCO0928/S.scabies hybrid. Once the hybrid containing the

disruption cassette has been successfully created and grown, it can be introduced to the tubercle

crop host and definitive answers to rmdA’s influence on species pathogenesis can be formulated.

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