INTRODUCTION

Pseudomonades are oxidative phosphorylation chemo-heterotrophic bacteria. The genus includes

species of ecological, economic and medical interest. Pseudomonas species, although some have
long been recognized as plant pathogens, are omnipresent in soil. Pseudomonades can affect our
lives and our environment in both positive and negative ways. Some of them cause illness.

Source of Isolate

To study, the soil was collected from the campus of the University of New Haven located in
West Haven, CT. The coordination location is 41.290° N - 72.962 W ° and performed
phylogenetic analyses based on 16S rRNA gene with the intention of relating these analyses to
the physiological characteristics. In this work, several genes were used to delineate the
phylogenetic status of species in the genus Pseudomonas. The 16S rDNA was included because,
as a universal marker, it permits the ascription of a strain to the genus and allows comparisons
between very divergent bacteria (Santos and Ochman, 2004).

Enrichment, Isolation and Identification of Pseudomonas

Isolation of Pseudomonas directly from the environmental is rather a difficult task. In environment
their numbers are generally quite low relative to those of other organisms present.
Direct application to an agar medium of these environmental samplesmedium will likely yield few to no
pseudomonad colonies. So the strategy here is to enrich pseudomonades in liquid media, increasing
their relative numbers, before obtaining isolates on solid media.

Enrichment A good microbial enrichment strategy is selectively promoting the growth of organism
and hindering the growth of the vast numbers of undesired microbes. A minimal medium with an
exotic carbon source like sodium benzoate is a better choice; it would restrict growth to only those
with the enzymes required for benzoate catabolism, including representatives of Pseudomonas.

The basal medium for the enrichment is:

1. 33 mM Na-K-phosphate, pH 6.8
2. 0.05% MgSO4

3. 0.005% FeCl3

4. 0.0005% CaCl2

called a mineral base because all of the components are inorganic.

By adding a carbon and nitrogen source to this mineral base, the enrichment medium is produced. T
here are two types of enrichment crops available: some incubated aerobically (containing an organic
source of carbon and nitrogen) and the other incubated an aerobically containing glycerol, KNO3, a
nd NH4Cl [1].

The Polymerase Chain Reaction (PCR)

The procedure of the polymerase chain reaction (PCR) amplifies a DNA fragment from a complex
mixture selectively. The reaction of the polymerase chain consists of three independent reactions se
quentially performed and then repeated 20-30 times (cycles). Thisis a chain reaction because each
cycle doubles the number of copies of the target DNA. These results from the double-stranded
nature of DNA; each strand and its complementary primer serve as a substrate for DNA
polymerase. A piece of DNA present once in a sample will be replicated in the first cycle yielding
two copies; after the second cycle, four copies will be present, and after 30 cycles, 230 copies (109
copies) could theoretically result.

It is important to model oligonucleotide primers to classify the Pseudomonas rRNA gene in a mixtu
re of DNA molecules extracted from the whole chromosome.

Dual-stranded DNA to be copied (the template) is heat denatured to single-stranded DNA in the
first step of each cycle (denaturation). It includes ~95 ° C temperatures. In the second step
(annealing), the temperature is lowered to 50-55 ° C to allow the oligonucleotide primers to bind on
the denatured template DNA to their complementary 12 sequences.
 Fig.1.Multiple sequence alignment of five bacterial rRNA genes

Complementary DNA strand. The DNA polymerase used in PCR derives from a thermopile as such
organisms have macromolecules that are stable at high temperatures. You will use Taq DNA
polymerase (pronounced “tack”) from Thermusaquaticus that works optimally at 72°C and can
withstand temperatures of 95°C without loss of activity.

As with all in vitro reactions, a source of energy is needed for the reaction to proceed. Each of the
three steps of PCR has specific energy sources: the denaturation step occurs by the energy from the
heat; the annealing step is a spontaneous reaction (reversing the denaturation step); and the
extension/elongation step is powered by the dNTPs, deoxynucleotide triphosphates, each of which
contains a high energy triphosphate that provides the energy for its incorporation into a growing
nucleotide chain [2].
 Fig.2.diagram illustrates the 16S rRNA gene of Pseudomonas positions of the two primers

Keep in mind the antiparallel nature of DNA – it is essential to know the 5' and 3' ends of each
DNA strand and each oligonucleotide primer in order to understand PCR. Unless otherwise
indicated, primer sequences are written in the 5' to 3' direction and the substrate for DNA
polymerase is a 3' end.

The following diagram illustrates the 16S rRNA gene of Pseudomonas (~1500 bp) and the arrows
indicate the approximate positions of the two primers that will be used for the PCR amplification.
The primers are centered at ~27 bp and ~1450 bp in the gene [3]..

The genomes of Pseudomonas species range from 5-7 megabase pairs (Mbp, 106 bp) and different
species have 4-7 copies of the 1500 bp 16S rRNA. A quick calculation illustrates the power of PCR:
If there are 4 copies of the rRNA gene (4 X 1500 bp = 6000 bp) in a 6 Mbp genome, then the rRNA
genes constitute about 6,000 bp/6,000,000 bp or ~0.1% of the genomic DNA. Following PCR, the
target (16S rRNA gene) constitutes >99% of the DNA present. It’s important to remember that PCR
is the selective copying of one region of the chromosomal DNA; there is no hydrolysis (cutting) of
the DNA in this process.
Purification of Genomic DNA from Pseudomonas

The bacterial genome is defined as all of the hereditary material (DNA) contained within a given
cell. This means that the bacterial genome is comprised of both the chromosome and any plasmid
DNA that may be present in the cytoplasm. To isolate and purify genomic DNA from a
Pseudomonas isolate ,the molecule will break during isolation into pieces of about 20-30 kb
(kilobase pairs, or 20,000-30,000 bp).

The bacterial culture is first centrifuged to isolate the cells from the growing medium and discard
the spent material. The cells will be resuspended in the lysis buffer because this buffer is optimized
for the initial reactions and the growth medium will likely have components that interact with these
reactions..Lysis buffer contains sodium dodecyl sulfate (SDS), a detergent that will solubilize
membranes.

The second step in the purification of genomic DNA is the addition of Proteinase K, an enzyme that
hydrolyzes most proteins). This enzymatic reaction is performed at 56°C, the optimal temperature
for this enzyme. Recall

The next steps in the purification of genomic DNA eliminate the remaining macromolecules until
only the DNA remains.

Guanidine hydrochloride, a chemical that denatures proteins, including Proteinase K and RNase A,
is added next. The addition of ethanol to the mixture causes the DNA to precipitate from solution.
This is a physical 16 change, in which the ethanol dehydrates the DNA and the individual DNA
molecules have a greater affinity for one another than they do for the solvent, and the result is
precipitation from solution. The entire mixture of precipitated DNA, hydrolyzed cellular
components, denatured proteins, and soluble metabolite pools is applied to the DNeasy mini
column. Only DNA binds to the column; all other components pass through, and the DNA has been
isolated.

The remaining steps serve to purify the isolated DNA, by washing with ethanol-based buffers
(AW1 and AW2). In each case, the DNA remains bound to the column matrix and contaminants are
washed through into the collection tube. Centrifugation provides the force to wash the solutions
over the bound DNA. The final step is elution of the DNA from the column matrix with an aqueous
elution buffer (AE). For this final centrifugation, the column should be placed into a clean tube, as
the wash-through will be the final pure DNA preparation

Agarose Gel Electrophoresis

Agarose gel electrophoresis is used to separate DNA fragments so that they may be visualized.
Agarose is a highly purified polysaccharide that is the primary component of the agar used in solid
bacterial media. The migration of molecules through an electric field is dependent on a combination
of size, overall charge, and shape.Because all molecules of DNA have an essentially identical
charge to mass ratio (i.e., identical charge densities, or one phosphate per nucleotide), the rate of
migration of linear molecules of DNA through an electric field is dependent almost entirely on the
size of the molecule. As the phosphate groups in DNA carry a negative charge, DNA is a polyanion
and migrates toward the anode (positive pole). For linear DNA, the mobility of a fragment is
inversely proportional to the size of the molecule; that is, smaller fragments move faster. So if DNA
samples are loaded into an agarose gel and allowed to migrate through an electric field, we can
separate the molecules based on their size.
 Fig.3.Agarose Gel Electrophoresis

16S rRNA genes and the Ribosomal Database Project (RDP)

Because the 16S rRNA molecule is an essential structural component of the ribosome, and bacterial
cells contain more than 10,000 ribosome’s, most bacteria have multiple copies of the 16S rRNA
gene in their genome. Pseudomonas genome sequences show that a single species has 4-6 genes that
encode 16S rRNA, and these genes are not identical in sequence.

For example, the image to the left shows the DNA sequencer output from a PCR product. Each
nucleotide is labeled with a different colored marker and the detector recognizes that color and
“calls” the base at that position. The peak stands out significantly from the baseline at most
positions, but when the DNA from the PCR contains a SNP, the sequencer detects both nucleotides
at a position. In this example, it is clear that position #141 has an equal mixture of C and T: both are
detected with equal frequency and that frequency is about half of the other peaks. The interpretation
is that there are two populations of DNA fragments within the PCR product, one with the sequence
GTAAGCTAATAC and the other with the sequence GTAAGTTAATAC. Note that the software
inserts a Y at this position, indicating a pYrimidine (C or T).

The existence of SNPs in the DNA sequences derived from Pseudomonas 16S rRNA gene raises a
question about how organisms can be classified when multiple (slightly different) copies of the 16S
rRNA exist in an organism. The Ribosomal Database Project (RDP) has a single entry (sequence)
for each organism, and is therefore incomplete. However, it is still a useful tool in that it can
identify close relatives. There are more than 100 forms of strains within the Pseudomonas genus
that were identified and validated in the literature and classified into "subgroups" as reported by
Mulet et al. 2010. (DNA sequence-based Pseudomonas genus review 12: 1513-1530.
Environmental Microbiology)

Bacterial Growth Media

1. Pseudomonas Minimal (Broth or Agar) Media

2. Pseudomonas Isolation Agar (PIA)

3. Pseudomonas F agar

4. Nitrate Broth

5. Starch Agar

Preparation of aerobic enrichment cultures for pseudomonads.

• For your aerobic enrichment culture, measure 25 ml of enrichment medium mineral base and add
it to a 250 ml flask. (It may have a slight precipitate – swirl to suspend.)

• The carbon and nitrogen sources will use in these enrichments are listed in the table below.
• Add the listed number of drops of your assigned carbon and nitrogen sources to the flask. Note
that some compounds, specifically histidine, arginine, and penicillin-G, act as both carbon and
nitrogen sources.

• Add 0.5 gram of soil to the flask. Record here the source of your inoculum:

• Cap the flask with two layers of aluminum foil and incubate at 30°C for a maximum of 2 days to
minimize growth of other organisms.

Preparation of an anaerobic enrichment culture for denitrifying pseudomonades.

• Label a reagent bottle and measure approximately 60 ml of enrichment medium mineral base. Add
some to the reagent bottle, leaving about 2 cm of space from the base of the neck of the bottle.

• Set the bottle in half of a Petri plate to catch any medium that overflows. Carefully add 0.60 ml of
50% glycerol, 3.0 ml of 20% KNO3 and 1 ml of 10% NH4Cl to the to the solution in the bottle.

• Add 0.5 gram of dry soil. Record the source of your inoculums:

• Top off the bottle with enrichment mineral base so that a bead of liquid forms at the top, above
the lip of the bottle (see illustration at left). Angle the glass stopper onto the bead of liquid and twist
the stopper to close the bottle snugly (trying not to trap any air, and letting the excess liquid fall
onto the Petri plate). Invert the bottle several times to mix the contents.

• Incubate the enrichment at 30°C for 2 days. Agitation is not necessary during this incubation.

Streaking pseudomonad enrichments for single colonies.

• Examine your two enrichment cultures (from both pairs in the group) under phase contrast
microscopy for the presence of motile, paired rods.

• For each of the eight different carbon sources used in the enrichment cultures in the class, three-
phase streak a loopful of the enrichment on a plate of minimal medium containing the identical
carbon source. For example, streak a loopful of the sodium benzoate enrichment onto a plate of
sodium benzoate minimal agar medium.

• Label each plate completely, including both the carbon source included in the medium as well as
the original source of inoculum (soil).

• Incubate all plates (except that derived from the anaerobic enrichment) at 30°C for 5 days. Store
your broth enrichment cultures at 4C.

• Incubate the glycerol-ammonium nitrate minimal medium plate in a GasPak® jar to generate an
anaerobic environment. The GasPak jar will be incubated at 30°C for 5 days.

Presumptive identification of pseudomonads and streaking on a selective medium.

Examine your minimal agar streak plates. Remember that these derived from liquid enrichments
that were not pure cultures.

• Prepare wet mounts by setting up a “reservoir slide” with a few drops of water. For each wet
mount, transfer a loopful of water from the reservoir to a portion of a microscope slide. Sample the
colony with the sterile inoculating needle. Prepare wet mounts of small portions of your putative
pseudomonad colonies for microscopic examination at 1000X, looking for paired rods.
Pseudomonas strains are short rods and may be mistaken for cocci. (Motility may not always occur
under these conditions.) If it appears to be a pseudomonad, you will need to use the remainder of
the colony to three-phase streak onto a plate for isolation. Use the space below to make notes on
your work[5].

• After confirmation by microscopic examination, streak putative pseudomonad colonies for

isolation on Pseudomonas isolation agar (PIA). This medium is designed to select for the growth of
Pseudomonas species by inhibiting others. It should therefore help narrow your search for putative
pseudomonads.

• Streak a total of ten PIA plates with different inocula. Diversity in this exercise comes from
different soil samples, not from different carbon sources. Use the table provided below to help keep
track of each isolate and assign each a number for future reference.

• Incubate the PIA plates at 30C for 24 hr. Store minimal agar plates at 4C.

Confirmation of pure cultures.

Examine your streaks of presumptive Pseudomonas strains on PIA. Not all of the strains that you
streaked are expected to grow, but your group should have at least one isolate per person. Of the
isolates that did grow on PIA, examine the single colonies carefully to ascertain whether it is a pure
culture. Subtle differences in colony morphology (opaqueness and pigmentation) may be apparent
in the colonies, and it’s quite possible at this stage in the enrichment to have a mixed culture.
Follow the flowchart on the next page to guide each isolate’s progress in the next two labs [6]..

Do not proceed with the Gram and oxidase tests until you are certain that your culture is pure. Each
foursome must have a minimum of four pure cultures that meet all of the criteria for Pseudomonas
species by the end of Lab 5. Restreak these putative Pseudomonas isolates onto separate LB agar
plates. Transferring these cultures to an all-purpose medium like LB helps avoid any problems
associated with carry-over from a selective medium like PIA that could affect growth and/or
metabolism on the media that will be used to identify these microbes.

Start a broth culture of a Pseudomonas isolate.

Inoculate with a single colony of Pseudomonas. Incubate at 30°C for 24 hours.

Prepare a glycerol stock of your isolate.

Bacterial strains are stored as glycerol stocks. Glycerol is a cryo-protectant and cultures mixed with
glycerol can be frozen and stored indefinitely at -70°C. These can easily be prepared by mixing a
liquid culture and glycerol and flash-freezing in liquid nitrogen. The strains are then revived as
needed by inoculating fresh media with a small nugget of the frozen culture. • Calculate how much
50% (v/v) glycerol should be mixed with your culture to get 1 ml of a suspension of cells in 15%
(v/v) glycerol. of culture +l of 50% glycerol • Label a screw cap tube, and in it mix sterile 50%
glycerol and a sample of your overnight culture. Vortex to mix. • Leave the mixtures in the
designated rack for the TA to freeze. • Save the remainder of your culture as it has two additional
uses today [7].

Phenotypic Testing of Pseudomonas: Inoculating the test media.

• Completely label each of the following plates and divide each plate into four sections, one for
each isolate in the group collection.

Pseudomonas minimal medium (PMM)

without a carbon source PMM with glycerol + nitrate

PMM with glucose Pseudomonas F agar

PMM with histidine TSA + starch

PMM with trehalose 3 LB plates, labeled for incubation at

4°C,

PMM with arginine 30°C, and 42°C

• Prepare the cells from your broth culture for inoculation by “washing” them in saline. This
eliminates any carryover of the rich broth medium to the minimal media plates that might allow
some growth. Transfer 1 ml of culture to a microcentrifuge tube and spin the tube in a
microcentrifuge for 1 minute. Discard the supernatant in the beaker provided and then aseptically
transfer 1 ml of saline to the tube. Vortex to resuspend the cells. Continue vortexing until there is no
visible pellet and the suspension has turbidity similar to the starting culture.

• Place the mini spin column of DNeasy in a new 2 ml collection tube and insert 500 l AW1 buffer.
Centrifuge for 1 min at the maximum speed. Discard the liquid stream.

• Place the mini spin column of DNeasy in a new 2 ml collection tube and insert 500 l AW2 buffer.
Centrifuge for 2 minutes at maximum speed. Discard the liquid stream.

• Replace the DNeasy mini spin column in the same tube (n)Examine the genomic DNA prep and
determine the presence of a plasmid.

The presence of plasmid DNA in the genome is a useful feature in the classification and
identification of Pseudomonas strains. When visualizing the genomic DNA on an agarose gel, this
can be measured. Throughout removal, the chromosomal DNA was broken into 20-30 kb linear
parts and will appear as a single band smaller than the largest band in the size norms. Plasmids are
going to be smaller [8].

• In a new style

• Store your genomic DNA preparation at 4°C.

Phenotypic testing on Pseudomonas strains: observing and interpreting results

• Observe and compare the relative growth at 4 ° C, 30C and 42C for each isolate on the specific
minimal media and LB agar. In the Summary Table on page 38, reported growth (+) or no growth-).
Using scope or multiple + to show the relative amount of growth observed as appropriate.

• Checking for the development of alcoholic amylase. Flood the TSA + iodine starch plate to test
for iodine production

Amplification of the 16S rRNA gene

Recover your genomic DNA preparation and tag a tiny PCR duct.
• Place the following ice components in the PCR tank: mix 38/l buffer mix 8.5/l template DNA
(genomic DNA preparation) 2/l Taq DNA polymerase 1/l

• Mix the components by setting the micropipettes to 25/l and gently pipetting up and down several
times.

• Use the thermal cycler to position the tube. A program known as "ICE".[9].

Complete phenotypic testing.

• Retrieve and compare the gelatinase trial crops with the control crops. Plastic strip gelatin clearing is an ind
icator of the activity of gelatinase

• Examine the pattern of growth on plates that were incubated for one week.

• Record (+) or () for each isolate in the Summary Table at the manual's back.

Examination of Pseudomonas rRNA PCR products on an agarose gel.

The effectiveness of the PCR reaction is determined by running a small sample on an agarose gel,
usually 10% of the reaction, and searching for a band of the desired width.

• Position the PCR tube momentarily (a few seconds) in a de-capped microcentrifuge tube and
rotate it to the bottom of the PCR tube.

• Switch 5 l of PCR to a fresh pipe and add 5 liters of water and 2 liters of 6X

PCR product purification.

The PCR product (DNA) has to be isolated from the other components of the reaction before its
sequence can be determined. Materials will be needed for this exercise after the agarose gels have
been finished.

• Mark the specimen with two microcentrifuge tubes and a spin QIAquick (pink) board.

•Switch the remaining PCR (~45 l) to a 1.5 ml microcentrifuge pipe and apply 5 volumes (225 l) of
guanidine hydrochlorideand mix well by vortexing.
• Apply this mixture to the spin column of QIAquick which was put in a collection pipe of 2 ml.
30-60 seconds centrifuge.

• The flow-through is removed and the spin column in the collection pipe is replaced.

• Apply 750 liters of ethanol wash buffer to the spin and centrifuge column for 30-60 sec. • Remove
the flow-through and install the spin column in the collection pipe.

• Again, the centrifuge

• Add 30 liters of water to the center of the membrane to elute the DNA, allow the column to stand
for 1 minute, then centrifuge for 1 minute.

• Remove the column of pink and close the microcentrifuge duct. Check that this tube is properly
labeled and place it in the designated rack to be submitted for DNA sequencing.
Download DNA sequence and compare it to the ribosomal database.

Download the DNA sequence, compare it to the RDP, and match the DNA sequences.

Fig 4. Molecular Size Standards for agrose gel electrophoresis

DNA sequence analysis of the Pseudomonas rRNA gene

DNA sequenceformatting for use in other programs

Before using your sequence, it must be edited, and this process requires judgment. Your sequence
will have A, G, C, T, and N, and perhaps some others (Y, R). N ‘any "nucleotide and the code could
not unambiguously classify the residue. The least reliable sequence is at the beginning and at the
end (closest to the primer and near the resolving power of the detector). There will be a string of Ns
at the beginning, usually 12-15. Delete those bases (Ns and other bases) from your sequence. [10].

because these Ns are placeholders. Near the end of the sequence you will see an increasing
frequency of Ns. This is a limitation of the technology, and you will probably need to eliminate
dozens of bases. When the sequence is mostly Ns, delete; when it is about half Ns, save it.

Interpreting N in the middle of the sequence, as well as bases labeled R or Y (or other) requires
examining the original data tracing from the sequencing reactions. Scan through the sequence,
identify potential SNPs, and adjust the sequence as needed.

Sequence must be in an appropriate format to prepare the data for comparison, and a useful sequenc
e format is called "fasta."Line 1 begins with the ">" symbol followed by a string of characters
identifying the sequence (but no spaces) and a "return." The sequence then follows (upper or lower
case) without gaps, as illustrated:

Comparing your 16S rRNA sequence to the sequences in the Ribosomal Database (RDP)

That's just what the Ribosomal Database is – a list of 16S rRNA sequences that can be checked for
matches or downloaded from different sequences. It is updated frequently to include sequences that
have recently been deposited in public databases. In May 2014, there were 2,929,433 sequences in
the database, and that number will certainly increase by the time you search for matches to your
Pseudomonas rRNA sequence. The homepage for the Ribosomal Database is
http://rdp.cme.msu.edu/.

From the RDP homepage you can search the database for those sequences most closely related to
yours. Click on the Sequence Match (SEQMATCH) link and paste your DNA sequence into the box
in fasta format. Below the box are some choices for which sequence you want to search within the
database. Choose "Type" strains (well-characterized standard strains), Source “isolates,” >1200 bp
sequences (fairly complete sequences, not fragments), and "good" quality. Set KNN matches to “5.”
Click on "submit."
Under the "hierarchy view", click on "View selectable matches." You will be presented with a list
of organisms clustered in taxonomic groups. As we're fairly certain that we are looking at
Pseudomonas species, focus on those sequences (in the genus Pseudomonas). Each taxonomic
group name is followed by three numbers in parentheses, for example, (0/20/116). The first number
is the number of organisms that the program has selected as good matches for your query sequence;
the second number is the number of somewhat reasonable matches; and the third number is the total
number of sequences in that taxonomic category in the database.

Within each genus, there is a list of species; for each species, the following information is
presented. The first number, in blue, is a clickable link to that organism's 16S rRNA gene sequence
file in Genbank. The similarity score is highlighted in pink, although it is often not calculated and
the words "not_calculated" will appear highlighted in pink. Highlighted in orange is the S_ab score,
a measure of the degree of relatedness of your query sequence and the sequence listed. The higher
the S_ab number, the more related the two sequences, and identical sequences have an S_ab score
of 1.000. The final number shown is the number of unique common oligomers (not relevant for our
purposes), followed by the genus and species name of the organism

Downloading sequences from the RDP for use in other programs

Click on the blue number to open another window with the GenBank file for the organism’s 16S
rRNA gene. The fourth line lists the source organism, and published references describing the
species of Pseudomonas are 40 listed next. Finally, the file contains the entire 16S rRNA gene
sequence. Remember that this is the DNA sequence for a structural RNA – it will have T's not U's.
The sequence is presented in groups of 10 nucleotides, 6 groups to a line, and each line is
numbered. To convert this sequence to fasta format and to lose the gaps and numbers and lower
case, go to a sequence conversion web site, e.g., http://www-bimas.cit.nih.gov/molbio/readseq/.
Simply copy the DNA sequence (with gaps and numbers), paste it in the box, and click "submit."
Copy the result to a Word file.

Aligning DNA sequences

The Clustal program accepts multiple sequences in fasta format and performs an alignment of those
sequences. The program can be found at http://www.ebi.ac.uk/Tools/msa/clustalw2/. Copy the
sequences from your Word document and paste them into the box. Accept all of the default settings
and click on "run." Wait a few seconds for the program to run, and the output of the program (scroll
down...) is an alignment. At each position where the sequences are identical, an asterisk appears
below the alignment; at positions where the sequences differ, there is no symbol. This allows you to
scan visually and locate differences between the sequences. The alignment can be copied and pasted
into Word for presentation (use landscape orientation of the page). Note that if one of your
sequences is upper case and the other is lower case, the program will not add asterisks.

Examining alignments and extracting meaning from them

The 16S rRNA bacterial gene is about 1500 bp long and an alignment of several Pseudomonas
genes will be extensive. Positions in the alignmentwith asterisks are not overly informative, as
those represent portions of the 16S rRNA that are identical in all Pseudomonas species. Look
instead at the positions without an asterisk, where there are differences between/among the
sequences.

Comparative Phylogeny Analysis

Our study aimed at conducting a comparative analysis of the phylogenies obtained from the 16S
rRNA gene in source isolates and investigating the physiological characteristics correlated with
phylogeny. Here we report a significant congruence between the 16S rRNA gene phylogenies.
Pseudomonas isolates are grouped into one cluster through phylogenetic analysis of 16S rRNA
genes.

Many methods were used to measure individual phylogenetic trees of the 16S rRNA gene: JukesCantor;
maximum probability; maximum parsimony; and minimum evolution; to compare the phylogenetic
relationships between the species of Pseudomonas. There were no significant differences between
trees, regardless of the method used for clustering. All the trees are clear and the key lines have been
shown.

The strain's 16S rRNA gene has also been amplified and sequenced. Sequences of the 16S rRNA gene
from 23 excellently-characterized reference strains comprising the main Pseudomonas phyla were used
to compare with previous studies. Analyzes were also carried out on strains in the database with 16S
rRNA sequences of the strain. As revealed by the Distance Matrix (DM) process, the resulting
phylogenetic tree is identical to the tree constructed by maximum parsimony (MP) and maximum
likelihood (ML) for the well-supported branches. We use clusters based on 16S rRNA gene data, r-
clusters, and those based to simplify comparisons. Data shows the names of the 16S rRNA sequence for
the various strains in which 16S rRNA was determined. The cluster of aeruginosa is closed and is
distinctly identical to the others. One of the strains in this cluster has a 16S rRNA gene that is very similar
to the reference strains of P. aeruginosa (percent). This finding is consistent with the findings of the API
and pigment development on growth media

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