Pseudomonas frederiksbergensis

About: Pseudomonas frederiksbergenis is a gram-negative bacteria that was originally identified in Frederiksberg, Copenhagen, Denmark at a coal gasification site. This bacteria is rod shaped, and colonies are known to be a pale yellow. Pseudomonas is known to be a species that is important for soil, food and water decomposition.

Date Collected: 2/8/2017

Methods of isolation and characterization: 

  • P. frederiksbergenis was isolated from a specific soil sample at the Environmental Education Center (EEC) located in Farmville, VA.
  • The soil sample was taken directly from Buffalo Creek, a small creek that flows into the Appomattox river.
  • Once we collected our sample we plated it onto three agar plates: direct sample, 1/10 and 1/100 dilution.
  • We observed growth at 24hrs and 48hrs and selected a specific colony to analyze that was not overlapping other colonies.
  • We ran DNA isolation and purification through Nano Drop to ensure we had a pure sample to then proceed to PCR amplification of 16s rRNA.
  • After isolating 16s rRNA of our colony we ran gel electrophoresis against MSp1 to ensure we isolated 16s rRNA to send off for sequencing.
  • Sadly we were unable to draw any conclusions from our gel due to clarity issues, never the less we went ahead and sent off our sample off to be sequenced.
  • Once sequenced, we than took our sequence and ran it through BLAST analysis which gave us a 99% match
  • Using our data analysis from BLAST as well as the color, form, and size of our colony we identified our sample to be P. frederiksbergenis.

Results:

 

Figure 1. Chromatogram of our Buffalo Creek soil sample, which we identified as P. frederiksbergenis.

 

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Figure 2. Buffalo Creek soil sample and location in which we isolated P. frederiksbergenis.

 

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Figure 3. Our colony of P. frederiksbergenis under a microscope. This photo highlights the pale yellow color that we used to further support our classification.

 

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Figure 4.   The 1/100 dilution plate of the Buffalo Creek soil shows the specific colony we isolated P. frederiksbergenis.

 

References:

Pseudomonas frederiksbergensis sp. nov., isolated from soil at a coal gasification site.
S. M. Andersen, K. Johnsen, J. Sørensen, P. Nielsen, C. S. Jacobsen
Int J Syst Evol Microbiol. 2000 Nov; 50(Pt 6): 1957–1964. doi: 10.1099/00207713-50-6-1957

 

Janthinobacterium lividum

Janthinobacterium lividum

Background

Janthinobacterium lividum is a gram-negative bacteria that has aerobic qualities.  It possess a pigment called violacein that gives the bacteria its dark purple color (Figure 1).

farm soil circle

Figure 1: Janthinobacterium lividum colony on agar plate

 Janthinobacterium lividum is commonly found in the skin of amphibians such as salamanders and expresses fungal and tumor fighting properties.

Date collected – February 18, 2017

References

Valdes, Natalia et al. (2015) “Draft genome sequence of Janthinobacterium livid strain MTR reveals its mechanism of capnophilic behavior.”

Rebiller, Eria et al. (2016) “Direct and Indirect Horizontal Transmission of the Antifungal Probiotic Bacterium Janthinobacterium livid on Green Frog (Lithobates clamitans) Tadpoles.”

Methods for Isolation and Identification

  • Soil was collected from Buffalo Creek close to a nearby farm (Figure 2)EEC siteellie collecting waterellie wit dirt

Figure 2: Collection site and the collection of surface water and bank soil

  • Sample was mixed with water, diluted, spread on agar plates, and incubated for 48 hours at room temperature
  • Colony was isolated and genomic DNA was extracted
  • PCR amplification was conducted to replicate the DNA and prepare for gel electrophoresis
  • Gel electrophoresis was used to compare the different nucleotide bases and attempt to identify the bacterial strain. However, the gel electrophoresis was unsuccessful
  • The sample was sent to be sequenced over Spring Break.  The sequences were uploaded into SnapGene Viewer, edited, and pasted into BLAST

Results

  • The sequences were compared to the matches given by BLAST.  Janthinobacterium lividum was a 99% match with few gaps, so it was concluded that this was the colony that was isolated was a fungus (Figure 3)

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Figure 3: Gel electrophoresis pictures show little success

  • The BLAST match came up with a 99% match, 1 gap, and 977 nucleotides (Figure 4).  This suggests that the bacteria isolated was almost guaranteed to be Janthinobacterium lividum

 

BLAST match

Figure 4: BLAST sequence versus sample sequence

 

Murriel Grimes, Alyssa Oppedisano, Elle Richardson

Psudomonas Frederiksbergenesis strain dsm 13022

Pseudomonas frederiksbergensis is a Gram-negative bacteria. It was discovered by from a coal gasification site in Frederiksberg, Copenhagen, Denmark. This bacteria was cultured by Andersen S, Johnsen K, Sorensen J, Nielsen P, Jacobsen C. in 2000.

References: 

  • Andersen S, Johnsen K, Sorensen J, Nielsen P, Jacobsen C. 2000. Pseudomonas fredricksbergensis sp. nov., isolated from soil at a coal gasification site. Int. J. of Syst. and Evol. Microbiol. 50:1957-1964.
Date collected: February 8, 2017
Methods for isolation and identification:
  • Soil was taken from the Appomattox River (Figure 1), diluted by 1:10, then placed on an agar plate and isolated at room temperature for 48 hours
  • A purple colony was taken from the plate for DNA isolation and PCR amplification
  • When the PCR product was produced, it was digested with MSP1 and run through a gel
  • Once the PCR was done and the DNA was purified it was sent of for sequencing so we could determine what bacteria it was
appomattoxFigure 1: Collection site at the Appomattox River
Results:
  • The sequenced PCR product for the 16s rRNA gene generated 714 bases that were used to identify the genus and species of the bacteria colony. NCBI BLAST analysis revealed 99% identity with the bases of the 16s rRNA gene of Pseudomonas Fredricksbergenesis. There was found to be no gaps and 714 out of 716 base pairs were matched. Below shows the Blast percentages (Figure 2). Figure 3 shows the base pairs. Attatched is a more clear visual of the Blast sequence: http://blogs.longwood.edu/pecmicrobes/files/2017/04/Sequenced-dna.pdf
Score Expect Identities Gaps Strand Frame
1312 bits(710) 0.0() 714/716(99%) 0/716(0%) Plus/Plus

Figure 2: Blast data

Sequence

Figure 3: The blast base pairs

Contributed by: Alyssa Oppedisano, Elle Richardson, Murriel Grimes BIOL 250 – Spring 2017, Longwood University

 

Bacillus cereus

Bacillus cereus is a Gram-positive, rod-shaped, aerobic, motile, beta hemolytic bacterium commonly found in soil and food.

Reference:

  • Been, M. De. “Comparative analysis of two-component signal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis.” Microbiology 152.10 (2006): 3035-048. Web.

Date collected: February 8, 2017

Methods for isolation and identification:

  1. Collection and Sampling of Environmental Samples at Environmental Education Center (Figure 1)
  2. Selection of bacteria from plate to be analyzed (Figure 2)
  3. Polymerase Chain Reaction to amplify 16S rRNA
  4. Restriction Enzyme Digestion & Gel Electrophoresis (Figure 3)
  5. DNA Sequencing Analysis/Identification

 

map

Figure 1. Sampling site locations in Lancer Park. The top site is Buffalo Creek and the lower site is the pond where samples were taken

PT10

Figure 2: picture of Bacillus cereus

Results:

  • MspI digestion (Figure 3): A 1,500 bp product was amplified by PCR. Upon digestion with a band at 500 bp digested and a 1000 bp undigested.

PW1 electrophoresis

Figure 3: Mspi digestion of Bacillus cereus PCR product

  • Sequence analysis (Figure 4): The sequenced PCR product generated 887 bases. NCBI BLAST analysis revealed 99% identity with 887 of the bases matching the 16s rRNA gene of Bacillus cereus (Figure 4).

PT10 sequence

  • Figure 4: The BLAST sequence match of the sample bacteria to Bacillus cereus

Contributed by: Matthew W. Bowman & Carly Carter, BIOL 250 – Spring 2017, Longwood University

Bacillus megaterium

Bacillus megaterium is a cytokinin promoting bacterium used to promote plant root overgrowth. The possible applications of Bacillus megaterium is being examined  in the deserts of Egypt for the promotion of desert agriculture by Köberl and others. It is a gram-positive, rod shaped, spore forming bacteria. It is used in the biocontrol of plant diseases and nitrogen fixation has been demonstrated in some strains (2011).

References:

Köberl, Martina ; Müller, Henry ; Ramadan, Elshahat M. ;  Berg, Gabriele. 2011. Desert Farming Benefits from Microbial Potential in Arid Soils and Promotes Diversity and Plant Health. PLOS. 6 (9):): e24452.

Date of Collection from Buffalo Creek Shore: February 8

Methods for isolation and identification:

  • 1.1 g of soil from the shore of Buffalo Creek (Figure 1) was combined with 12 mL of sterilized water. 20 uL of the fluid was then added to 180 uL sterilized water to produce the Shore 1:10 dilution sample. This dilution was spread onto a nutrient agar plate using a sterile plastic hook. The plate was left to grow at room temperature for 48 hours.
  • A bright yellow colony was chosen for DNA sequencing and isolation (Figure 2). The 16s rRNA of the colony was amplified using PCR whose product was then digested via MspI.
  • Identity of bacteria was determined using DNA sequence.

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Figure 1: Shore Collection Site

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Figure 2: Colony Isolated for Identification

Results:

  • Gel Electrophoresis for MspI Digestion (Figure 3): Purity of product was indicated by prominence of base-pair marker. Both bars were prominent indicating purity of product was high. MspI digestion product was lodged at the start of the lane and unable to be run. 1550 base pairs was determined to be the amount of base pairs in the undigested PCR products.

Screenshot (10)

Figure 3: Gel Electrophoresis Plate for Shore Bacteria.

  • Identification of Bacteria using BLAST (Figure 4): The sequence of Bacillus megaterium was compared at the gaps to our DNA sequence. This file contains the DNA sequence of the isolated bacteria: SETAS_PREMIX_JF7529_1. The closest match to our bacterium was Bacillus megaterium.

mkhb

Figure 4: BLAST Query For Bacteria Identified as Bacillus megaterium.

Contributed to by: Sandra Elsakr and Taylor Alvey Biology 250, Spring 2017.

Pseudomonas Arsenicoxydans

Pseudomonas Arsenicoxydans was isolated from the soil of the Appomattox River. It was first discovered in sediment samples from the Atacama Desert in Chile, which has a high arsenic concentration in both the water and sediments (Campos et al. 2010).

References:

Campos V, Valenzuela C, Yarza P, Kampfer P, Vidal R, Zaror C, Mondaca M, Lopez-Lopez A, Rossello-Mora R. Pseudomonas arsenicoxydans sp nov., an arsenite-oxidizing strain isolated from the Atacama desert. Systematic and Applied Microbiology. 2010;33:193-197.

Date Collected: February 8, 2017

Methods for isolation and identification:

  • A soil sample was taken where the shore met the water at the Appomattox River (Figure 1). 100μl of the sample was put on an agar plate and was incubated at 25 degrees Celsius for 48 hours.
  • A light yellow-greenish, circular colony (Figure 2) was chosen for 16s rRNA gene sequencing by PCR amplification.
  • The PCR product was sent for DNA sequencing to identify the unknown bacteria of the colony selected.

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Figure 1. Site of collection.

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Figure 2. Colony from the Appomattox River sample selected for identification.

Results:

  • MspI digestion (Figure 3): Bands can be seen at the 1550, 600, and 500 marker.

gel

Figure 3. Results of PCR amplification and MspI digestion for Appomattox River.

  • DNA Sequence: The sequenced PCR product produced 862 quality base pairs that were used to identify the bacteria as Pseudomonas Arsenicoxydans. Here is the link to the full sequence: https://app.box.com/file/143282805134. NCBI BLAST revealed 98% similarity with 3 gaps out of 862 base pairs (Figure 4).

seq

Figure 4. BLAST Alignment of Pseudomonas Arsenicoxydans.

Contributed by: Andrea Soles, BIOL 250, Spring 2017

Bacillus pumilus colony 13A

Bacillus pumilus is a gram-positive motile purple pigmented bacteria that has been found from penaeid shrimp that is resistant to marine pathogens.

References:

  •  J E Hill, J C F Baiano, A C Barnes, Isolation of a novel strain of Bacillus pumilus from penaeid shrimp that is inhibitory against marine pathogens; Journal of Fish Diseases, 2009, Vol 32:1007-1016

Date Collected: February 9, 2017

Methods for Isolation and Identification:

  • A soil sample was collected 11.1 inches into Buffalo Creek (Figure 1).  A nutrient broth was added to the sample and it was spread onto an LB Agar using a sterile hockey stick. The agar was incubated at 30 degrees Celsius.
  • A small purple colony (Figure 2) was selected for 16S rRNA gene sequencing by PCR amplification.
  • The PCR product was digested and sequenced in order to identify the bacteria.

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Figure 1. Collection site

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Figure 2. Selected colony

Results:

  • MspI digestion (Figure 3) : The amplification of our PCR product was about 1500 bp long. Upon digestion with MspI, about 3 bands appeared at about 500 bp, 350 bp, and 200 bp were identified.

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Figure 3. Gel electrophoresis of PCR amplification and MspI digestion

  • Sequence Analysis (Figure 4): The PCR product generated 1167 bases of high quality reads. NCBI Blast analysis revealed that 98% identify with the bacteria Bacillus pumilus. 

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Figure 4. NCBI Blast analysis of colony 13A

Contributed by: Dayna Rouse and Jada Russell, BIOL 250 Spring 2017, Group 13