Planococcus sp. Isolated from sub-artic decomposing wood.

Morgen Southwood April 26, 2017

 

Introduction

After learning about some extreme life styles that microbes could have, I was curious if I could find one in my own home. I sampled two places, a piece of wood from my fire wood stack (which is exposed to the extreme cold temperatures of Alaskan winters) and the ashes from my stove. If any microbes grew from the firewood it would mean that they were able to withstand temperatures below -50 degrees Celsius and be psycrophiles, if any microbes grew in my wood stove they would have had to be thermophiles. I was unable to grow any microbes from the wood stove but the growth from the previously frozen wood was abundant. Each of those colonies represented a microbe that had the physiological capability of surviving those conditions, and I was interested in observing them in the lab.  The identification of this microbe would add to the depth of knowledge of what bacteria are and can be found on decomposing wood in the sub-artic.

The firewood pile was slowly decomposing, and it is likely that the microbe that was isolated from it was a part of the community that was utilizing the wood as an energy source. Decomposing communities frequently have a mix of fungi and bacteria. These communities emit extracellular enzymes to decay the wood, this results in an environment with high acidity and enzymes that are producing free radical oxygen species⁷_. To thrive in this community it would be helpful to have mechanisms to process radical oxygen species. One such mechanism is the use of Catalase to catalyze the transformation of the reactive species hydrogen peroxide into O₂. It is natural for bacteria in these community’s to be adapted to oxidative stress⁸

Throughout the course of this study I isolated one colony and performed many physiological and genomic tests to identify what species my isolate was. Considering the environment in which it was found, there were a few expected results: such as the microbe’s oxygen class and catalase test results. Once identified, I could then research the species and compare it’s physiology and genomic data to current literature to confirm the taxonomic assignment.

 

Methods

Collecting sample and isolation process

The sample was collected from a dry piece of wood. Sterile water was used to moisten the wood and a sterile swab was rolled along the wood’s surface.     The sample was streaked using the quadratic method to grow pure isolated colonies onto a tryptic soy agar (TSA) plate. To control growth rates the plates were grown in incubators for 2-4 days at 37 degrees Celsius then moved to a refrigerator to ensure colonies did not grow large enough to touch. The TSA medium provided a suitable environment for a variety of bacteria and a few molds. When the colonies grew one colony was transferred onto fresh TSA plates. The quadratic streak was used 4 times to ensure a pure culture. At this time the culture was transferred into an agar slant. Tests were performed on the isolate by obtaining cells from either the TSA plates or from the slant agar.

 

Physiological Characteristics

The gram stain was performed to assess the thickness of the peptidoglycan wall. The sample was stained using the provided protocol, gram-positives and negative controls were compared to the isolate to determine its gram-state (lab 4). While the cells were prepped in slides some characteristics were observed with the microscope, such as cell alignment shape and length. Colony morphology including color, size, margin and elevation was observed from growth on the TSA agar.

Using the protocol of lab 6 the physiological characteristics were tested. A suspension of the microbe in broth was tested simultaneously in all 21 of the tests included in the API 20E test strip. Additionally the isolate underwent the thioglycolate test- for oxygen class, oxidase test- for the presence of cytochrome c-oxidase, and lastly the catalase test- for the presence of catalase enzyme.

The cells inoculated onto Eosin Methylene Blue (EMB) and MacConkey Agar (MAC) plates. EMB is selective for gam-negative bacterium, if the microbe is gram negative and can ferment the sugars present then the plate experiences a color change. MAC plates are also selective for Gram-negative microbes and differentiates between lactose fermenters (agar turns pink) and non-lactose-fermenters (agar turns colorless).

The susceptibility of the isolate to 8 different antibiotics was tested using the Kirby-Bauer Method agar and the disk diffusion test per the protocol of Lab 9. The antibiotics tested were Amikacin, Cefazolin, Cefoperazone, Gentamicin, Piperacillin, Tobramycin, Trimethoprim and Vancomycin. After a two-day waiting period while the microbe was given the opportunity to develop where it could, the diameter of prohibited growth was measured and compared to standard antibiotic specific diameters and then classified as either susceptible, intermediate or resistant.

 

Bioinformatics

                      Dna was extracted and purified from the isolates cells using protocol from lab 5. Cell lysis was accomplished through adding sodium dodecyl sulfate (SDS), which is a surfactant that chemically weakened the cell walls, and the use of PowerBeads and the centrifuge to mechanically break the cell walls. A series of patented solutions C1-4 were added following the protocol to purify and remove inhibitors and proteins until all that remained was a pure solution of DNA. The DNA was then sent to the UAF’s DNA Core Lab sequenced using the Illumina MiSeq DNA sequencer which utilized the next-generation sequencing methodology.

Using Illumina’s BaseSpace dashboard to handle the data, the isolate’s genome was analyzed and compared to an existing database to search for similar sequences of DNA. BaseSpace has many apps that provide different information about the genomic data. The SPAdes Genome Assembler provided the overall result of the genomes assembly. Of this information, only the number of contigs that were greater than 1000 base pairs long [# contigs (>=1000bp)], The total length of the assembled genome, the longest contig and the guanine and cytosine percentage in the genome (GC%) were observed. The next app that was used was the Kraken Metagenomics, which provided taxonomic assignment. This app provided sample information such as the total number of reads that were used for classification and the percent of reads that were classified. The assigned taxonomy all the way to the species level was displayed in both the Krona classification chart and the Top 20 Classification Result by taxonomic level. Both sources have specific classification percentages that confer certainty. The final app Prokka Genome Annotation assigned gene regions that likely code for either tRNAs, rRNAs, CRISPRs and coding genes (CDs) and the abundance of them. The APP provides an extensive list of regions that were specifically identified as the above elements.

Further analysis of the genomic information was necessary. The contig.fasta file from the SPAdes Genome Assembly produced by Illumina’s BaseSpace was analyzed by BLAST. BLAST had a different database and could possibly have the genome of the isolates species if base space did not.     BLAST suggested Genus and species with associated query cover percentage and identity percentage.

 

Results

Physiological Characteristics

The Isolate appeared to be a pure culture by the third quadratic streak; a fourth was performed for certainty. The Microbes transfer to the agar slant was successful: Image 1, and was successfully re-cultured from the slant when necessary.

Image 1: Microbe growing on TSA agar

Image 2: Gram-stained microbe

The gram-stain was repeated three times before a successful stain was accomplished. The gram-stain revealed that the isolate was a gram-positive coccid: Image 2. While under the microscope it was observed that the cells could be aligned linearly, in pairs or quads but not in a longer strep chain. The purity of the isolate was confirmed with the microscopic observation of a mono-culture. The cells were measured to be 1 micrometer in diameter.  Colony morphology on the TSA agar revealed a yellow colony that’s size increased with time, a smooth round margin and the colony was an elevated domed.

Image 3: API 20E test results

The API 20e tests had negative results for all for the 21 tests: Image 3. Considering that the API 20e test is designed for enteric gram negative microbes these results are confirmation that the microbe is gram-positive. The thioglycolate test had growth throughout the agar with thicker growth at the surface, which revealed that the microbe’s oxygen class was facultative aerobe. The catalase test produced bubbles and the oxidase test produced a blue color change on the strip indicating that the microbe was positive in both tests. These results reveal that the cells contained both cytochrome c-oxidase and the catalase enzyme respectively. The isolate failed to grow on EMB or MAC plates, considering that both plates are selective for gram negative, these results confirm the results of the gram positive test: Image 4.

 

 

Physiological Tests	Results
Gram	Positive
Cell morphology	1 micrometer diameter, coccus, cells divide linearly
Colony Morphology	Yellow colonies that’s size increases with time, a smooth round margin, elevated dome.
API 20e	All negative results
Fluid thyioglycolate	Facultative anaerobe
Oxidase	Positive result
Catalase	Positive result
MAC	No growth
EMB	No growth

Table 1: Physiological Test Results

The microbe was susceptible to all eight antibiotics tested. The Inhibition Zones exceed the antibiotic specific threshold diameter for susceptibility see table 2 for details.

Antibiotic Name	Minimum Inhibition zone for susceptibility (mm)	Microbe exceed inhibition zone (yes/no)
Amakicin	>=17	Yes
Cefazolin	>=18	Yes
Cefoperazone	>=21	Yes
Gentamicin	>=15	Yes
Piperacillin     Enterobacteriaeae     S. pneumoniae	>=21 >=18	Yes Yes
Tobramycin	>=15	Yes
Trimethoprim	>=16	Yes
Vancomycin	>=17	Yes

Table 2: Antibiotic Susceptibility Results

Bionformatics

The analysis of the DNA with BaseSpace revealed unreliable data. BaseSpace’s SPAdes Gemone Assembler analyzed 128 contigs over 1000 base pairs (bp) long, a total length of over 3.7 million bp, the longest contig  was 196 thousand bp, and the GC% was 44.4%.     Kraken Metagenomics app was only able to classify 2.67% of reads, and of those reads only 17% of those reads identified the microbe as Lysinibacillus sphaericus. The Prokka Genome Annotation app identified 64 tRNAs, 0 rRNAs, 1 CRISPR, and 3739 CDS.     Image 5 and 6 are from the Kraken app.

Image 5: Krona Classification Chart from BaseSpace displays the large portion of unclassified reads.

Image 6 To 20 Classification Results by Taxonomic Level from BaseSpace

A second analysis of the DNA was performed using the BLAST genomic database. BLAST classified the organism as Planoccus donghaensis with a query cover of 77% and a 78% identity. Several other species had similar query cover, including Planococcus antarticus: Image 7.

Image 7: BLAST Genomic Results

Discussion

The genotypic test results for this microbe were vague, the results of BaseSpace had a low number of reads that could be classified and of those reads there was little consensus. The results of the SPAdes Genome Assembler were above required values; # contigs >=1000 bp should be in the hundreds, total length should be at least a million bp and the longest contig should be at least 100000 bp. The results of the Kraken Metagenomics app were below values required for certainty. The percent of contigs read needed to be greater than 80% and the percent of those reads that needed to agree upon a classification to trust that classification to the genus level was 80% and to the genus level was 60%. The results did not meet these thresholds, and this is the reason that the BaseSpace indentified taxanomy of Lysinibacillus sphaericus was rejected.

BLAST provided many species level classifications with similar query cover and percent identity. The species classification with the highest query cover is poorly documented in literature. The first publication of a species under the genus Planococcus was P. citreus. It was identified in 1894, and was only approved in 1980⁶. This genus is relatively young and is currently growing with many of the species being described within the last ten years. Of the few documents that relate to Planococcus donhaensis, there is one that notes the origin of the sample, the South Korean Sea. There are some qualities that are consistent between my sample and P. donghaensis, they are both, gram-positive, aerobic, coccus and oxidase positive ¹. However these similarities are not enough to convince me that they are the same species. A hallmark of the genus Planococcus is that they are usually halo-tolerant gram-positive bacteria that frequently inhabit Antartica⁴. The isolates holotolerance was not tested but the similar cellular membrane composition and habitat conditions indicate that this genus is likely correct.

The negative results and lack of growth in the API 20e, MAC, and EMB are all consistent with the microbe’s gram-positive cell wall. These tests reveal nothing more than a conformation that it is indeed a gram-positive microbe. The oxygen class determined by the fluid thioglycolate test and the positive results of the Oxidase and Catalase test are both consistent with the oxygenic environment that the microbe was isolated from. These tests proved that the microbe could thrive in an oxygenated as well as an anoxic environment, that it contained cytochrome c oxidase which is a part of the electron transport chain found in microbes that utilize oxygen, and that it contained the catalase mechanism for dealing with oxidative damage, respectively.

At the genus level, morphology can vary greatly. Many of the morphological and physiological analyses made on the isolate are not held by every species in the Planoccocus genus. The morphological characteristics that are consistent with the isolate and across the genus are: gram-positive membranes, cocci cell shape, colonies are yellow orange in color, catalase positive and an aerobic oxygen class⁵. My isolate was not just aerobic but a facultative aerobe, which is inconsistent with two articles that state the genus is strictly aerobic⁵. The isolate was found to be susceptible to all antibiotics tested with the disk diffusion test. This is consistent with a study that found, to the level of the genus, that Planococcus’ were susceptible to all antibiotics tested³.

The second most likely species suggested by was P. antarticus, at least this species shares a similar environment. P. antarticus thrived in an Antarctica, my sample would likely have similar mechanisms for surviving temperatures around -45 degrees Celsius in Faribanks winters. P. donghaensis may have had the ability to deal with these temperatures in its genome, but it is not certain since it’s environment doesn’t select for such characteristics.

The literature on the Planococcus species like donghaensis, kocurii, halocryophilus and antarticus often notes how closely related the species are and how further analysis like G-C content, fatty acid strains, DNA-DNA hybridization etc^1,2 are needed to differentiate the species. To conclusively Identify the taxonomy of this isolated microbe, it would be advisable to repeat DNA isolation and genomic analysis that was preformed in this project, and to additionally perform other genotypic analyses like, DNA-DNA hybridization and 16s rRNA analysis and fatty acid identity. Considering the limit of species identified to this date, there is the potential that this microbe could be a new species.

 

 

 

Work Cited

1. Jeong-Hwa C, Wan-Taek I, Qing-Mei L, Jae-Soo Y, Jae-Ho S, Sung-Keun R, Dong-Hyun R. Planococcus donghaensis nov, a starch-degrading bacterium isolated from the East Sea, South Korea. Int J Syst Evol Microbiol. 2007;57:2645—2650. doi: 10.1099/ijs.0.65036-0.

 

2. Reddy G, Prakash J, Vairamani M, Prabhakar S, Matsumoto G. Shivaji S. Planococcus antarcticus and Planococcus psychrophilus nov. isolated from cyanobacterial mat samples collected from ponds in Antarctica. Extremophiles. 2002;6:253—261.

 

3. Tuncer, I. (2016). Antibiotic resistance of bacterial isolates from sediments of eastern Mediterranean Sea in association with environmental parameters. Journal of Bacteriology and Parasitology, 7(6), 51. https://doi.org/10.4172/2155-9597.C1.025

 

4. See-Too, W. S., Ee, R., Lim, Y.-L., Convey, P., Pearce, D. A., Yin, W.-F., & Chan, K.-G. (2017). AidP, a novel N-Acyl homoserine lactonase gene from Antarctic Planococcus Scientific Reports, 7, 42968. https://doi.org/10.1038/srep42968

 

5.         Fackrell, H. (n.d.). Planococcus. Retrieved April 11, 2017, from Uwindsor.ca website: https://web2.uwindsor.ca/courses/biology/fackrell/Microbes/17375.htm

6. Euzeby, J. P., & Parte, A. C. (2017, April 1). Genus planococcus. Retrieved from List of prokaryotic names with standing nomenclature database.

 

7. Valášková V., de Boer W., Gunnewiek P. J. K., Pospíšek M., Baldrian P. (2009).  Phylogenetic composition and properties of bacteria coexisting with the fungus  Hypholoma fascicularein decaying wood.  ISME J.  3  1218—1221. 10.1038/ismej.2009.64
8. de Boer W., van der Wal A. (2008).  “Interactions between saprotrophic basidiomycetes and bacteria,’ in  British Mycological Society Symposia Series 8  eds Lynne Boddy J. C. F., van Pieter W., editors. (Cambridge, MA: Academic Press; )  28  143—153.