1

1 R. Christian McDonald

2 BIOL-515, Spring 2018

3 February 27, 2018

4 Introduction

5 Metagenomics is the process by which entire communities of microorganisms are

6studied in mass in order to investigate the underlying abundance of microbial biodiversity and

7emergent ecology (Handelsman et al. 1998). Traditionally, these communal sources of

8microorganisms have included soil (Kakirde et al., 2010), water (Venter et al., 2004), and the

9human gut (Qin et al., 2010). However, metagenomics can technically be applied to any

10community of microorganisms. In contrast to traditional cultivation-based genomics, which

11intentionally cultures and targets very specific microbial colonies for analysis, metagenomics

12offers a fundamentally more comprehensive overview. Due to the rapid commercialization of

13ever increasingly more affordable “next-generation” sequencing technologies, metagenomic

14investigations have become a quick and economical means of performing bulk explorative

15studies on microbial biodiversity.

16 Two principle metagenomic techniques exist for exploring microbial biodiversity: 16S

17ribosomal RNA (rRNA) amplicon sequencing and whole-genome “shotgun” sequencing

18(Sharpton, 2014). Carl Woese (1977) pioneered the use of the 16S rRNA locus as an informative

19phylogenetically marker in the classification and identification of microorganisms. Woese

20defined bacterial species on the premise of differences in variable regions of the 16S rRNA locus

21(≈3%). The study of microbiota on the basis of 16S amplicon sequencing has revealed an
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22abundant and explosive microbial biodiversity across an expanse of communal sources

23(Sharpton, 2014). However, 16S amplicon sequencing is limited to the scope of taxonomic-

24based investigations. In contrast, whole-genome “shotgun” metagenomics capitalizes on those

25economical “next-generation” sequencing technologies in order to ask deeper, functional-

26based questions concerning microbial genomes.

27 In this short study, we investigated the microbial biodiversity of two soil samples

28collected at two distinct points along the southern bank of the St. Joseph River on the campus

29of Andrews University, Berrien Springs, Michigan (Fig. 1-2). Cultivation-based 16S amplicon

30sequencing and BLAST analysis revealed two distinct bacterial species: Pseudomonas koreensis

31and Bacillus sp. However, a true metagenomic 16S amplicon sequencing analysis has not yet

32been performed.

33 Methods and Results

34 Two surface soil samples were collected along the southern bank of the St. Joseph River

35on the campus of Andrews University, Berrien Springs, Michigan (Fig. 1-2). Each sample was

36collected in a sterile collection tube on January 15, 2018 and stored at 4°C for approximately 24

37hours. Each soil sample was plated on LB agar plates (Fig. 3). Positive (with water) and negative

38(without water) control plates were also prepared (Fig. 3). The plates were incubated at room

39temperature for approximately 72 hours before being subsequently wrapped with Parafilm and

40stored upside-down at 4°C.

41 The 16S rRNA loci from two specific bacterial genomes (one from each soil plate) and

42from the both soil metagenomes (S1 and S2) were amplified using conventional PCR and
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43visualized using gel electrophoresis (Fig. 4). We were successful in amplifying the 16S rRNA loci

44in both soil samples and the first bacterial colony (C1). However, we were unsuccessful in

45amplifying the 16S rRNA locus in C2. The unsuccessful amplification could be caused by

46procedural error or by the erroneous isolation of a non-bacterial colony. The successful

47amplification of both soil samples does suggests that both soil samples do contain copious

48amounts of bacteria. However, the faint band in S2 suggest less metagenomic 16S rRNA

49amplification when compared to S1 (C1) (Fig. 4).

50 The C1 and S1 gel bands were isolated and purified via gel extraction techniques.

51Quantitative PCR (qPCR) was performed in order to quantify 16S rRNA copy number and the

52proportion of bacterial-derived DNA from both soil samples (S1-2) and from both isolated

53colonies (C1-2). The qPCR results were grossly inconclusive and likely reflect some form of

54procedural error (Table 1). The number of 16S copies that were determined by qPCR failed to

55coincide with the general trend that we expected based on the DNA dilutions that we prepared.

56However, the similarities between the various melting curves and peak melting temperatures

57(Fig. 5) do suggest that our samples at least contain fragments of similar size and chemistry.

58 The 16S rRNA amplicons of both colonial samples (C1 and C2) were Sanger sequenced

59by a commercially available, third-party sequencing service. The C2 amplification, which was

60previously unsuccessful, was repeated by Dr. Peter Lyons prior to Sanger sequencing. The

61sequencing results were trimmed in order to eliminate potentially erroneous data. Bases 1-33

62and 539-639 were removed from the C1 sequence; and bases 1-20 and 544-564 were removed

63from the C2 sequence. Each trimmed sequence was analyzed using Nucleotide BLAST analysis

64via the NCBI. Each sequence revealed confident alignment matches. C1 aligned highly
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65confidently with Pseudomonas koreensis. However, C2 aligned with similar confidence against

66several dozens of strains of Bacillus sp. (with Bacillus wiedmanni being the top hit) (Fig. 7). The

67trimmed Sanger sequences and two BLAST-matched sequences (P. koreensis. and Bacillus sp.)

68were aligned using MUSCLE in order to highlight sequence similarities and differences (Fig. 6).

69 Discussion

70 Both Pseudomonas sp. and Bacillus sp. are highly ubiquitous in nature and can occupy a

71wide variety of ecological niches. Therefore, the inclusion of P. koreensis. and Bacillus sp. in our

72soil samples is not particularly all that revealing. Kwon et al. (2003) identified P. koreensis. as a

73novel gram-negative, non-spore-forming strain isolated in agriculturally-derived soil in Korea.

74Conversely, members of Bacillus sp. are gram-positive and spore-forming. Both Bacillus sp. and

75P. koreensis. are both rod-shaped.

76 Although P. koreensis. lacks highly detailed characterization, Bacillus sp. has significant

77roles in industry and scientific research. However, much work is still being done in order to

78characterize the properties of P. koreensis. For example, Anbu (2013) characterized an

79extracellular lipase produced by P. koreensis. Furthermore, Priest (1977) recognized the

80relevance of Bacillus sp. in the production of industrially, medically, and scientifically relevant

81extracellular enzymes (e.g. for brewing, for medicine, and for timber preservation, etc.).

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85

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87 Tables and Figures

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89Figure 1: A map of the St. Joseph River on the campus of Andrews University, Berrien Springs,

90Michigan. Samples (1 and 2) were collected on January 15, 2018.

91
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92Figure 2: Coordinates from where the two soil samples were collected. Sample 1 is shown on

93the left and sample 2 is shown on the right.

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95

96Figure 3: Cultivation of soil microbes on agar plates. The top plates are positive and negative

97controls (with water and without water). The bottom plates are soil samples 1 and 2. One

98colony (circled) from each soil plate was harvested for cultivation-based 16S rRNA sequencing.

99
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100Figure 4: Gel electrophoresis of 16S rRNA amplification from four specimens: 2 bacterial

101colonies (C1-2) and 2 soil samples (S1-2). Sample numbers indicate sample source (Fig. 1 & 2).

102GeneRuler 1000bp Plus DNA ladder was used in the first lane (R).
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103

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105
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106Figure 5: Quantitative PCR (SYBR Green) was used to quantify the 16S rRNA copy number and

107the proportion of bacterial-derived DNA from both soil samples and both colonial samples.

108

109Figure 6: Truncated MUSCLE alignment results highlighting the relationship between C1 and

110Pseudomonas koreensis; and C2 and Bacillus sp.

111>C1
112GCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAA-
113GGGTTGTAGATTAATACTCTGCAATTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGG
114TAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAA
115ATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGT
116AGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGC
117GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCT
118TGAGCTC-TTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTA
119>C2
120GCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACC
121TTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCC
122GGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTC
123ATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGA
124GGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTA
125GATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACG
126CATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTG
127>Pseudomonas koreensis
128GCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAA-
129GGGTTGTAGATTAATACTCTGCAATTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGG
130TAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAA
131ATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGT
132AGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGC
133GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCT
134TGAGCTC-TTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTA
135>Bacillus sp.
136CGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCT
137TGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCG
138GAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCA
139TTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATATGGAG
140GAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAG
141ATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGC
142ATTAAGCACTCCGCCTGGGGAGTACGGCCGCAAGGCTGA
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143Figure 7: FASTA format of both colonial 16S rRNA sequences determined via Sanger Sequencing

144(C1 and C2) and two relevant bacterial species identified via Nucleotide BLAST analysis.

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qPCR Quantification Data

Sample Ct log(Copy #) Copy # (10^) 1/8
C1 (1/10) 19.93 49.49 3.11E+49 3.89E+48
C2 (1/10) 21.35 52.46 2.92E+52 3.64E+51
S1 (1/10) 18.15 45.77 5.86E+45 7.32E+44
S2 (1/20) 17.6 44.62 4.14E+44 5.17E+43
Control 16S (1/10) 20.34 50.35 2.24E+50 2.80E+49
C2 (1/80) 20.15 49.95 8.98E+49 1.12E+49
C2 (1/80) 20.35 50.37 2.35E+50 2.94E+49
S1 (1/80) 19.31 48.20 1.57E+48 1.96E+49
S2 (1/80) 18.21 45.89 7.82E+45 9.78E+44
Control 16S (1/80) 18.45 46.40 2.49E+46 3.11E+45
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150Table 1: Quantitative PCR results.

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160 Literature Cited

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