Two of the ribosomal sequence from the bacterial isolates matched the representative
sequence of 2 of 3 dominant OTUs assembled from the 454 pyrosequencing data. DNA
from the isolates was isolated and amplified using the standard 27F -1492R primer pair.
PCR conditions were as follows 5 minutes 94°C, 30 cycles of 94°C for 30 sec, 57°C for 45
sec and 72°C for 90 sec with the final elongation step at 72°C for 7 minutes. The PCR
product was purified and sequenced using Sanger technology. Achromobacter isolated
from the M. truncatula rhizosphere, when compared using BLAST, showed 99 %
similarity against a representative sequence from OTU 19 (score of 544, e value = 1e159). Arthrobacter sp. isolated from the B. distachyon rhizosphere, when compared
using BLAST, showed 100 % similarity against a representative sequence from OTU 39
(score of 556, e value = 7e-163). Alignments were based on the V1-V2 16S rRNA gene
only. Genomic DNA was isolated from both strains and they were sequenced using half a
plate of 454Flx each. Achromobacter gDNA sequencing produced 50 contigs (of which
14 were smaller than 100 bp in length). Arthrobacter gDNA sequencing produced 48
contigs (of which 17 were smaller than 100 bp in length).
The bacterium isolated from the rhizosphere of Medicago was identified as
Achromobacter xylosoxidans (top hit in the NCBI database – accession number:
NR_074754.1, based on the full 16S rRNA sequence and the colony isolated from the
Brachypodium rhizosphere was identified as Arthrobacter sp. (top hit in the NCBI
database – accession number EF110914.1, based on the full 16S rRNA sequence – gDNA
sequencing (Figure 1). Full 16S rRNA gene sequences were obtained from the genomic
DNA FASTA file using online RNAmmer 1.2 server (a part of CBS prediction server)
(Lagesen et al 2007). However, using the full 16S rRNA gene sequences would skew the
results towards fully sequenced strains of these species, so in order to fully understand
the phylogeny of these strains a partial 16S rRNA gene (1460 and 1486 bp of sequence
starting at 27 bp from the start of the gene for Achromobacter and Arthrobacter,
respectively) was compared (BLAST) against the GenBank database.
A
Achromobacter xylosoxidans
Achromobacter xylosoxidans subsp. xylosoxidans strain A19 16S ribosomal RNA
isolate 16S rRNA gene
Achromobacter xylosoxidans A8 complete genome
Achromobacter xylosoxidans A8 complete genome
Achromobacter xylosoxidans A8 strain A8 16S ribosomal RNA
Achromobacter sp. F32 16S
Achromobacter sp. R-46660
Arsenite-oxidizing bacterium Alcaligenes fecalis (HLE)
Alcaligenes sp. 16S rRNA
Achromobacter spp.
Alcaligenes faecalis strain N05
Achromobacter spp.
Achromobacter spp.
Achromobacter spp.
Achromobacter spp.
Achromobacter spp.
Bordetella
Pigmentiphaga daeguensis strain ML-3
Pusillimonas sp. YC6271
Rhodobacter
Ochrobactrum pseudogrignonense strain 21-6PIN
Tetrathiobacter kashmirensis strain 3T5F
Denitrobacter sp. CHNCT17
Burkholderia sp.
Kerstersia gyiorum strain HF2
Burkholderiales
Limnobacter thiooxidans strain CS-K2
Cupriavidus sp. ASC-9842
Pseudomonas lemoignei strain ATCC 17989T
Undibacterium sp. CMJ-15
Oxalobacteraceae
Achromobacter isolate 16S rRNA sequence used for comparison:
ACGCTAGCGGGATGCCTTACACATGCAAGTCGAACGGCAGCACGGACTTCGGTCTGGTGGCGAGTGGCGAACGGGTGAGTAAT
GTATCGGAACGTGCCTAGTAGCGGGGGATAACTACGCGAAAGCGTAGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGG
ATCGCAAGACCTTGCACTATTAGAGCGGCCGATATCGGATTAGCTAGTTGGTGGGGTAACGGCTCACCAAGGCGACGATCCGT
AGCTGGTTTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATTTTGG
ACAATGGGGGAAACCCTGATCCAGCCATCCCGCGTGTGCGATGAAGGCCTTCGGGTTGTAAAGCACTTTTGGCAGGAAAGAAA
CGTCATGGGCTAATACCCCGTGAAACTGACGGTACCTGCAGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACG
TAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTCGGAAAGAAAGATGTGAAATCCCAGAGCTC
AACTTTGGAACTGCATTTTTAACTACCGGGCTAGAGTGTGTCAGAGGGAGGTGGAATTCCGCGTGTAGCAGTGAAATGCGTAG
ATATGCGGAGGAACACCGATGGCGAAGGCAGCCTCCTGGGATAACACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGG
ATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTAGCTGTTGGGGCCTTCGGGCCTTAGTAGCGCAGCTAACGCGTG
AAGTTGACCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGATGATGTGG
ATTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTCTGGAATTCCGAAGAGATTTGGAAGTGCTCGCAAGAGA
ACCGGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTC
ATTAGTTGCTACGAAAGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGC
CCTTATGGGTAGGGCTTCACACGTCATACAATGGTCGGGACAGAGGGTCGCCAACCCGCGAGGGGGAGCCAATCCCAGAAACCC
GATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGTCGCGGTGAA
TACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTTTACCAGAAGTAGTTAGCCTAACCGTAAGGGGGG
CGATTACCACGGTAGGATTCATGACTGGGGTGAAGTCGTAACAA
Arthrobacter sp.
Arthrobacter sp. S21003
Arthrobacter sp. B1033
Arthrobacter sp. S21004
Arthrobacter sp. J3.31
Arthrobacter sp. S22236
B
Arthrobacter humicola strain KV-653
Arthrobacter spp.
Arthrobacter sp. J3.46
isolate 16S rRNA gene
Arthrobacter sp. J3.16
Arthrobacter sp. J3.33
Actinobacterium EC5
Microbacterium sp.
Arthrobacter spp.
Arthrobacter sp.
Arthrobacter spp.
Actinobacterium MES16
Arthrobacter sp. bD37(2011)
Arthrobacter spp.
Bacterium W10
Arthrobacter spp.
Arthrobacter spp.
Arthrobacter globiformis
Arthrobacter spp.
Arthrobacter sp. RS-33Arthrobacter spp.
Arthrobacter
spp.
Renibacterium
Salmoninarum
Zhihengliuella alba
Arthrobacter globiformis
Arthrobacter sp. RS-33
Psychrophilic marine bacterium
PS32 Salmoninarum
Renibacterium
Citricoccus sp. PL13f S6
Zhihengliuella alba
Psychrophilic marine bacterium PS32
Micrococcus lylae
Citricoccus sp. PL13f S6
Antarctic bacterium R-9183
Micrococcus lylae
Antarctic
bacterium
Nesterenkonia
sp.
AC84 R-9183
sp. AC84
Rothia sp.Nesterenkonia
RV13
Rothia sp. RV13
Kocuria sp. IARI-R-30
Kocuria sp. IARI-R-30
Arthrobacter isolate 16S rRNA sequence used for comparison:
AGAGTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGATGATCCGGTGCTTGCACCGGG
GATTAGTGGCGAACGGGTGAGTAACACGTGAGTAACCTGCCCTTAACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGG
ATATGACTCCTCATCGCATGGTGGGGGGTGGAAAGCTTTATTGTGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTGGTGA
GGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCC
TACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGG
TTGTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGC
GGTAATACGTAGGGCGCAAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGCCGTGAAAGT
CCGGGGCTCAACTCCGGATCTGCGGTGGGTACGGGCAGACTAGAGTGATGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGA
AATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCATTAACTGACGCTGAGGAGCGAAAGCATGGGGA
GCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGGACATTCCACGTTTTCCGCGCCGT
AGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGG
CGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGGACCGGACCGGGCTGGAAACAGTCCTT
CCCCTTTGGGGCCGGTTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC
AACCCTCGTTCCATGTTGCCAGCGCGTAATGGCGGGGACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGACGA
CGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAAAGGGTTGCGATACTGTGAGGTGG
AGCTAATCCCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGA
TCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCACGAAAGTTGGTAACACCCGAAGCCGGT
GGCCTAACCCCTTGTGGGAGGGAGCTGTCGAAGGTGGGACTGGCGATTGGG-ACTAAGTCGTAACAAGGTA
Figure 1. Tree alignment of fragments of 16S rRNA genes. A) Achromobacter isolate 16S
rRNA gene trimmed to 1460 bp (16S rRNA gene sequence based on gDNA sequencing)
aligned against the top 100 hits in the GenBank NCBI database and other
Burkholderiales species (in order to root the tree). B) Arthrobacter isolate 16S rRNA
gene trimmed to 1486 bp (16S rRNA gene sequence based on gDNA sequencing) aligned
against the top 100 hits in the GenBank NCBI database and other Actinomycetales
species (in order to root the tree).
Sequences were aligned using ClustalW and trees were constructed using MEGA5
software using Neighbor-Joining with Bootstrap of 1000 replicates (bootstrap values
are shown on next to the tree nodes) (Felsenstein 1985, Saitou and Nei 1987, Tamura et
al 2004, Tamura et al 2011). A Neighbor-Joining tree is commonly used for this kind of
analysis (Micallef et al 2009). For the Arthrobacter alignment the node containing the
isolates 16S rRNA gene sequence is resolved next to the main tree (black arrow).
The genome of Achromobacter xylosoxidans is 7.01 Mbp and that of Arthrobacter
sp. is 3.815 Mbp. These are minimum sizes because the contigs have not been
closed and there may be missing DNA.
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