Should Azomonas insignis be
reclassified?
S. Schach1 , J. Brown2 , M. Lee-Brown3
Northwest Guilford1 , NC State2 , Guilford
Results
Introduction
The Azotobacteria, including species of the
genera Azotobacter and Azomonas, are a
common and important group of free-living
nitrogen-fixers in the Family
Pseudomonadaceae. Azotobacter are rod
shaped, gram negative bacteria that can fix
aerobic nitrogen. Azomonas insignis is a
relatively unknown species. The reason for this
study is that a previous strand, ATCC 12523, of
A. insignis was identified to be in the genus
Acinetobacter and in the family Moraxellaceae,
which are not nitrogen fixers. Due to this
previous identification of A. insignis (ATCC
12523) the Lee-Brown Lab ordered another
sample of A. insignis (ATCC 29360), to repeat
the identification process and see if this sample
also identified in the family Moraxellaceae.
College3
1. Kb DNA Ladder
1234
1234567
1. 1 Kb DNA Ladder
2. A. macrocytogenes
genomic DNA
3. A. insignis
genomic DNA
4. PCR Marker
515 Forward
GCGGATCCTCTAGACTGCAG
TGCCAGCAGCCGCGGTAA
GGCTCGAGCGGCCGCCCGG
GTTACCTTGTTACGACTT
TGYACACACCGCCCGTC
GCCWAGGCATCCACC
CGGGATCCGIIGAGGAAAGTC
CIIGC
CGGAATTCRTAAGCCGGRTT
CTGT
1492 Reverse
16S-23S ITS 1391 Forward
23 Reverse
RNase P
59 Forward
RNA
347 Reverse
•Key: Y=C,T; R=A,G; N=A,C,G,T; D=A,G,T; I=Inosine
•Agarose Gel Electrophoresis: Used to confirm PCR
products. 1 X TAE buffer used, 40mM Tris, 1mM acetic
acid, 20mM EDTA.
•Phylogenetic Analysis: PCR sequences were
sequenced by MWG Biotech (High Point, NC).
Sequences confirmed through BLASTn, and Ribosomal
Database Project. Sequences were aligned through SeAl. Phylogenetic trees were produced through Phylip
and TreeView. The 16S-23S ITS sequences were
identified through tRNAscan-SE.
5. PCR Marker
0.1
Estimated evolutionary distance
Serratia marcescens
79
0.1
Evolutionary distance
29
Haemophilus nfluenzae
89
100
100
60
87
Acinetobacter lwoffii
“Azomonas insignis” ATCC 12523
72
56
24
49
100
55
33
Azotobacter beijerinckii
Azotobacter vinelandii
17
Pseudomonas aeruginosa
Pseudomonas stutzeri
Pseudomonas putida
Azomonas insignis ATCC 29360
63
Pseudomonas azotoformans
Pseudomonas fluorescens
100
Allochromatium vinosum
Acidothiobacillus ferrooxidans
Xylella fastidiosa
Agrobacterium tumefaciens
58
100
Alkanindiges illinoisensis
85
54
51
Buchnera aphidicola
Vibrio cholera
environmental sequence vHge2
Acinetobacter sp. ADP1
environmental sequence ESH7-9
92
37
“Azomonas insignis” ATCC 12523
Haemophilus influenzae
40
Shewanella putrefaciens
Xylella fastidiosus
Acidothiobacillus ferrooxidans
100
Acidothiobacillus ferrooxidans
95 Azotobacter paspali
69
Azotobacter vinlandii
32
Azotobacter salinestris
58
Azotobacter chroococcum
Pseudomonas aeruginosa
Azomonas macrocytogenes
61
Pseudomonas stutzeri
70
Azomonas insignis ATCC 29360
Pseudomonas fluorescens
100
70
Pseudomonas azotoformans
Pseudomonas putida
Allochromatium vinosum
Agrobacterium tumefaciens
Caulobacter crescentus
40
Acinetobacter calcoaceticus
52
57
43
99
Yersina pestis
Vibrio cholerae
49
66
Erwinia agglomerulans
Klebsiella pneumoniae
Escherichia coli
Salmonella typhi
98
Salmonella typhimurium
Lecleria adecarboxylata
93
Serratia marcescens
53
46
Serratia liquefaciens
Yersinia pestis
Plesiomonas shigelloides
Proteus vulgaris
Providencia alcalifaciens
100
93
Escherichia coli
74
100
•Culturing: Burk’s Nitrogen Free Medium (Strandberg
1968)
16S rRNA
4. A. insignis NifH
7. PCR Marker
•Species Source: ATCC 29360
Primer Sequences
3. + Control
6. A. insignis 16S23S ITS
Methods and
Materials
Primers
1. 1 Kb DNA Ladder
2. + Control
5. A. insignis
RNase P RNA
Shewanella putrefaciens
Gene
345
4. A. insignis 16S
rRNA
100
•Polymerase Chain Reaction (PCR): The amplification
of 16S rRNA , RNase P RNA, 16S-23S ITS, and NifH.
Then sent for sequencing at MWG Biotech (High Point,
NC)
2
3. - Control
97
•Gram Staining: Confirm that cells were gram negative
rods
1
2. + Control
29
Caulobacter crescentus
Phylogenetic tree based on 16S rRNA
Phylogenetic tree based on RNase P RNA
Conclusions
•Azomonas insignis (ATCC 29360) is not misclassified proving that it is a valid
species. However, A. insignis (ATCC 12523) is related to Acinetobacter and was
originally misidentified.
•Phylogenetic trees show that Azomonas insignis (ATCC 29360) is related to
Azomonas.
•Azomonas insignis (ATCC 29360) contains an azotobacterial nifH gene
References:
• Strandberg,G.W. and P.W. Wilson.1968. Formation of the nitrogen-fixing enzyme system in
Azotobacter vinelandii. Can. J. Microbiol. 14:25-31
•Kennedy, Christina; Paul Rudnick; Melanie L. Macdonald; and Thoyd Melton. “Azotobacter.” The
Gammaproteobacteria. Part B. The Proteobacteria. Vol. 2. Bergey’s Manual of Systematic
Bacteriology 2nd Edition. East Lansing, MI: 2005. 384-402.
Acknowledgements: Rice Strange of Northwest Guilford High School, and the NC International Science Challenge