The document summarizes genomic analysis of Clostridium solventogenic bacteria strains. It finds that the strains are closely related to Clostridium butyricum based on average nucleotide identity and tetranucleotide frequency. Pan-genome analysis shows Clostridium has a large, open pan-genome. Single-copy ortholog detection is influenced by sequence quality and produces comparable results between different detection methods. Overall, the analysis helps characterize the taxonomy and metabolism of the solventogenic Clostridium strains.

1. Análisis genómico comparativo de
Clostridium solventogénicos: Una mirada al
metabolismo fermentativo y a su taxonomía
Juan Pablo Rosas M.
18 de Septiembre 2015

2. 0
0,03
0,06
0,09
0,12
0,15
0,18
0,21
IBUN158B
IBUN18A
IBUN22A
IBUN13A
IBUN140B
IBUN18S
IBUN137K
IBUN62B
IBUN62F
IBUN95B
IBUN64A
IBUN18Q
IBUN125C
DSM5431
DSM2478
g1,3Propanediol/L-h
Q24h (g 13PD/L-h)
GLYCEROL
F ERMENTAT I ON
Aragón; 2010
Strains
B a c k g r o u n d

3. Background - 16S rRNA gene sequence based phylogeny
rnammer
ssu-align
RAxML (BS1000)

4. N e x t - G e n e r a t i o n
S e q u e n c i n g ( N G S )
tRNA rRNACDS
C o n t i g s
d e n o v o As s e m b l y
Tr i m m i n g / F i l t e r i n g
Functional
anotation
F a s t Q C ,
C l e a n R e a d s ,
F a s t X t o o l k i t
V e l v e t , S p a d e s , S O A P d e n o v o
– PA GIT
G i m m e r, P r o d i g a l ,
G e n e M a r k M A K E R 2
B L A S T x ,
B l a s t 2 G O ,
I n t e r p r o S c a n ,
K A A S
Phylogenetic
analysis
S S U - a l i g n ,
j M o d e l Te s t ,
R a x M L
I l l u m i n a / S o l e x a H i s e q
2 0 0 0 p a i r e d - e n d
R N A m m e r,
t R N A s c a - S E
T O O L S
B a c k g r o u n d - N G S p r o j e c t

5. GENOME STATISTICS
B a c k g r o u n d - N G S p r o j e c t r e s u l t s
Attribute
STRAINS
IBUN 13A IBUN 22A IBUN 62F IBUN 125C
Crop Potatoes Potatoes Grass Soy
Reads
2.810.165
(82%)
1.516.402
(74%)
1.986.017
(73%)
3.442.170
(74%)
Genome size (bp) 4.643.590 4.607.385 3.836.807 4.596.888
% GC content 28.65 28.67 28.77 28.67
No. of contigs 261 208 85 71
No. of genes 4086 4124 3356 3959
No. of proteins 4022 4063 3288 3888
No. of RNA genes 64 61 68 71
Genes assigned to COGs 2727 2677 2290 2740
Protein coding genes with
function prediction
3428 3424 2818 3346

6. G O A L
Explore the genomic variation among
the native strains, and extend to
closely related Clostridia and genus
level, through genomic comparative
analysis.

7. M E T H O D S
STRAIN INDUSTRIAL SPECIES GENUS
Whole Genome
Alignment
Nucmer
MAUVE
Orthologous
Clusters
Comparison
OrthoMCL
OrthoVenn
DDH in silico
Average Nucleotide
Identity (ANI)
TETRA frequencies
Pan-genome
Core-genome
Analysis
GET_HOMOLOGUES
Tettelin, et al; 2005
Single-copy
Orthologous Detection
GET_HOMOLOGUES
OrthoMCL
Phyla_AMPHORA
G=4 G=21 G=98

8. R E S U L T S
How different are the Clostridium strains at
the genomic level?
Figure 1. Nucmer dotplot alignment of C. IBUN13A and C. IBUN22A (top).
Mauve alignment of four strains. (right)
STRAIN
Clostridium sp. IBUN 13A
Clostridiumsp.IBUN62F

9. R E S U L T S
How are gene families (orthologous clusters) distributed
among native strains?
Figure 2. Venn diagram showing the distribution of shared gene families among C. IBUN 13A,
C. IBUN62F, C. IBUN125C and C. IBUN22A.
STRAIN

10. dhaD dhaB1 dhaB2 dhaTdhaAdhaSdhaK3dhaK2dhaK1dhaY
O x i d a t i v e p a t h w ay Re d u c t i v e p a t h w ay
H2O
NAD+
NADH
NAD+
NADH
ATP
ADP
Glycerol 3-hydroxy
propionaldehyde
1,3-PropanediolDihydrooxyacetoneDihydrooxyacetone -P
R E S U L T S
Which structure of the genes is involved in the
metabolism of glycerol?
Figure 3. dha regulon organization in C.IBUN 13A, C. IBUN125C and C. IBUN22A
STRAIN

11. R E S U L T S
What is the relationship between the native and industrial
interest Clostridia strains, using in silico DDH measures?
0,7 0,95
Average Nucleotide Identity (ANI)
Figure 4. Heatmap for ANI values between each genome retrieved by pairwise alignments with MUMmer.
INDUSTRIAL SPECIES

12. R E S U L T S
Tetranucleotide frequency (TETRA)
Figure 5. Heatmap for TETRA values between each genome.
INDUSTRIAL SPECIES

13. R E S U L T S
Figure 6. ANI and TETRA correlation values for 21 Clostridia genomes (left) and Richter, et al (2009) paper
(right).
ANI vs TETRA
INDUSTRIAL SPECIES

14. Pan-genome
Core-genome
Study # genomes Status Study level pan core
Zhou; et al 40 Complete genus ~23357 ~169
present 98 Both genus ~43852 ~140
R E S U L T S
Pan-genome analysis
Figure 7. Clostridium pan-genome (left) and core-genome (right). Estimated pan and core genome size
(table).
GENUS

15. R E S U L T S
Figure 8. Number of orthologous clusters (single-copy Orthologous blue and inparalogues red) in all
taxa for industrial strains and Clostridium ssp. (left). Number of species per orthologous groups vs number
of genes per orthologous groups (right).
Single-copy Orthologous Gene Detection in all taxa
GENUS

16. R E S U L T S
Figure 9. Number of sequences per Firmicutes markers in 98 Clostridium ssp. genomes sequences.
Single-copy Orthologous Gene Detection using Pre-defined markers
Phyla_AMPHORA
C. ultunense
GENUS

17. R E S U L T S
sets
Number of
Taxa
Single-copy
Orthologous
Complete set 98 1
without C. ultunense 97 13
Complete + IBUN 33 15
Complete 28 15
Drafts 69 14
Industrial strains 21 316
Phyla_AMPHORA 97 22
Figure 10. Relationship between number of single-copy orthologous and number of taxa.
GENUS

18. I n s u m m a r y
• Genomic analysis agree with phenotypic results, showing that
Clostridium sp. IBUN62F can be different to the other three strains
• Solventogenic Clostridia strains are closely related to Clostridium
butyricum.
• Clostridium is a large and diverse prokaryotic genus, with an open
pan-genome.
• The Single Copy Orthologues detection is highly influenced by the
genome sequence quality.
• Single Copy Orthologous detection is comparable between
Get_homologues and Phyla_AMPHORA.

19. GRACIAS !! Acknowledgements to
Dolly Montoya
Liliana López Kleine
Ximena Pérez Mancilla
Diego Mauricio Riaño Pachón