Comparative GenomicsPreliminary Results

Outline● Species Delineation Results

○ Average Nucleotide Identity (ANI)○ Alignment-Free Method (Kmer-based)

● MLST Results● Serogroup/Serotype (Cap locus)● Molecular Characterization Theory

○ Teichoic Acid○ Urease ○ Lipase○ Alkaline Phosphatase○ Beta-galactosidase○ Ornithase Decarboxylase○ Proline Arylamidase○ Gamma Gilutamyl Transferase○ Indole production

● Pipeline

ANI Results (ANIb)(BLAST)

ANI Results (ANIm)(MUMmer)

tetra Results

Alignment-Free Methodk-mer size selection:● The goal is to maximize the number of distinct k-mers identified.● Only k-mers with count greater than 1 are selected to ensure that

sequencing errors do not contaminate the analysis.● It is generally seen that the number of distinct k-mers peak at a given k-

mer length, this is selected as the optimal k-mer length.● The highest number of distinct k-mers were found at k-mer length of 11 for

all the assemblies.

Alignment-Free Method

Alignment-Free Method

Alignment-Free Method

Outline● Species Delineation Results

○ Average Nucleotide Identity (ANI)○ Alignment-Free Method (Kmer-based)

● MLST Results● Serogroup/Serotype (Cap locus)● Molecular Characterization Theory

○ Teichoic Acid○ Urease ○ Lipase○ Alkaline Phosphatase○ Beta-galactosidase○ Ornithase Decarboxylase○ Proline Arylamidase○ Gamma Gilutamyl Transferase○ Indole production

● Pipeline

MLST Results● MLST performed using MLST profiles for N.meningitidis

and H.influenzae from PubMLST.● Done using python toolkit for MLST written by Leighton

Pritchard.● Next goal is to setup a local implementation of BIGSdb

and evaluate which of the two methods to use.

MLST Results

pgm fumC aroE gdh pdhC adk abcZ ST

gi_385337120_ref_NC_017512.1_ 3 1 2 3 2 1 1 5

gi_15793034_ref_NC_003116.1_ 3 1 3 4 2 3 1 4

gi_121633901_ref_NC_008767.1_ 6 3 4 8 4 3 2 11

gi_385339062_ref_NC_017513.1_ 2 2 7 8 5 3 2 8

gi_385340991_ref_NC_017514.1_ 9 5 9 9 6 6 3 41

gi_77358697_ref_NC_003112.2_ 2 4 5 5 3 10 4 74

gi_385852231_ref_NC_017516.1_ 8 4 5 6 3 10 4 32

gi_385850283_ref_NC_017515.1_ 9 9 15 8 11 10 4 269

MLST Results for N.meningitidis references

MLST Resultspgm fumC aroE gdh pdhC adk abcZ ST

NODE_1275_length_56_cov_1480_ID_2549

19 1 2 3 2 1 1 7

NODE_1235_length_56_cov_10703_ID_2469

19 1 2 3 2 3 1 2859

NODE_188_length_130_cov_204.333_ID_375

3 53 13 26 41 5 1 162

NODE_1342_length_56_cov_249_ID_2683

6 3 4 8 4 3 2 11

NODE_192_length_128_cov_1337_ID_383

8 10 6 9 9 7 2 9997

NODE_169_length_128_cov_884_ID_337

9 5 9 9 6 6 3 41

NODE_202_length_128_cov_737_ID_403

70 5 9 9 6 6 3 414

MLST results for Assemblies (Nm)

Outline● Species Delineation Results

○ Average Nucleotide Identity (ANI)○ Alignment-Free Method (Kmer-based)

● MLST Results● Serogroup/Serotype (Cap locus)● Molecular Characterization Theory

○ Teichoic Acid○ Urease ○ Lipase○ Alkaline Phosphatase○ Beta-galactosidase○ Ornithase Decarboxylase○ Proline Arylamidase○ Gamma Gilutamyl Transferase○ Indole production

● Pipeline

Serogroup/Serotype Determination (Cap locus)

From Dr.Xin Wang

Serotype DeterminationFrom Dr.Xin Wang

Outline● Species Delineation Results

○ Average Nucleotide Identity (ANI)○ Alignment-Free Method (Kmer-based)

● MLST Results● Serogroup/Serotype (Cap locus)● Molecular Characterization Theory

○ Teichoic Acid○ Urease ○ Lipase○ Alkaline Phosphatase○ Beta-galactosidase○ Ornithase Decarboxylase○ Proline Arylamidase○ Gamma Gilutamyl Transferase○ Indole production

● Pipeline

Molecular Characterization● Neisseria and Haemophilus strains have different enzyme profiles● Pathways of Interest to the CDC:

○ Teichoic Acid○ Urease○ Lipase○ Alkaline Phosphatase○ Beta-galactosidase○ Proline Arylamidase○ Ornithase Decarboxylase○ Gamma Gilutamyl Transferase○ Indole Production

● Identify genes associated with each pathway and make use of functional annotations in order to characterize the sequences

Teichoic Acid Biosynthesis● Bacterial polysaccharide found within the cell wall of

Gram-positive bacterium● Provide rigidity to the cell-wall by attracting cations● Not found within Gram-negative bacteria

UreaseBacterial ureases are often the mode of pathogenesis for many medical conditions. They are associated with hepatic coma, infection stones, and peptic ulceration.

The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme Urease.

Two units of ammonia are formed with resulting alkalinity in the presence of the enzyme, and the increased pH is detected by a pH indicator.

N.meningitidis and H.influenzae are negative for Urease test.

UreaseBacterial ureases are often the mode of pathogenesis for many medical conditions. They are associated with hepatic coma, infection stones, and peptic ulceration.

The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme Urease.

Two units of ammonia are formed with resulting alkalinity in the presence of the enzyme, and the increased pH is detected by a pH indicator.

N.meningitidis and H.influenzae are negative for Urease test.

UreaseBacterial ureases are often the mode of pathogenesis for many medical conditions. They are associated with hepatic coma, infection stones, and peptic ulceration.

The urease test is used to determine the ability of an organism to split urea, through the production of the enzyme Urease.

Two units of ammonia are formed with resulting alkalinity in the presence of the enzyme, and the increased pH is detected by a pH indicator.

N.meningitidis and H.influenzae are negative for Urease test.

Lipase> Lipolysis is carried out by lipases. In this process triglycerides are broken down into fatty acids.> H. influenzae is negative for the lipase test> Pathway for N. meningitidis is:

Lipase

Alkaline phosphatase● Hydrolase responsible for removing

phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids.

● Role in bacteria○ active only during phosphate starvation○ dephosphorylating organic compounds

at the membrane for bacterial uptake● Both bacteria test negative for alkaline

phosphatase activity

Beta-Galactosidase● β-galactosidase is an enzyme which hydrolyzes the

β-glycosidic bond formed between a galactose and its organic moiety

● Detected using X-gal which is a sugar that forms an intense blue product after cleavage by β-galactosidase

● Neisseria meningitidis or Haemophilus influenzae test negative for this enzyme[1]

Proline Arylamidase

Proline ArylamidaseProline arylamidase has several names: prolyliminopeptidase (PIP),

hydroxyproline aminopeptidase, or proline aminopeptidase.

Lab tests are available to distinguish between pathogenic Neisseria species via

detection of this enzyme.

Proline Arylamidase

Proline ArylamidaseProline arylamidase appears to be produced by at least some strains of N.

meningitidis (Unemo et al., 2007).

Presently, our strongest N. meningitidis candidate for this region of interest is

NMC0342 (FAM18).

Ornithine DecarboxylaseH.influenzae biotyping (associated gene speF)

H. influenzae Biotype Ornithine Decarboxylase

I Positive

II Negative

III Negative

IV Positive

V Positive

VI Positive

VII Negative

VIII Negative*Laboratory methods for detection of Meningitis, CDC

Gamma Glutamyl Transferase

Biological process: glutathion metabolic process

EC number: EC 2.3.2.2

Reaction: a 5-L-glutamyl-[peptide] + an amino acid < -- > a 5-L-glutamyl-amino acid + a peptide

Component: two subunits

Gamma Glutamyl TransferasePathway

Gamma Glutamyl Transferaseannotation results

N.meningitidis H.influenzae

Indole production- Converts Tryptophan to Indole- Indole can act as an extracellular signal to

regulate biofilm formation in indole producing bacteria

Indole production test (Nm)Indole production test - Determines whether the microbe produces indole from the amino acid tryptophan

- If indole is produced, it will react with a chemical reagent added after incubation to produce a color change

- N. meningitidis : Kovac’s oxidase test

(Positive : Pink, Negative : Yellow)

Indole production test(Nm)

Figure 3. Flow chart for identification and characterization of a N. meningitidis isolate

Indole production(Nm)

Genes Present(Nm)

Indole production(Nm)Tryptophan synthase: enzyme that catalyzes the final two steps in the biosynthesis of tryptophan - trpA(tryptophan synthase subunit alpha unit) :catalyze the reversible formation of indole and glyceraldehyde-3-phosphate (G3P) from indole-3-glycerol phosphate (IGP)

- trpB(tryptophan synthase subunit beta) :catalyze the irreversible condensation of indole and serine to form a pyridoxal phosphate (PLP) dependent reaction

Indole-3-glycerol phosphate synthase(trpC) :catalyses the fourth step in the biosynthesis of tryptophan(ring closure)

Outline● Species Delineation Results

○ Average Nucleotide Identity (ANI)○ Alignment-Free Method (Kmer-based)

● MLST Results● Serogroup/Serotype (Cap locus)● Molecular Characterization Theory

○ Teichoic Acid○ Urease ○ Lipase○ Alkaline Phosphatase○ Beta-galactosidase○ Ornithase Decarboxylase○ Proline Arylamidase○ Gamma Gilutamyl Transferase○ Indole production

● Pipeline

Preliminary Pipeline

References- D'Amato, RICHARD F., et al. "Rapid identification of Neisseria gonorrhoeae and Neisseria meningitidis by using enzymatic profiles."

Journal of clinical microbiology 7.1 (1978): 77-81.- Richter M, & Rosselló-Móra R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA

106(45):19126-31.- Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. (2007) DNA-DNA hybridization values and their

relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol. 57(Pt 1):81-91- Konstantinidis, Konstantinos T., and James M. Tiedje. “Genomic Insights That Advance the Species Definition for Prokaryotes.”

Proceedings of the National Academy of Sciences of the United States of America 102.7 (2005): 2567–2572. PMC. Web. 1 Apr. 2015.- Kanehisa, M., Goto, S., Sato, Y., Kawashima, M., Furumichi, M., and Tanabe, M.; Data, information, knowledge and principle: back to

metabolism in KEGG. Nucleic Acids Res. 42, D199–D205 (2014)- Kanehisa, M. and Goto, S.; KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27-30 (2000). - Hideyuki Takahashi, Toshiro kuroki, Yuko watanabe Reliability of the Detection of Meningococcal γ-Glutamyl Transpeptidase as an

Identification Marker for Neisseria meningitidis; Microbiol. Immunol., 48(6),485-487,2004- Hideyuki Takahash, Haruo Watanabe.; Post-translational processing of Neisseria meningitidis c-glutamyl aminopeptidase and its association with inner membrane facing to the cytoplasmic space; FEMS Microbiology Letters 234 (2004) 27–

35

Homework1) Which method is more computationally

intensive for species delineation? (20 pts)2) What is the difference between serogroup

and serotype?(20 pts)3) What is the criteria for selection of k-mers?

(20 pts)

Homework4) Identify the species of bacteria (40pts)a. The value of ANI between x and H. influenzae is 0.9732 while between x

and N. meningitidis is 0.6534.Which species does x belong to?b. y passes the Kovac’s Oxidative test and tests positive for glucose and

maltose production but negative for sucrose or lactose. Which species does y belong to?

c. z is found to have the gene speF. Which species(Nm,Hi, or Hh) does z belong to?

Please complete the assignment and submit it to vvenkat@gatech.edu with the subject as Comp_genomics_lastname by 15 April 2015,11:55 pm EST.