Evaluation of Functional Gene for Classification of Pathogenic Bacteria

Department of Microbiology Regeneration and Advanced Medical Science , Gifu University, Graduate school of Medicine Yanagido 1-1, Gifu, Japan

MLST (multilocus sequence typing) analysis information is rapidly accumulated, but we need more conveincing data to satisfy medical community and veterinarian to change BSL3 classification.
It is no doubt that DNA based identification and

detection of pathogens have a powerful potential to control infectious diseases because of the rapiness and sensitiveness.

However, current taxonomy of BSL3 pathogens have many problems to use genetic identification and detection methods.

Problems of classification of BSL3 Pathogens

Examples: 1. Bacillus cereus group:B.anthracis,B.cereus, B thuringiensis, and B.mycoides carry identical 16S rRNA sequences.
They were classified different organisms because of their pathogenic factors.

2. Escherichia coli and Shigella species shares identical 16S rRNA. They were classified different organisms
because of their pathogenic factors.

3. Clostridium botulinum species is clusters of phylogenetically different organisms.

They only shares common feature to produce botulinum toxins.

Historical Errors
Strain which does not produce gas from glucose, lactose negative and nonmotile and etc.
No Biochemically identified as Escherichia group Classied as Shigella species and serologically identified as Shigella species

Yes

cc
Later, none motile, lactose negative, were found and described as Pathogenic E.coli
E.coliO7 =S.boydii 12 E.coli 28a,c=S.boydii13 E.coli O112a,c=S.dysenteriae 2 E.coli O124=S.dysenteriae 3 E.coli O143=S.boydii 8 E.coli O144=S.dysenteriae 10 E.coli O152=S.dysenteriae12

E.coli O antigen: O1 to O180

Average 16S rRNA SNP within a species is less than 1.3%Constantinidis and Tiedje2005
Species cut off

16S rDNA cannot differentiate BSL3 pathogens and Plant pathogens from their closely related species because their sequence are related more than 99.5%.

Need complete protein sequence analysis to use housekeeping genes as a taxonomic tool

Whole genome sequenced strains(1230str.)

Million species>7000species>1400gensus>280Family

1. Only 500 dollar to determine whole genome of one strain. 2. Shot gun sequencing produced many full sequenced genes and protein information. Strain level information is bursting. In medical micobiology, pathogenic species are well defined and their differentiation methods from nonpathogenics are well established in detail through 100 year history of pathogenic bacterial history.

Cannot reach to Common ancester

Higher taxon

Species level :pick up only specific genes, pathogenic factors among species

Common ancester
Higher taxon

Species level: compare commonly conserved hose keeping genes

Commonly Conserved Genes among whole genome sequenced strains

[J] COG0048 Ribosomal protein S12 [J] COG0049 Ribosomal protein S7 [J] COG0051 Ribosomal protein S10 [J] COG0052 Ribosomal protein S2 [J] COG0088 Ribosomal protein L4 [J] COG0089 Ribosomal protein L23 [J] COG0090 Ribosomal protein L2 [J] COG0091 Ribosomal protein L22 [J] COG0100 Ribosomal protein S11 [J] COG0102 Ribosomal protein L13 [J] COG0103 Ribosomal protein S9 [J] COG0244 Ribosomal protein L10 [J] COG0255 Ribosomal protein L29 [J] COG0256 Ribosomal protein L18 [J] COG0231 Translation elongation factor P (EF-P)/translation initiation factor 5A (eIF-5A) [J] COG0361 Translation initiation factor 1 (IF-1) [J] COG0532 Translation initiation factor 2 (IF-2; GTPase) [L] COG0550 Topoisomerase IA [J] COG0124 Histidyl-tRNA synthetase [J] COG0143 Methionyl-tRNA synthetase [J] COG0162 Tyrosyl-tRNA synthetase [J] COG0441 Threonyl-tRNA synthetase [J] COG0442 Prolyl-tRNA synthetase [J] COG0495 Leucyl-tRNA synthetase [J] COG0522 Ribosomal protein S4 [J] COG0525 Valyl-tRNA synthetase [E] COG0006 Xaa-Pro aminopeptidase1 [E] COG0006 Xaa-Pro aminopeptidase2 [O] COG0492 Thioredoxin reductase [E] COG0112 Glycine/serine hydroxymethyltransferase [F] COG0504 CTP synthase (UTP-ammonia lyase) [G] COG0126 3-phosphoglycerate kinase [G] COG0148 Enolase [G] COG0149 Triosephosphate isomerase [G] COG1109 Phosphomannomutase [J] COG0030 Dimethyladenosine transferase (rRNA methylation) glycine tRNA synthetase, alpha subunit glycine tRNA synthetase, beta subunit [J] COG0008 Glutaminyl-tRNA synthetases [J] COG0013 Alanyl-tRNA synthetase [J] COG0016 Phenylalanine tRNA synthetase, alpha subunit [J] COG0072 Phenylalanine tRNA synthetase, beta subunit [J] COG0018 Arginyl-tRNA synthetase [J] COG0024 Methionine aminopeptidase [J] COG0060 Isoleucyl-tRNA synthetase 124 32 103 241 201 100 273 110 129 142 130 165 63 117 188 72 890 653 414 677 424 642 572 891 206 951 361 441 321 417 545 387 432 255 456 273 303 689 471 876 327 795 577 264 938

Ribosome Proteins, Replication Modification, Protein maturation

T-RNA Synthesis

House keeping Protein SAP in Escherichia (N=44strains)

0,000% 0,500% 1,000% 1,500% 2,000% 2,500% 3,000% 3,500%

coli

43
41 39 37 35 33 31 29 27 25 23

Ribosome Proteins

21
19 17 15 13 11 9 7 5 3 1

T-RNA

synthesis

DNA Extension, Modificatio n,

Ribosome protein SAP in E.coli(N=44 strains)

Ribosome protein
Ribosomal protein L10 Ribosomal protein L13 Ribosomal protein L18 Ribosomal protein L2 Ribosomal protein L22 Ribosomal protein L23 Ribosomal protein L4 Ribosomal protein S10 Ribosomal protein S11 Ribosomal protein S4 Ribosomal protein S7 Ribosomal protein S12 Ribosomal protein L29 Ribosomal protein S9 Ribosomal protein S2

Amino
165 142 117 273 110 100 201 103 129 206 32 124 63 130 241

Averag Range e
0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.009% 0.020% 0.063% 0.156% 0.000% 0.000% 0-0.8% 0-0.3% 0-0.3% 0-1.0% 0-0.5% 0.000% 0.000% 0.000% 0.000% 0-0.8% 0-1.6% 0-0.8% 0-0.4%

SD
0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.219% 0.198%

Ribosome Proteins

Ribosome protein SNP within a species was less than 1.6%

T-RNA and Replication associated Protein SNP

Escherichia coli 44 (COG genes) Translation elongation factor P (EF-P)/translation initiation factor 5A (eIF-5A) Translation initiation factor 1 (IF-1) Triosephosphate isomerase Translation initiation factor 2 (IF-2; GTPase) Phosphoglycerate kinase Thioredoxin reductase CTP synthase (UTP-ammonia lyase) Topoisomerase IA
Phosphomannomutase Glycine/serine hydroxymethyltransferase Glycine tRNA synthetase Dimethyladenosine transferase (rRNA methylation) Threonyl-tRNA synthetase Valyl-tRNA synthetase Phenylalanine tRNA synthetase, beta subunit Alanyl-tRNA synthetase Methionine aminopeptidase Methionyl-tRNA synthetase Phenylalanine tRNA synthetase, alpha subunit Arginyl-tRNA synthetase Glutaminyl-tRNA synthetases Isoleucyl-tRNA synthetase Leucyl-tRNA synthetase Tyrosyl-tRNA synthetase Xaa-Pro aminopeptidase1 Prolyl-tRNA synthetase Histidyl-tRNA synthetase Xaa-Pro aminopeptidase2

Amino Average
188 72 255 890 387 321 545 653 456 417 689 273 642 951 795 876 264 677 327 577 471 938 891 424 361 572 414 441 0.000% 0.000% 0.000% 0.014% 0.144% 0.475% 0.059% 1.498% 3.479% 0.070% 0.197% 0.269% 0.269% 0.351% 0.400% 0.400% 0.419% 0.422% 0.471% 0.479% 0.500% 0.503% 0.584% 0.685% 0.694% 0.810% 0.942% 3.248%

Range
0-0.5% 0.000% 0-0.4% 0-0.1% 0-0.3% 0-1.2% 0-0.6% 0-4.6% 0-24.0% 0-0.2% 0-0.4% 0-0.7% 0-1.1% 0-0.7% 0-1.0% 0-2.7% 0-1.5% 0-1.5% 0-0.9% 0-1.9% 0-1.1% 0-1.0% 0-1.0% 0-4.7% 0-1.8% 0-1.2% 0-6.8% 0-6.1%

SD
0.000% 0.000% 0.000% 0.000% 0.153% 0.321% 0.148% 1.243% 3.502% 0.098% 0.182% 0.280% 0.084% 0.124% 0.272% 0.185% 0.276% 0.182% 0.209% 0.179% 0.262% 0.227% 0.172% 0.282% 0.806% 0.203% 0.409% 1.634%

DNA

T-RNA

t-RNA Ile-tRNA Ala-tRNA Asp-tRNA Thr-tRNA Arg-tRNA Gln-tRNA Leu-tRNA Met-tRNA Lys-tRNA Val-tRNA Ser-tRNA Tyr-tRNA Val-tRNA Cys-tRNA Gly-tRNA Asn-tRNA Pro-tRNA Glu-tRNA Phe-tRNA His-tRNA Trp-tRNA Sec-tRNA Xaa-tRNA

Str.K-12 W3110 5 5 5 2 4 4 8 6 6 5 5 2 2 1 5 4 3 3 2 2 1 1

Str.K-12 DH10B 6 6 2 4 6 4 8 7 6 7 2 3 2 1 6 4 4 3 2 1

Str.K-12 MG1655 6 5 3 4 7 4 7 7 6 2 6 3 5 1 6 4 4 4 2 1 1 1 3

Copy Numbers of T-RNA gene within E.coli K-12 strains

Different among strain level:

Not suitable for classification

House keeping genes (dnaJ, rpoB, gyrB) are usually exit only one on their chromosome only exit one copy on their chromosome

Genus Escherichia

E. coli E. furgsonii E. arbertii Shigella spp. E. vulneri E. hurmanii E. blattae

16S rRNA phylogeny

dnaJ sequence
E.ictaruliT

Citrobacter
E hosinae E hosinae T C _ lapgei 1132 C _ neteri 1133 E tarda E tardaT

E. (Animal source)

hermannii GTC347
C _ sedlakiiGTC 1322

Citrobacter (Human source)

E ictaluri -

C C _ _ werkmanii wermanii 1141 1141 C _ koseri 1138 C _ rodentium 1139 C _ amalonaticus 1135 Ci . fr 9 574 C _ youngae 1142 C _ braakii 1136 Ci . fr 9517 C . fr 9635 Ci . fr 9 502 Ci . frDNA C . fr 612 1

E dwardsiella

E.vulneris1164
Leclercia
L.adecarboxylata L.adecarboxylata

E. blattae1161

E. albertii TW 07627

Sh . boy dys 1912 1913 1914 1930 E . Sh coliDNA . dys 1929 1385 1386 E . coli 11096 E . coli 9508 E . coli 11097 E . coli 9531 9563 9572 Sh . son 1910 1911 Sh Sh . . flex fle 1919 1921 Sh E _ . fergusonii dys 1057 1162 Sh . son 1909 Sh Sh . . flex fle 1924 1925 E . coli 9521

E.coli E.urgsonii Shigella spp.

0.1

E.coli serovars (O1 to 180) and Shigella sp.

DnaJ sequence

DnaJ Protein SAP( E.coli )

E. coli-Shigella spp.(<0.53%)

Other Escherichia (2.1%) Citrobacter (3.2%-3.7%)

Salmonella(4.3%)

RpoB Protein SAP

E. coliShigella(<0.005%)
Other Escherichia
Citrobacter

Salmonella

GryB Protein SAP

E. coli-Shigella (0-0.25%) Other Escherichia (0.87-2.o%) Citrobacter (1.49-4.35%) Salmonell a (3.2%)

House keeping Protein SAP

Escherichia
5,00% 4,00%

Citorbacter Salmonella

376 AA

3,00% 2,00% 1,00%

DnaJ<0.53%
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

0,00%

5,00% 4,00%

1342 AA

3,00%
2,00% 1,00% 0,00%

RopB<0.005%
1 3 5 7 9 11 13 15 17 19 21 23 25

5,00% 4,00%

804 AA

3,00%

2,00%
1,00% 0,00% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

GryB<0.25 %

E. vulneri, E. hurmanii, E. blattae should be reclassified as different genera

Genus level DnaJ Amino acid diversities

Reclassification of Shigella and E.coli

Escherichia coli Enterohaemorrhagic Engeroinvasive Enterotoxigenic Enteoaggregative Enteropathogenic E. furgsonii-> nonpathogenic
E.coli

Shigella dysenteiae Serovar O1 Serovar all others

Shigella flexneri Shigella sonnei Shigella boydii

E. arbartii-> independent species in the genus Escherichia E. vulneris E. blattae Citrobacter or new genera

E. hurmanii

* E.blattae is already proposed to transfer to a new genus

Bacillus cereus group

B.anthracis B.cereus B.thuringiensis
B.mycoides B.pseudomycoides B.weihenstephanensis

These shares more than 99.5% 16S rRNA similarities with B.anthracis

99.3%-95% similarities to B.anthracis

DnaJ sequence of Out break strains of B.cereus was more than 99% related to B.anthracis DnaJ variation within B.cereus (9.6 is bigger than E.coli(1.5%)

dnaJ sequence

Diversities among B.cereus isolated from Blood culture B.cereus divided into 4major groups.

DnaJ sequence isolated from severe pneumoniae was closely related to B.anthracis

Pathogenic factors to diffeentiate B.cereus group

DnaJ sequence SNP

B.pseudomycoides GTC 02831T

B.cereus group3 (n=21 )

B.cereus group1 (n= 4 )

B.mycoidesB.weihenstephanensis Group(n=18)

B.cereus thuringiensis Group2 (n= 39 ) Cereus (n=22), Thuringiesis(n=17)

Outbreak strains of B.cereus (n=4) Anthracis group (n=38 strains)

0.1

Outbreak strains make typical B.cereus colonies

Suspect B.cereus , B.thuringiensis

Suspect B.subtilis , B.anthracis

Suspect B.mycoides

When does a strain spin out from a species ?

House keeping Protein variation (SAP)

Use complete amino acid sequence to discuss Species border or genus border. Until amino acid of a protein changes, the function is same among strains with different coden usage.
House keeping Gene Sequence variation (SNP) Analyze strain evolution within a species. Different coden usage among strains in a species is useful to analyze whether a strain is ready to spin out from a species.