Identification of alkA gene related to virulence of Shigella flexneri 2a by mutational analysis

Table 1 Strains and plasmids

Strains and plasmids	Characteristics	Source (reference)
Strains
DH5α	endA1 hsdR17(rk-mk+) supE44 thi-1 recA1 gyrA(Nalr)	Our lab
	RelA1 △ lacIZYA-argF) U169 deoR (φ80dlac △(lacZ)M15)
S17-1λpir	thi-1 thr leu tonA lacY supE recA::RP4-2-Tc::Mu, Smr, λpir	Guzman CA
MC1061	hsdR2 mcrB araD139 △ (ara ABC-leu)7679 △ lacX74 galU galK rpsL thi, Smr	Our lab
2457T	S. flexneri 2a wild type, Nalr	Maurelli AT
2457T05	a derivative strain of 2457T, contained araBp-gam-bet-exo, Nalr	This study
2457T028D	△ alkA, derivative strain of 2457T, Nalr, Kmr	This study
Plasmids
pMD18-T	a derivative constructed from pUC18, Apr	TaKaRa
pXL275	a mobile and suicide plasmid, ori R6K, mob RK2, sacBR, Cmr	Rui et al[17]
pKD46	oriR101, repA101(ts), araBp-gam-bet-exo, Apr	CGSCa
pXLkd46	pXL275 derivative, inserted a fragment containing araBp-gam-bet-exo, Cmr	This study
pMDKm05	pMD18-T derivative, inserted Kmr gene, Kmr, Apr	Our lab
pMD028	pMD18-T derivative, inserted alkA gene, Apr	This study
pMD028pKm	pMD18-T derivative, inserted a cassette (5’alkA end-Kmr-3’alkA end), Kmr, Apr	This study

^aCGSC: E. coli Genetic Stock Center.

Table 2 BLAST analysis of S. flexneri 2a alkA gene

Bacteria	Gene product	Gene function	Identities (%)
Escherichia coli K12	3-methyl-adenine DNA glycosylase II	DNA - replication, repair, restriction/modification	97%
Escherichia coli O157:H7	3-methyl-adenine DNA glycosylase II	Macromolecule synthesis, modification: DNA - replication, repair, restriction/modification	97%
Salmonella typhimurium	3-methyl-adenine DNA glycosylase II	Unknown	74%
Ralstonia solanacearum	putative transcription regulator protein	Miscellaneous; not classified regulator	38%
Pseudomonas aeruginosa	DNA-3-methyladenine glycosidase II	Unknown	37%
Xanthomonas axonopodis	DNA methylation and regulatory protein	Unknown	35%
Mycobacterium bovis	Methylated-DNA—protein-cysteine methyltransferase	Adaptative response	34%
Mycobacterium tuberculosis	alkA protein	Unknown	34%
Vibrio cholerae	ada regulatory protein	Unknown	32%
Bacillus anthracis	DNA-3-methyladenine glycosidase	Unknown	32%
Archaeoglobus fulgidus	3-methyladenine DNA glycosylase	DNA repair at suboptimal and maybe even mesophilic temperatures	30%

Table 3 Competitive analysis of different strains of S. flexneri 2a

Strains	Competitive index (CI)a
	BALB/c mouse infection	HeLa cells infection
2457T05	0.96 ± 0.27	1.07 ± 0.35
2457T028D	0.21 ± 0.09b	0.25 ± 0.11b
MC1061	0.03 ± 0.02b	0.02 ± 0.01b

^aCompetitive index (CI) is the ratio of mutant CFU to wild-type CFU after correcting the ratios for deviations of the in-oculum ratio from a value of 1.

^bP < 0.0078.