Download Construction of nanA mutants

Mutation of NanA
The nanA gene sequence was originally obtained from TIGR4 genome (SP_1693) from the
TIGR CMR but the start site as for R6 of MSY was used. The following sequence, and the
2.5 kb upstream and downstream of this sequence, to construct the nanA mutants, as follows:
Primers (5’
SP1693_Janus1
SP1693_Janus2
SP1693_Janus3
SP1693_Janus4
SP1693_Janus5
3’):
GAC GAT GTT TGC TAT CCC ATT ACC
CAG GTA CCT TAT CTA CCA GGA CAC CGC TGC
CGC GAG CTC AAG GAG ATA GCA GGT TTT CAA GCC
TCC AAG TTC CCC ATT CTT CTG TC
CGC GGT ACC AAG GAG ATA GCA GGT TTT CAA GCC
Janus_kpnF
CAG GTA CCG TTT GAT TTT TAA TGG
Janus_sacR
CAG AGC TCG GGC CCC TTT CCT TAT GCT TTT GG
SKH302_kanR
ACC TTA GCA GGA GAC ATT CC
SKH303_rpsLF
ACA TCC CAG GTA TCG GAC AC
Construction of nanA mutants
This was carried out by the Janus cassette mutagenesis essentially as described by Sung, et al,
2001 (Appl. Environ. Microbiol., Nov. 2001, p. 5190–5196) , as follows.
Construction of in-frame, insertion of the Janus cassette (KanR rpsL) in replacement of
the nanA gene.
Using TIGR4 chromosomal DNA as template, the upstream and downstream flanking regions
of nanA were amplified using primers SP1693_Janus1 vs SP1693_Janus2, and primers
SP1693_Janus3 vs SP1693_Janus4, respectively, generating approx. 1 kb PCR products in
each case. The 1500 bp Janus Cassette was amplified with primers Janus KpnF and Janus
SacIR. The PCR products generated from the 3 individual reactions were then cleaned and
digested with the appropriate enzymes, cleaned again, and then ligated. The ligation mix was
used as template for an extended PCR using KOD polymerase and primers SP1693_Janus1
and SP1693_Janus4. This PCR product was then used to transform competent TIGR4_SR1
and transformants were selected on blood agar plates containing 200 µg/ml Kan. Selected
transformants were screened for SmS. The correct junction fragments were verified by PCR
with primers internal to the Janus cassette and the two outer primers outside of the nanA gene.
Construction of in-frame, unmarked deletion of nanA.
Using TIGR4 chromosomal DNA as template, unmarked nanA mutant was constructed by
amplifying the upstream and downstream flanking regions of nanA using primers
SP1693_Janus1 vs SP1693_Janus2, and primers SP1693_Janus5 vs SP1693_Janus4,
respectively, generating approx. 1 kb PCR products in each case. The PCR products generated
were then cleaned and digested with KpnI, cleaned again, and then ligated. The ligation mix
was then used to transform competent TIGR4 nanA deletion replacement mutant generated
above and the transformants were plated on on blood agar plates containing 1000 µg/ml Sm.
Selected transformants were then screened for loss of kanamycin resistance by replica plating
onto blood agar. The flanking regions of putative mutants were PCR-amplified and the inframe deletions of the nanA gene were confirmed by sequencing.
All mutants above were confirmed to correct by sequencing, optochin sensitivity, quellung
reaction (with type 4 serum) and negative for the expression of NanA by Western blot.
Construction of TIGR4 mutants expressing site-specific mutations in the active site
residues of the nanA gene.
Using TIGR4 chromosomal DNA as template, the upstream and downstream flanking regions
of nanA were amplified using primers SP1693_Janus1 vs SP1693_Janus2, and primers
SP1693_Janus3 vs SP1693_Janus4, respectively, generating approx. 1 kb PCR products in
each case. The wild-type, E647T, or Y752F nana genes were amplified with primers NanAF
and NanAR described for the plasmids (pNanA571, pNanA_E647T and pNanA_Y752F) and
using those plasmid DNAs, respectively, as template. The two flanking region PCR products
were cleaned and digested with the appropriate enzymes. Similarly, the gene products were
also cleaned, digested. After a final cleaning, three products for each gene replacement were
then ligated. The ligation mix was used as template for an extended PCR using KOD
polymerase and primers SP1693_Janus1 and SP1693_Janus4. The ligation mix was then used
to transform competent TIGR4 nanA deletion replacement mutant generated above and the
transformants were plated on on blood agar plates containing 1000 µg/ml Sm. Selected
transformants were then screened for loss of kanamycin resistance by replica plating onto
blood agar. The correct insertion was verified by PCR and the presence of the nanA sitespecific active site residue mutations were confirmed by sequencing.
The gene replacement mutants were confirmed to be positive for the expression of NanA by
Western blot. The active sialidase activity of the wild type replacement and loss of such
activity for the mutant NanA was confirmed by enzyme activity assays.
All mutations in NanA were also transferred to the strain EF3030 by transformations.