Table 2 Primers used in this study

ZR165

GATCTCCCGGGCTAGCGGCCGCAATTGACGTCTCGAGA

Annealed oligonucleotides ZR165/ZR166 ligated to BglII-SpeI digested pRL278 to produce pZR824

ZR166

ctagTCTCGAGACGTCAATTGCGGCCGCTAGCCCGGGA

ZR241

tggaTCCAACTCTACAGGAATTGTCTG

ZR241,242 primer pair amplifying alr5283–84 ORF (2.7 kb); in knockout mutant with GFP-Sp^r cassette, primers amplify 4.7 kb

ZR242

tcctAGGAATTGGTATTGGGGATTG

ZR243

CAAAAAGCGGCcGCTGAAGGTAAAATC

ZR243,244 primer pair for site-directed mutagenesis to introduce NotI site at 997 bp of alr5283–84 region

ZR244

TTTACCTTCAGCgGCCGCTTTTTGG

ZR1584

atgcatatgctagcgacgtcggATCCCTTAACTTACTTATTAAATAATTTATAG

Primers ZR1584/ZR1585 PCR amplified 1921 bp Cm^r/Em^r cassette (NsiI/NdeI-NheI/BamHI-Em^r/Cm^r cassette-EcoRV/BglII/XhoI/XmaI/SmaI) from pRL271 ligated to pCR2.1-TOPO to produce pZR2222

ZR1585

tccCGGGAAGTATCCAGCTCGAGATC

ZR261

CAAGAATTGGGACAACTCCAGTG

ZR261, 1602 primer pair for verifying insertion of P-alr5283-P-alr5284 in pZR2223 shuttle vector (construction pZR2239)

ZR1602

tgacgtcGACTCCAAAAATCAGCAGATTTCC

ZR1603

tcccgggtacCTAAACCAGCAGTGGTCTAAACC

ZR1602, 1603 primer pair amplifying P-alr5283-P-84 (2.2 kb); amplification retains promoters for both genes

ZR1606

ZR1607

ZR1608

ZR1609

atgcatATGCAGCAGGTTGCAGCCGATTTAG

tggatCCTAAACCAGCAGTGGTCTAAACC

CATGGtatatctccttcttTTAAGCTGCTGTAAGTCCGTAGGCT

GACTTACAGCAGCTTAAaagaaggagatataCCATGTTTGGTCTAATTGG

ZR1606,1607 primer pair amplifying alr5283–84 (2 kb); amplification includes promoter for alr5284 only

ZR1606-1608-1609-1607 overlap PCR (1.7 kb)

(1) Primer pair ZR1606, 1608 amplify alr5283 ORF

(2) Primer pair ZR1609, 1607 amplify alr5284 ORF

(3) Primer pair ZR1606, 1607 using template fragments from steps 1 and 2 for PCR overlap, combining the fragments (1.7 kb)

LK2406

TTAAGGGCCCGGGAGATCTAGACCGGTACTAGTC

Annealed oligonucleotides LK2406/LK2407 ligated to AflII digested pZR606 to produce pZR666; ApaI-XmaI-BglII-XbaI-AgeI-SpeI multiple cloning sites (MCS)

LK2407

TTAAGACTAGTACCGGTCTAGATCTCCCGGGCCC