Article Figures & Data
Figures
- Fig. 1.
Genetic organization of the ∼4.7-kb region separatingctrA and galE in serogroup A meningococcal strain F8229. Open arrows, drawn to scale, represent the locations and orientations of ORF1 to -4. Dotted open arrows indicate the flanking genes ctrA and galE only, portions of which are shown. Open triangles point to the locations of insertion mutations created in ORF1 to ORF4 (see text). Locations of primers (Table 1) are shown by solid arrows.
- Fig. 2.
RT-PCR of mRNA prepared from wild-type serogroup A strain F8229 to detect an ORF1 to ORF4 polycistronic transcript. Primer SE56 (Table 1; Fig. 1), which is complementary to the 3′ end of ORF4, was used as the RT extension primer to generate cDNA in the RT reaction [RT (+)]. A control without RT added [RT (−)] was included under otherwise identical conditions. Primers SE46 and SE61 were used for PCR amplification of an approximately 2.8-kb ORF1-to-ORF4 product. Lane 1, 1-kb ladder (Gibco-BRL); lane 2, positive control using F8229 chromosomal DNA as the template; lane 3, PCR result using the RT (+) template; lane 4, negative control using the RT (−) template. DNA size standards (in base pairs) are indicated.
- Fig. 3.
Autoradiographs showing primer extension products for the serogroup A meningococcal genes ctrA and ORF1. Primer extension reactions were loaded alongside standard double-stranded DNA-sequencing reactions (load orientation of G, A, T, C) obtained by sequencing ctrA and ORF1 control DNA templates with the extension primers SE40 (ctrA) and SE41 (ORF1). The DNA sequence surrounding the primer extension bands has been expanded. The nucleotides corresponding to the putative start points of transcription are circled.
- Fig. 4.
Nucleotide sequence of the 218-bp intergenic region separating the start codons for the serogroup A ctrA and ORF1 loci. The start points and directions of transcription of the ORF1 and ctrA mRNAs are indicated by ti and arrows, respectively. Predicted −10 and −35 promoter binding sequences are indicated, as well as the putative Shine-Dalgarno ribosome binding sites (RBS). The predicted initiation codons for ctrA and ORF1 are shown in boxes.
- Fig. 5.
Colony immunoblots of wild-type serogroup A meningococcal strain FA8229, its ORF1 to -4 mutants, and unencapsulated variant F8239. Strains were grown overnight on GC base agar, transferred to nitrocellulose, and probed with anti-serogroup A monoclonal antibody 14-1-A (45). (A) Wild-type encapsulated strain F8229; (B) unencapsulated variant F8239; (C) F8229-ORF1Ω; (D) F8229-ORF2Ω; (E) F8229-ORF2aphA-3; (F) F8229-ORF3Ω; (G) F8229-ORF4Ω.
Tables
- Table 1.
Oligonucleotide primers used in this study
Primer Nucleotide sequence (5′→3′) LJ4 CCACCACCAAACAATACTGCCG SE26 GCGTTTAACAAGCGTTCTAAGCC SE33 GTCAACTCAGAAGATAAGAATTGG SE40 GAATAGCACTACATGCACTTCCC SE41 CAGGGCGAGTGCCAAAGACG SE46 GAAGCTGTAGCTGCAGGAACTG SE56 AATCATTTCAATATCTTCACAGCC SE57 TTACCTGAATTTGAGTTGAATGGC SE61 CAAAGGAAGTTACTGTTGTCTGC SE63 TTCATATAACTTGCGGAAAAGATG JS102 GAGCCTATTCGAAATCAAAGCTG JS103 AGATACCATTAGTGCATCTATGAC JS104 CATGAAACTCAGCACAGATAGAAC JS105 GTTATTTAAATCTAGCCATGCTGG GalE1 CGTGGCAGGATATTGATGCTGG
