Sean D. Reid, Corinne Herbelin, Alyssa C. Bumbaugh, Robert K. Selander &
Thomas S. Whittam
Institute of Molecular Evolutionary Genetics,
Pennsylvania State University, University Park, Pennsylvania 16802, USA
MLST Strains
New enterohemolysin results from Peter Feng (June 1, 2000)
Table of MLST PCR
Table of MLST primers
Download sequences
We chose 7 genes involved in housekeeping functions that are spaced around the E. coli chromosome for nucleotide sequencing. Five loci have been shown previously to be polymorphic for multiple alleles as detected by protein electrophoresis. In addition, we examined arcA, a gene encoding a protein involved in the control of aerobic respiration. We have also sequenced rpoS, a gene encoding the sigma factor involved in stress response, and we are now developing a reduced set of new primers for sequencing.
Location of 7 genes on the 100 minute map of the E. coli K-12 chromosome. The genes are clockwise (protein, centisome) as follows: icd (isocitrate dehydrogenase, 25.8) rpoS (sigma factor 38, 61.8), mdh (malate dehydrogenase, 72.9 ), aroE (shikimate dehydrogenase, 73.9), mtlD (mannitol 1-phosphate dehydrogenase, 81.3), pgi (phosphoglucose isomerase, 91.2), and arcA (aerobic respiratory control protein , 100). The number of codons sequenced is given next to each locus.
Individual loci (21 E. coli, 1 Typhimurium)
Combined data sets
DNA preparation. E. coli strains were grown on MacConkey’s agar overnight at 37 C. A single colony of each strain was transferred to 10 ml of Luria broth and grown overnight under agitation at 37 C. Chromosomal DNA was isolated from each strain in accordance with the instructions in the Puregene DNA Isolation Kit from Gentra Systems, Inc. (Minneapolis, Minn.). Isolated DNA was stored at 4 C.
PCR. Oligonucleotide primers were designed based upon published sequences for rpoS (Compan, 1994) , mdh (Boyd et al. 1994), aroE (Anton, 1988) , mtlD (Davis, 1988) , pgi (Froman, 1989) , arcA (Compan, 1994) , and icd (Wang et al. 1997). Primers were synthesized by a Beckman 1000 oligonucleotide synthesizer (Beckman, Fullerton, Calif.) Template DNA for cycle sequencing was obtained through amplification for 30 cycles as follows: 94 C for 1 min, 53 C for 2 min, and 72 C for 3 min with an initial denaturing step of 94 C for 5 min. The PCR products were purified with Qiaquick spin columns (QIAGEN Inc., Valencia, Calif.) and suspended in the supplied elution buffer to suitable concentrations, as determined by agarose gel electrophoresis.
Cycle Sequencing. Cycle sequencing was performed with a Prism Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit from Applied Biosystems. Sequencing gels were run on an Applied Biosystems 373A automated sequencer. Raw sequences of both DNA strands were analyzed and concatenated by DNASTAR with additional internal sequencing primers designed based on the generated sequence data. All conflicting and putative polymorphic sites were sequenced at least three times to reduce sequencing error.