Question

1. In the PCR exercise you described why we amplify the 16s ribosomal RNA gene.  What other genes might help us to identify specific bacteria?  In other words, what traits could be sequenced that are unique to bacteria?

Answer 1

We amplify the 16s ribosomal RNA gene to identify microorganisms. The 16S rRNA genes are *ubiquitous*; ribosomes can't translate mRNA without their 16S rRNA component, so all bacteria have it. Because these genes are essential, they are also very *highly conserved*. This high conservation also makes it possible to construct *universal primers* that can amplify 16S rRNA genes from widely divergent bacteria. A nearly complete 16S rRNA gene sequence is therefore very easy to obtain for a novel bacterial isolate, and it provides enough phylogenetic information to identify the isolate at least down to the genus level, thanks to the huge database of 16S rRNA gene sequence information that is publicly available and easily searchable.

Multilocus sequence typing (MLST) is another technique to identify microorganisms. The procedure characterizes isolates of microbial species using the DNA sequences of internal fragments of multiple housekeeping genes.

MLST directly measures the DNA sequence variations in a set of housekeeping genes and characterizes strains by their unique allelic profiles. The principle of MLST is simple: the technique involves PCR amplification followed by DNA sequencing. Nucleotide differences between strains can be checked at a variable number of genes depending on the degree of discrimination desired.

About seven to eight house-keeping genes are commonly used in the laboratories. Quoting Staphylococcus aureus as an example, seven housekeeping genes are used in MLST typing. These genes include carbamate kinase (arcC), shikimate dehydrogenase (aroE), glycerol kinase (glpF), guanylate kinase (gmk), phosphate acetyltransferase (pta), triosephosphate isomerase (tpi) and acetyl coenzyme A acetyltransferase (yqiL) as specified by the MLST website. However, it is not uncommon for up to ten housekeeping genes to be used.