GENETIC ANALYSIS AND POST TRANSLATIONAL MODIFICATIONS OF TYPE IV PILI IN METHANOCOCCUS MARIPALUDIS
Balachandran Nair, Divya
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Methanococcus maripaludis has two different surface appendages that are considered to be type IV pili-like structures: the well-studied archaella as well as type IV pili. Though prevalent among various archaea, studies on type IV pili in any archaeon are extremely limited. In this thesis, many novel essential components necessary for type IV pili formation in M. maripaludis have been identified by gene deletion analysis coupled with electron microscopy. Several of these essential components were found in a previously identified eleven gene locus (mmp0231-mmp0241) containing three previously identified pilin-like genes and a prepilin peptidase. The genes in this locus were shown in this work, by reverse transcriptase-polymerase chain reaction experiments, to be arranged in a single operon. The remaining unstudied genes, except for mmp0231, were deleted and, with the exception of mmp0235 and mmp0238, found to be essential for piliation. Outside this locus, the conserved assembly ATPase (mmp0040; epdL) and, unusually, two tandem versions of the conserved type IV pilus platform protein (mmp0038 and mmp0039; epdJ and epdK) were identified. All three were shown to be essential for piliation. Furthermore, the gene encoding the major pilin structural protein (mmp1685; epdE) was identified at a separate locus. In addition, six other pilin-like genes (mmp0528, mmp0600, mmp0601, mmp0709, mmp0903, mmp1283) scattered around the genome were targeted for deletion, with electron microscopy of the individual deletion strains revealing that normal piliation was only affected when mmp1283 (epdD) was missing, suggesting it was a fourth minor pilin. Lastly, studies were conducted to examine the order of the two posttranslational modifications of pilins and archaellins. While it was determined that signal peptide cleavage and N-linked glycosylation of archaellins could occur independently of each other, this was not the case for pilins. Detection of epitope-tagged pilins expressed in various mutant backgrounds unable to carry out one, the other, or both posttranslational modifications, strongly suggested that pilins cannot be N-glycosylated unless the signal peptide is first removed. These studies reveal that the type IV pili of M. maripaludis are more complex than those of other studied archaea and also raise questions about how the cell is able to distinguish between the two types of type IV-pilin-like proteins, archaellins and pilins.