Functional Characterization of the Cell Division Gene Cluster of the Wall-less Bacterium Mycoplasma genitalium

Abstract

It is well-established that FtsZ drives peptidoglycan synthesis at the division site in walled bacteria. However, the function and conservation of FtsZ in wall-less prokaryotes such as mycoplasmas are less clear. In the genome-reduced bacterium Mycoplasma genitalium, the cell division gene cluster is limited to four genes: mraZ, mraW, MG_223, and ftsZ. In a previous study, we demonstrated that ftsZ was dispensable for growth of M. genitalium under laboratory culture conditions. Herein, we show that the entire cell division gene cluster of M. genitalium is non-essential for growth in vitro. Our analyses indicate that loss of the mraZ gene alone is more detrimental for growth of M. genitalium than deletion of ftsZ or the entire cell division gene cluster. Transcriptional analysis revealed a marked upregulation of ftsZ in the mraZ mutant. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics confirmed the overexpression of FtsZ in MraZ-deprived cells. Of note, we found that ftsZ expression was upregulated in non-adherent cells of M. genitalium, which arise spontaneously at relatively high rates. Single cell analysis using fluorescent markers showed that FtsZ localization varied throughout the cell cycle of M. genitalium in a coordinated manner with the chromosome and the terminal organelle (TMO). In addition, our results indicate a possible role for the RNA methyltransferase MraW in the regulation of FtsZ expression at the post-transcriptional level. Altogether, this study provides an extensive characterization of the cell division gene cluster of M. genitalium and demonstrates the existence of regulatory elements controlling FtsZ expression at the temporal and spatial level in mycoplasmas.

MraZ is a transcriptional inhibitor of cell division in Bacillus subtilis

“…In this report, we show that similar to previous studies conducted in other organisms, MraZ is a transcriptional regulator in B. subtilis (11-13, 16). Specifically, it is important in repressing the expression of two essential cell division genes pbpB and ftsL , in addition to the non-essential mraW gene described above, which does not appear to have a direct role in cell division (10) ( Fig.…”

“…Previous work in E. coli and Mycoplasma showed that overexpression of mraZ results in a change in transcriptional regulation of its own operon causing lethal filamentation in E. coli and cell enlargement in Mycoplasma (11)(12)(13). In E. coli, this phenotype could be resolved by co-expression of the gene immediately downstream of mraZ, mraW, a putative 16S rRNA (and possibly DNA) methyltransferase (12,14).…”

Toward the Complete Functional Characterization of a Minimal Bacterial Proteome

“… 26 Of this group, one gene, JCVISYN3A_0520 (that from now on will be referred to as /0520, and likewise for the other JCVI-syn3A genes discussed) is adjacent to genes of the highly conserved division and cell wall ( dcw ) cluster 51 − 53 and has been previously characterized for Mycoplasma genitalium . 54 Another of these genes (/0527) immediately precedes the dcw cluster on the reverse strand of the circular genome. From previous bioinformatic investigations, 12 , 19 /0520 is characterized as a putative member of the α–β hydrolase superfamily while /0527 is annotated as a protein of unknown function containing a domain of the DUF177 family whose members have been posited to participate in membrane protein biosynthesis among other roles.…”

Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas

“…To date, only a few studies have used immunofluorescence to study protein localization in mycoplasmas, and all of them have focused on ascertaining the polar distribution of proteins in the cells of Mycoplasma mobile , M. pneumoniae , and M. genitalium , which all exhibit highly polarized shapes ( 32 – 35 ). Similarly, a small number of studies have reported the polar localization or colocalization of proteins fused to the fluorescent proteins mCherry and enhanced yellow fluorescent protein (EYFP), again in highly polarized cells ( 36 – 40 ). Other fluorescent proteins have been expressed successfully in several Mycoplasma species, including green fluorescent protein (GFP) ( 41 ), Venus ( 42 ), mNeonGreen, and mKO2 ( 43 ), but have only been used as expression reporters or transformation markers.…”