Transcriptional response to metal starvation in the emerging pathogen Mycoplasma genitalium is mediated by Fur-dependent and –independent regulatory pathways

Abstract

Transition metals participate in numerous enzymatic reactions and they are essential for survival in all living organisms. For this reason, bacterial pathogens have evolved dedicated machineries to effectively compete with their hosts and scavenge metals at the site of infection. In this study, we investigated the mechanisms controlling metal acquisition in the emerging human pathogen Mycoplasma genitalium. We observed a robust transcriptional response to metal starvation, and many genes coding for predicted lipoproteins and ABC-transporters were significantly up-regulated. Transcriptional analysis of a mutant strain lacking a metalloregulator of the Fur family revealed the activation of a full operon encoding a putative metal transporter system and a gene coding for a Histidine-rich lipoprotein (Hrl). We recognized a conserved sequence with dyad symmetry within the promoter region of the Fur-regulated genes. Mutagenesis of the predicted Fur operator within the hrl promoter abrogated Fur-and metal-dependent expression of a reporter gene. Metal starvation still impelled a strong transcriptional response in the fur mutant, demonstrating the existence of Fur-independent regulatory pathways controlling metal homeostasis. Finally, analysis of metal accumulation in the wild-type strain and the fur mutant by ICP-MS revealed an important role of Fur in nickel acquisition.

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

“…RNA-Seq analyses were performed as previously described (Torres-Puig et al, 2018;Martínez-Torró et al, 2020). Mid-log phase cultures of M. genitalium were scraped off in 1 ml of fresh SP-4 and reinoculated in two new 25 cm 2 tissue culture flasks with fresh SP-4 medium for 6 h. Then, cells were lysed, and total RNA was extracted using the miRNeasy Mini Kit (Qiagen).…”

Metal utilization in genome-reduced bacteria: Do human mycoplasmas rely on iron?

“…2 A). Unlike canonical group II ECF modules which are expressed constitutively [68] , the shared ECF module encoded by the MG302-MG303-MG304 operon is overexpressed more than two times under iron deprivation, which is also associated with a nickel (4-fold) and cobalt (2-fold) acquisition increase [24] . This mechanism should be similar in Mpn , the only mollicutes that contains this second ECF operon.…”

“…This mechanism should be similar in Mpn , the only mollicutes that contains this second ECF operon. The putative MG521 S-component is also overexpressed 1.3-fold upon metal depletion [24] , but from a basic phylogenetic analysis it is likely a vitamin B 2 importer ( Fig. 2 A), rather than a transition metal transporter, and it is present in Mge (MG521) and Mpn (MPN527), but also in Mpe (MYPE910) (but not in Mho ).…”

“…The MG302-MG303-MG304 (MPN431-MPN432-MPN433) complex seems likely to work together with a protein which is solely present in Mge and Mpn , but not in Mpe . MG149 (MPN162) is a distinctive protein of the minimal genomes of Mge and Mpn , which shows a 2.1-fold overexpression during iron deprivation [24] , and it that has been described as a histidine-rich lipoprotein with a N-terminal signal peptide, which will be anchored to the extracellular side of the membrane through palmitoylation of a cysteine residue (Uniprot entry: P47395). These molecular and structural features resemble the soluble substrate binding protein (SBP) of ABC importers [85] , which are not present in ECF transport systems.…”

“…Indeed, class I ribonucleotide reductase (RNR), an essential enzyme canonically iron- and/or manganese-dependent, has overcome the requirement of metals in mycoplasmas [23] . Furthermore, despite recent studies reporting the existence of a transcriptional regulator of the Fur (ferric uptake regulator) family in Mge and Mpn [24] , [25] , the specific role of the Fur regulated genes in metal acquisition is unclear. In this review, we aim to discuss the current understanding of iron utilization in four human-infecting mycoplasmas, Mge , Mpn , Mho and Mpe , and describe putative iron-dependent enzymes, transporters, and regulatory systems.…”