RESUMEN
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.
RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Mycoplasma genitalium/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , 2,2'-Dipiridil/farmacología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Homeostasis , Modelos Moleculares , Mycoplasma genitalium/genética , Regiones Promotoras Genéticas , Proteómica , Proteínas Represoras/química , Transcripción Genética/efectos de los fármacosRESUMEN
Mycoplasma genitalium is a human pathogen adhering to host target epithelial cells and causing urethritis, cervicitis and pelvic inflammatory disease. Essential for infectivity is a transmembrane adhesion complex called Nap comprising proteins P110 and P140. Here we report the crystal structure of P140 both alone and in complex with the N-terminal domain of P110. By cryo-electron microscopy (cryo-EM) and tomography (cryo-ET) we find closed and open Nap conformations, determined at 9.8 and 15 Å, respectively. Both crystal structures and the cryo-EM structure are found in a closed conformation, where the sialic acid binding site in P110 is occluded. By contrast, the cryo-ET structure shows an open conformation, where the binding site is accessible. Structural information, in combination with functional studies, suggests a mechanism for attachment and release of M. genitalium to and from the host cell receptor, in which Nap conformations alternate to sustain motility and guarantee infectivity.