RESUMO
RodZ of rod-shaped bacteria functions to link MreB filaments to the Rod peptidoglycan (PG) synthase complex that moves circumferentially perpendicular to the long cell axis, creating hoop-like sidewall PG. Ovoid-shaped bacteria, such as Streptococcus pneumoniae (pneumococcus; Spn) that lack MreB, use a different modality for peripheral PG elongation that emanates from the midcell of dividing cells. Yet, S. pneumoniae encodes a RodZ homolog similar to RodZ in rod-shaped bacteria. We show here that the helix-turn-helix and transmembrane domains of RodZ(Spn) are essential for growth at 37°C. ΔrodZ mutations are suppressed by Δpbp1a, mpgA(Y488D), and ΔkhpA mutations that suppress ΔmreC, but not ΔcozE. Consistent with a role in PG elongation, RodZ(Spn) co-localizes with MreC and aPBP1a throughout the cell cycle and forms complexes and interacts with PG elongasome proteins and regulators. Depletion of RodZ(Spn) results in aberrantly shaped, non-growing cells and mislocalization of elongasome proteins MreC, PBP2b, and RodA. Moreover, Tn-seq reveals that RodZ(Spn), but not MreCD(Spn), displays a specific synthetic-viable genetic relationship with aPBP1b, whose function is unknown. We conclude that RodZ(Spn) acts as a scaffolding protein required for elongasome assembly and function and that aPBP1b, like aPBP1a, plays a role in elongasome regulation and possibly peripheral PG synthesis.
Assuntos
Peptidoglicano , Streptococcus pneumoniae , Peptidoglicano/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Divisão Celular/genéticaRESUMO
Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Proteínas de Ligação às Penicilinas/genética , Infecções Pneumocócicas/microbiologia , Streptococcus pneumoniae/genética , Divisão Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/ultraestrutura , Citoesqueleto/genética , Citoesqueleto/ultraestrutura , Escherichia coli/genética , GTP Fosfo-Hidrolases/genética , Humanos , Microscopia de Fluorescência , Peptidoglicano/biossíntese , Peptidoglicano/genética , Infecções Pneumocócicas/genética , Streptococcus pneumoniae/patogenicidade , Streptococcus pneumoniae/ultraestruturaRESUMO
HSV-1 is a human pathogen that establishes a lifelong infection in the host. HSV-1 is transported by retrograde axonal transport to sensory neurons in the peripheral nervous system where latent viral genomes can reactivate. The resulting virus travels via anterograde axonal transport to the periphery and can cause clinical disease. CTCF insulators flank the LAT and IE regions of HSV-1 and during latency and maintain the integrity of transcriptional domains through a myriad of functions, including enhancer-blocking or barrier-insulator functions. Importantly, during reactivation, CTCF protein is evicted from the HSV-1 genome, especially from the CTRL2 insulator. CTRL2 is a functional insulator downstream of the 5'exon region of the LAT, so these results suggest that the disruption of this insulator may be required for efficient HSV-1 reactivation. To further explore this, we used a recombinant virus containing a deletion of the CTRL2 insulator (ΔCTRL2) in a rabbit ocular model of HSV-1 infection and induced reactivation. We show that, in the absence of the CTRL2 insulator, HSV-1 established an equivalent latent infection in rabbits, but those rabbits failed to efficiently reactivate from latency. Furthermore, we found a significant decrease in the expression of the gene Us9-, a gene that codes for a type II membrane protein that has been shown to be required for anterograde transport in neurons. Taken together, these results suggest that the functions of the CTRL2 insulator and Us9 activation in reactivating neurons are intrinsically linked through the regulation of a gene responsible for the axonal transport of HSV-1 to the periphery.