SummaryThe positions of DNA regions close to the chromosome replication origin and terminus in growing cells of Escherichia coli have been visualized simultaneously, using new widely applicable reagents. Furthermore, the positions of these regions with respect to a replication factory-associated protein have been analysed. Time-lapse analysis has allowed the fate of origins, termini and the FtsZ ring to be followed in a lineage-specific manner during the formation of microcolonies. These experiments reveal new aspects of the E. coli cell cycle and demonstrate that the replication terminus region is frequently located asymmetrically, on the new pole side of mid-cell. This asymmetry could provide a mechanism by which the chromosome segregation protein FtsK, located at the division septum, can act directionally to ensure that the septal region is free of DNA before the completion of cell division.
The cell wall of Staphylococcus aureus is characterized by an extremely high degree of cross-linking within its peptidoglycan (PGN). Penicillin-binding protein 4 (PBP4) is required for the synthesis of this highly cross-linked peptidoglycan. We found that wall teichoic acids, glycopolymers attached to the peptidoglycan and important for virulence in Gram-positive bacteria, act as temporal and spatial regulators of PGN metabolism, controlling the level of cross-linking by regulating PBP4 localization. PBP4 normally localizes at the division septum, but in the absence of wall teichoic acids synthesis, it becomes dispersed throughout the entire cell membrane and is unable to function normally. As a consequence, the peptidoglycan of TagO null mutants, impaired in wall teichoic acid biosynthesis, has a decreased degree of cross-linking, which renders it more susceptible to the action of lysozyme, an enzyme produced by different host organisms as an initial defense against bacterial infection.
Staphylococcus aureus is an aggressive pathogen and a model organism to study cell division in sequential orthogonal planes in spherical bacteria. However, the small size of staphylococcal cells has impaired analysis of changes in morphology during the cell cycle. Here we use super-resolution microscopy and determine that S. aureus cells are not spherical throughout the cell cycle, but elongate during specific time windows, through peptidoglycan synthesis and remodelling. Both peptidoglycan hydrolysis and turgor pressure are required during division for reshaping the flat division septum into a curved surface. In this process, the septum generates less than one hemisphere of each daughter cell, a trait we show is common to other cocci. Therefore, cell surface scars of previous divisions do not divide the cells in quadrants, generating asymmetry in the daughter cells. Our results introduce a need to reassess the models for division plane selection in cocci.
The essential function of penicillin-binding protein 2 (PBP2) in methicillin-susceptible Staphylococcus aureus RN4220 was clearly established by placing the pbp2 gene under control of the inducible P spac promoter; the resulting bacteria were unable to grow in the absence of inducer. In contrast, the deficit in PBP2 caused by inhibition of transcription of the pbp2 gene did not block growth of a methicillin-resistant S. aureus strain expressing the extra penicillin-binding protein PBP2A, a protein of extraspecies origin that is central to the mechanism of methicillin resistance. Several lines of evidence indicate that the essential function of PBP2 that can be compensated for by PBP2A is the transpeptidase activity. This provides direct genetic evidence that PBP2A has transpeptidase activity.The wide-spectrum resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all -lactam antibiotics has had a devastating impact on the chemotherapy of staphylococcal infections ever since the first appearance of MRSA in clinical specimens in the early 1960s. The key component of this resistance mechanism is an acquired penicillin-binding protein (PBP), PBP2A, which has unusually low affinity for all -lactam antibiotics. The genetic determinant of PBP2A, the mecA gene, is not native to S. aureus but was imported from an as-yet-unidentified extraspecies source (2). Until recently, the accepted model of this resistance mechanism implied that in the presence of -lactam antibiotics the only PBP that remains functional is the low-affinity PBP2A, since the other four staphylococcal PBPs had high enough affinities to penicillin type antibiotics to become rapidly and fully acylated and inactivated even at low drug concentrations that were several orders of magnitude below the concentration needed to inhibit the growth of many MRSA strains. In this model, PBP2A is a surrogate enzyme capable of taking over the normal functions of staphylococcal PBPs in cell wall biosynthesis.However, some new observations require basic revision of this model. The structural determinant of staphylococcal PBP2 was shown to be an auxiliary gene; transposon inactivation of pbp2 resulted in a massive reduction in the level of resistance in an MRSA strain despite the fact that the bacteria continued to produce normal amounts of PBP2A (20). Recent experiments showed that expression of high-level resistance required the cooperative functioning of PBP2A and the penicillin-insensitive transglycosylase (TGase) domain of PBP2 (17).These observations brought back into focus the possibility that native staphylococcal PBPs, primarily PBP2, are participants in the mechanism of -lactam resistance. The purpose of the study described in this paper was to examine in more detail the role(s) PBP2 plays in staphylococcal wall synthesis in antibiotic-susceptible bacteria as well as resistant bacteria (i.e., both in the absence and in the presence of acquired PBP2A). To do this, pbp2 was put under control of the inducible promoter P spac , which allowed testing ...
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