Biofilms can act as plasmid reserves in the absence of plasmid specific selection.

Plasmids facilitate rapid bacterial adaptation by shuttling a wide variety of beneficial traits across microbial communities. However, under non-selective conditions, maintaining a plasmid can be costly to the host cell. Nonetheless, plasmids are ubiquitous in nature where bacteria adopt their dominant mode of life - biofilms. Here, we demonstrate that biofilms can act as spatiotemporal reserves for plasmids, allowing them to persist even under non-selective conditions. However, under these conditions, spatial stratification of plasmid-carrying cells may promote the dispersal of cells without plasmids, and biofilms may thus act as plasmid sinks.

Staphylococcus aureus ClpX localizes at the division septum and impacts transcription of genes involved in cell division, T7-secretion, and SaPI5-excision.

In all living cells, molecular chaperones are essential for facilitating folding and unfolding of proteins. ClpX is a highly conserved ATP-dependent chaperone that besides functioning as a classical chaperone can associate with ClpP to form the ClpXP protease. To investigate the relative impact of the ClpXP protease and the ClpX chaperone in cell physiology of the important pathogenic bacterium Staphylococcus aureus, we assessed the transcriptional changes induced by inactivating only ClpXP, or by completely deleting ClpX. This analysis revealed that ClpX has a profound impact on S. aureus cell physiology that is mediated primarily via ClpXP-dependent pathways. As an example, ClpX impacts expression of virulence genes entirely via ClpXP-dependent mechanisms. Furthermore, ClpX controls a high number of genes and sRNAs via pathways involving both ClpXP protease and ClpX chaperone activities; an interesting example being genes promoting excision and replication of the pathogenicity island SaPI5. Independently of ClpP, ClpX, impacts transcription of only a restricted number of genes involved in peptidoglycan synthesis, cell division, and type seven secretion. Finally, we demonstrate that ClpX localizes in single foci in close proximity to the division septum lending support to the idea that ClpX plays a role in S. aureus cell division.

The Sle1 Cell Wall Amidase Is Essential for ß-Lactam Resistance in Community-Acquired Methicillin-Resistant Staphylococcus aureus USA300.

Most clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) strains have become resistant to ß-lactams antibiotics through horizontal acquisition of the mecA gene encoding PBP2a, a peptidoglycan transpeptidase with low affinity for ß-lactams. The level of resistance conferred by mecA is, however, strain dependent, and the mechanisms underlying this phenomenon remain poorly understood. We show here that ß-lactam resistance correlates to expression of the Sle1 cell wall amidase in the fast-spreading and highly virulent community-acquired MRSA USA300 clone. Sle1 is a substrate of the ClpXP protease, and while the high Sle1 levels in cells lacking ClpXP activity confer ß-lactam hyper-resistance, USA300 cells lacking Sle1 are as susceptible to ß-lactams as cells lacking mecA This finding prompted us to assess the cellular roles of Sle1 in more detail, and we demonstrate that high Sle1 levels accelerate the onset of daughter cells splitting and decrease cell size. Vice versa, oxacillin decreases the Sle1 level and imposes a cell separation defect that is antagonized by high Sle1 levels, suggesting that high Sle1 levels increase tolerance to oxacillin by promoting cell separation. In contrast, increased oxacillin sensitivity of sle1 cells appears linked to a synthetic lethal effect on septum synthesis. In conclusion, this study demonstrates that Sle1 is a key factor in resistance to ß-lactam antibiotics in the JE2 USA300 model strain and that PBP2a is required for the expression of Sle1 in JE2 cells exposed to oxacillin.

The ClpX chaperone controls autolytic splitting of Staphylococcus aureus daughter cells, but is bypassed by ß-lactam antibiotics or inhibitors of WTA biosynthesis.

ß-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of ß-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of ß-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with ß-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by ß-lactams or by inhibiting an early step in WTA biosynthesis. The finding that ß-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that ß-lactams do not kill S. aureus simply by weakening the cell wall.