LON DELETION IMPAIRS PERSISTER CELL RESUSCITATION IN ESCHERICHIA COLI

Bacterial persisters are non-growing cells that are highly tolerant to bactericidal antibiotics. However, this tolerance is reversible and not mediated by heritable genetic changes. Lon, an ATP-dependent protease, has repeatedly been shown to play a critical role in fluoroquinolone persistence. Although lon deletion (Δlon) is thought to kill persister cells via accumulation of the cell division inhibitor protein SulA, the exact mechanism underlying this phenomenon has yet to be elucidated. Here, we show that Lon is an important regulatory protein for the resuscitation of the fluoroquinolone persisters in Escherichia coli, and lon deletion impairs the ability of persister cells to form colonies during recovery, without killing these cells, through a sulA- and ftsZ-dependent mechanism. Notably, this observed non-culturable state of antibiotic-tolerant Δlon cells is transient, as environmental conditions, such as starvation, can restore their culturability. Our data further indicate that starvation-induced SulA degradation or expression of Lon during recovery facilitates Z-ring formation in Δlon persisters. Calculating the ratio of the cell length (L in µm) to the number of Z-rings (Z) for each ofloxacin-treated intact cell analyzed has revealed a strong correlation between persister resuscitation and calculated L/Z values, which represents a potential biomarker for Δlon persisters that are transitioning to the normal cell state under the conditions studied here.

Antistaphylococcal Activity of the FtsZ Inhibitor C109

Staphylococcus aureus infections represent a great concern due to their versatility and involvement in different types of diseases. The shortage of available clinical options, especially to treat multiresistant strains, makes the discovery of new effective compounds essential. Here we describe the activity of the previously described cell division inhibitor C109 against methicillin-sensitive and -resistant S. aureus strains. Antibiofilm activity was assessed using microtiter plates, confocal microscopy, and in an in vitro biofilm wound model. The ability of C109 to block FtsZ GTPase activity and polymerization was tested in vitro. Altogether, the results show that the FtsZ inhibitor C109 has activity against a wide range of S. aureus strains and support its use as an antistaphylococcal compound.

A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

Rod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple alternative division inhibition pathways exist during the SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism. Importance: Filamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

A newly identified prophage-encoded gene, ymfM, causes SOS-inducible filamentation in Escherichia coli

Rod-shaped bacteria such as Escherichia coli can regulate cell division in response to stress, leading to filamentation, a process where cell growth and DNA replication continues in the absence of division, resulting in elongated cells. The classic example of stress is DNA damage which results in the activation of the SOS response. While the inhibition of cell division during SOS has traditionally been attributed to SulA in E. coli, a previous report suggests that the e14 prophage may also encode an SOS-inducible cell division inhibitor, previously named SfiC. However, the exact gene responsible for this division inhibition has remained unknown for over 35 years. A recent high-throughput over-expression screen in E. coli identified the e14 prophage gene, ymfM, as a potential cell division inhibitor. In this study, we show that the inducible expression of ymfM from a plasmid causes filamentation. We show that this expression of ymfM results in the inhibition of Z ring formation and is independent of the well characterised inhibitors of FtsZ ring assembly in E. coli, SulA, SlmA and MinC. We confirm that ymfM is the gene responsible for the SfiC+ phenotype as it contributes to the filamentation observed during the SOS response. This function is independent of SulA, highlighting that multiple division inhibition pathways exist during the stress-induced SOS response. Our data also highlight that our current understanding of cell division regulation during the SOS response is incomplete and raises many questions regarding how many inhibitors there actually are and their purpose for the survival of the organism.ImportanceFilamentation is an important biological mechanism which aids in the survival, pathogenesis and antibiotic resistance of bacteria within different environments, including pathogenic bacteria such as uropathogenic Escherichia coli. Here we have identified a bacteriophage-encoded cell division inhibitor which contributes to the filamentation that occurs during the SOS response. Our work highlights that there are multiple pathways that inhibit cell division during stress. Identifying and characterising these pathways is a critical step in understanding survival tactics of bacteria which become important when combating the development of bacterial resistance to antibiotics and their pathogenicity.

DdcA antagonizes a bacterial DNA damage checkpoint

Bacteria coordinate DNA replication and cell division, ensuring that a complete set of genetic material is passed onto the next generation. When bacteria encounter DNA damage or impediments to DNA replication, a cell cycle checkpoint is activated to delay cell division by expressing a cell division inhibitor. The prevailing model for bacterial DNA damage checkpoints is that activation of the DNA damage response and protease mediated degradation of the cell division inhibitor is sufficient to regulate the checkpoint process. Our recent genome-wide screens identified the geneddcAas critical for surviving exposure to a broad spectrum of DNA damage. TheddcAdeletion phenotypes are dependent on the checkpoint enforcement protein YneA. We found that expression of the checkpoint recovery proteases could not compensate forddcAdeletion. Similarly, expression ifddcAcould not compensate for the absence of the checkpoint recovery proteases, indicating that DdcA function is distinct from the checkpoint recovery step. Deletion ofddcAresulted in sensitivity toyneAoverexpression independent of YneA protein levels or stability, further supporting the conclusion that DdcA regulates YneA through a proteolysis independent mechanism. Using a functional GFP-YneA we found that DdcA inhibits YneA activity independent of YneA localization, suggesting that DdcA may regulate YneA access to its target. These results uncover a regulatory step that is important for controlling the DNA damage checkpoint in bacteria, and suggests that the typical mechanism of degrading the checkpoint enforcement protein is insufficient to control the rate of cell division in response to DNA damage.Author SummaryAll cells coordinate DNA replication and cell division. When cells encounter DNA damage, the process of DNA replication is slowed and the cell must also delay cell division. In bacteria, the process has long been thought to occur using two principle modes of regulation. The first, is RecA coated ssDNA transmits the signal of DNA damage through inactivation of the repressor of the DNA damage (SOS) response regulon, which results in expression of a cell division inhibitor establishing the checkpoint. The second principle step is protease mediated degradation of the cell division inhibitor relieving the checkpoint. Recent work by our lab and others has suggested that this process may be more complex than originally thought. Here, we investigated a gene of unknown function that we previously identified as important for survival when the bacteriumBacillus subtilisis exposed to DNA damage. We found that this gene negatively regulates the cell division inhibitor, but is functionally distinct from the checkpoint recovery process. We provide evidence that this gene functions as an antagonist to establishing the DNA damage checkpoint. Our study uncovers a novel layer of regulation in the bacterial DNA damage checkpoint process challenging the longstanding models established in the bacterial DNA damage response field.

SosA inhibits cell division inStaphylococcus aureusin response to DNA damage

Inhibition of cell division is critical for cell viability under DNA damaging conditions. In bacterial cells, DNA damage induces the SOS response, a process that inhibits cell division while repairs are being made. In coccoid bacteria, such as the human pathogenStaphylococcus aureus, the process remains poorly understood. Here we have characterized an SOS-induced cell-division inhibitor, SosA, inS. aureus. We find that in contrast to the wildtype,sosAmutant cells continue division under DNA damaging conditions with decreased viability as a consequence. Conversely, overproduction of SosA leads to cell division inhibition and reduced growth. The SosA protein is localized in the bacterial membrane and mutation of an extracellular amino acid, conserved between homologs of other staphylococcal species, abolished the inhibitory activity as did truncation of the C-terminal 30 amino acids. In contrast, C-terminal truncation of 10 amino acids lead to SosA accumulation and a strong cell division inhibitory activity. A similar phenotype was observed upon expression of wildtype SosA in a mutant lacking the membrane protease, CtpA. Thus, the extracellular C-terminus of SosA is required both for cell-division inhibition and for turnover of the protein. Functional studies showed that SosA is likely to interact with one or more divisome components and, without interfering with early cell-division events, halts cell division at a point where septum formation is initiated yet being unable to progress to septum closure. Our findings provide important insights into cell-division regulation in staphylococci that may foster development of new classes of antibiotics targeting this essential process.ImportanceStaphylococcus aureusis a serious human pathogen and a model organism for cell-division studies in spherical bacteria. We show that SosA is the DNA-damage-inducible cell-division inhibitor inS. aureusthat upon expression causes cell swelling and cessation of the cell cycle at a characteristic stage post septum initiation but prior to division plate completion. SosA appears to function via an extracellular activity and is likely to do so by interfering with the essential membrane-associated division proteins, while at the same time being negatively regulated by the membrane protease CtpA. This report represents the first description of the process behind cell-division inhibition in coccoid bacteria. As several pathogens are included in this category, uncovering the molecular details of SosA activity and control can lead to identification of new targets for development of valuable anti-bacterial drugs.