The WalR-WalK signaling pathway modulates the activities of both CwlO and LytE through control of the peptidoglycan deacetylase PdaC in Bacillus subtilis

The WalR-WalK two component signaling system in Bacillus subtilis functions in the homeostatic control of the peptidoglycan (PG) hydrolases LytE and CwlO that are required for cell growth. When the activities of these enzymes are low, WalR activates transcription of lytE and cwlO and represses transcription of iseA , a secreted inhibitor of LytE. Conversely, when PG hydrolases activity is too high, WalR-dependent expression of lytE and cwlO is reduced and iseA is de-repressed. In a screen for additional factors that regulate this signaling pathway, we discovered that over-expression of the membrane-anchored PG deacetylase PdaC increases WalR-dependent gene expression. We show that increased expression of PdaC, but not catalytic mutants, prevents cell wall cleavage by both LytE and CwlO, explaining the WalR activation. Importantly, the pdaC gene, like iseA , is repressed by active WalR. We propose that de-repression of pdaC when PG hydrolase activity is too high results in modification of the membrane-proximal layers of the PG, protecting the wall from excessive cleavage by the membrane-tethered CwlO. Thus, the WalR-WalK system homeostatically controls the levels and activities of both elongation-specific cell wall hydrolases. Importance: Bacterial growth and division requires a delicate balance between the synthesis and remodeling of the cell wall exoskeleton. How bacteria regulate the potentially autolytic enzymes that remodel the cell wall peptidoglycan remains incompletely understood. Here, we provide evidence that the broadly conserved WalR-WalK two-component signaling system homeostatically controls both the levels and activities of two cell wall hydrolases that are critical for cell growth.

An Exhaustive Multiple Knockout Approach to Understanding Cell Wall Hydrolase Function in Bacillus subtilis

ABSTRACTMost bacteria are surrounded by their cell wall, a highly crosslinked protective envelope of peptidoglycan. To grow, bacteria must continuously remodel their wall, inserting new material and breaking old bonds. Bond cleavage is performed by cell wall hydrolases, allowing the wall to expand. Understanding the functions of individual hydrolases has been impeded by their redundancy: single knockouts usually present no phenotype. We used an exhaustive multiple-knockout approach to determine the minimal set of hydrolases required for growth in Bacillus subtilis. We identified 42 candidate cell wall hydrolases. Strikingly, we were able to remove all but two of these genes in a single strain; this “Δ40” strain shows a normal growth rate, indicating that none of the 40 hydrolases are necessary for cell growth. The Δ40 strain does not shed old cell wall, demonstrating that turnover is not essential for growth.The remaining two hydrolases in the Δ40 strain are LytE and CwlO, previously shown to be synthetically lethal. Either can be knocked out in Δ40, indicating that either hydrolase alone is sufficient for cell growth. Environmental screening and zymography revealed that LytE activity is inhibited by Mg2+ and that RlpA-like proteins may stimulate LytE activity. Together, these results demonstrate that the only essential function of cell wall hydrolases in B. subtilis is to enable cell growth by expanding the wall and that LytE or CwlO alone is sufficient for this function. These experiments introduce the Δ40 strain as a tool to study hydrolase activity and regulation in B. subtilis.IMPORTANCEIn order to grow, bacterial cells must both create and break down their cell wall. The enzymes that are responsible for these processes are the target of some of our best antibiotics. Our understanding of the proteins that break down the wall – cell wall hydrolases – has been limited by redundancy among the large number of hydrolases many bacteria contain. To solve this problem, we identified 42 cell wall hydrolases in Bacillus subtilis and created a strain lacking 40 of them. We show that cells can survive using only a single cell wall hydrolase; this means that to understand the growth of B. subtilis in standard laboratory conditions, it is only necessary to study a very limited number of proteins, simplifying the problem substantially. We additionally show that the Δ40 strain is a research tool to characterize hydrolases, using it to identify 3 ‘helper’ hydrolases that act in certain stress conditions.

Cell-Wall Hydrolases as Antimicrobials against Staphylococcus Species: Focus on Sle1

Some staphylococcal species are opportunistic pathogens of humans and/or animals with Staphylococcus epidermidis as one of the most important. It causes a broad spectrum of diseases in humans and animals. This species is able to form biofilms and has developed antibiotic resistance, which has motivated research on new antibacterial agents. Cell-wall hydrolases (CWHs) can constitute a potential alternative. Following a hijacking strategy, we inventoried the CWHs of S. epidermidis. The lytic potential of representative CWHs that could be turned against staphylococci was explored by turbidity assays which revealed that cell wall glycosidases were not efficient, while cell wall amidases and cell wall peptidases were able to lyse S. epidermidis. Sle1, which is encoded by chromosomal gene and composed of three anchoring LysM domains and a C-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain, was one of the most active CWHs. The phylogeny of Sle1 revealed seven clusters mostly identified among staphylococci. Sle1 was able to lyse several staphylococcal species, including Staphylococcus aureus, both in planktonic and sessile forms, but not Micrococcus.

Cell wall hydrolases act in concert during aerenchyma development in sugarcane roots

Background and Aims Cell wall disassembly occurs naturally in plants by the action of several glycosyl-hydrolases during different developmental processes such as lysigenous and constitutive aerenchyma formation in sugarcane roots. Wall degradation has been reported in aerenchyma development in different species, but little is known about the action of glycosyl-hydrolases in this process. Methods In this work, gene expression, protein levels and enzymatic activity of cell wall hydrolases were assessed. Since aerenchyma formation is constitutive in sugarcane roots, they were assessed in segments corresponding to the first 5 cm from the root tip where aerenchyma develops. Key Results Our results indicate that the wall degradation starts with a partial attack on pectins (by acetyl esterases, endopolygalacturonases, β-galactosidases and α-arabinofuranosidases) followed by the action of β-glucan-/callose-hydrolysing enzymes. At the same time, there are modifications in arabinoxylan (by α-arabinofuranosidases), xyloglucan (by XTH), xyloglucan–cellulose interactions (by expansins) and partial hydrolysis of cellulose. Saccharification revealed that access to the cell wall varies among segments, consistent with an increase in recalcitrance and composite formation during aerenchyma development. Conclusion Our findings corroborate the hypothesis that hydrolases are synchronically synthesized, leading to cell wall modifications that are modulated by the fine structure of cell wall polymers during aerenchyma formation in the cortex of sugarcane roots.

Exploring the diverse functions of peptidoglycan hydrolases in the plant pathogen, Agrobacterium tumefaciens

The plant pathogen Agrobacterium tumefaciens displays an atypical form of unipolar elongation, followed by incipient pole synthesis during cell division and cell separation. Currently, how polar growing bacteria modulate cell wall hydrolysis during growth and division remains largely unknown. This work includes the comprehensive analysis and characterization of the role of cell wall hydrolyses involved in bacterial growth, division, recycling and beta-lactam resistance in A. tumefaciens. First, we performed bioinformatic analyses and used reverse genetics to better understand the role cell wall hydrolases in A. tumefaciens. Inactivation of most cell wall hydrolases, led to no phenotypic defects suggesting a high degree of redundancy. However, inactivation of the amidase, AmiD, and the lytic transglycosylase Atu3779, revealed significant changes in beta-lactam resistance suggesting that these proteins are involved in the activation beta-lactamases and outer-membrane integrity. Next, we developed a tool (Figueroa-Cuilan et al., 2016) to dissect the role of essential genes, which enabled characterization of the essential regulator of cell division, DipM, a LytM-containing factor. Absence of DipM causes severe cell division defects, including increased cell length, mid-cell width and lysis. A cell wall composition analysis of cells devoid of DipM shows an increase in the activity of the PG hydrolases, lytic transglycosylases, suggesting that DipM may inhibit the activity of these enzymes. Lastly, we find that deletion of individual lytic transglycolsylases (LTs) from the DipM depletion strain delays the onset of the DipM depletion phenotype. Overall, this research provides mechanistic insights about the roles of peptidoglycan hydrolases and their regulators in cell growth and division. Understanding how bacterial cell wall hydrolysis is spatiotemporally regulated and coordinated with cell wall synthesis and cell division (Figueroa-Cuilan and Brown, 2018), will be applicable to other closely related polar-growing bacteria.