Katherine R. Parzych

Significance: Autophagy is a highly conserved eukaryotic cellular recycling process.Through the degradation of cytoplasmic organelles, proteins, and macromolecules, and the recycling of the breakdown products, autophagy plays important roles in cell survival and maintenance.Accordingly, dysfunction of this process contributes to the pathologies of many human diseases.Recent Advances: Extensive research is currently being done to better understand the process of autophagy.In this review, we describe current knowledge of the morphology, molecular mechanism, and regulation of mammalian autophagy.Critical Issues: At the mechanistic and regulatory levels, there are still many unanswered questions and points of confusion that have yet to be resolved.Future Directions: Through further research, a more complete and accurate picture of the molecular mechanism and regulation of autophagy will not only strengthen our understanding of this significant cellular process, but will aid in the development of new treatments for human diseases in which autophagy is not functioning properly.

Article18 March 2010free access Daughter cell separation is controlled by cytokinetic ring-activated cell wall hydrolysis Tsuyoshi Uehara Tsuyoshi Uehara Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Katherine R Parzych Katherine R Parzych Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Thuy Dinh Thuy Dinh Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Thomas G Bernhardt Corresponding Author Thomas G Bernhardt Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Tsuyoshi Uehara Tsuyoshi Uehara Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Katherine R Parzych Katherine R Parzych Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Thuy Dinh Thuy Dinh Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Thomas G Bernhardt Corresponding Author Thomas G Bernhardt Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Author Information Tsuyoshi Uehara1, Katherine R Parzych1,‡, Thuy Dinh1,‡ and Thomas G Bernhardt 1 1Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA, USA ‡These authors contributed equally to this work *Corresponding author. Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Armenise Building, Room 302A, Boston, MA 02115, USA. Tel.: +1 617 432 6971; Fax: +1 617 738 7664; E-mail: [email protected] The EMBO Journal (2010)29:1412-1422https://doi.org/10.1038/emboj.2010.36 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info During bacterial cytokinesis, hydrolytic enzymes are used to split wall material shared by adjacent daughter cells to promote their separation. Precise control over these enzymes is critical to prevent breaches in wall integrity that can cause cell lysis. How these potentially lethal hydrolases are regulated has remained unknown. Here, we investigate the regulation of cell wall turnover at the Escherichia coli division site. We show that two components of the division machinery with LytM domains (EnvC and NlpD) are direct regulators of the cell wall hydrolases (amidases) responsible for cell separation (AmiA, AmiB and AmiC). Using in vitro cell wall cleavage assays, we show that EnvC activates AmiA and AmiB, whereas NlpD activates AmiC. Consistent with these findings, we show that an unregulated EnvC mutant requires functional AmiA or AmiB but not AmiC to induce cell lysis, and that the loss of NlpD phenocopies an AmiC− defect. Overall, our results suggest that cellular amidase activity is regulated spatially and temporally by coupling their activation to the assembly of the cytokinetic ring. Introduction Most bacteria surround themselves with a complex cell envelope that serves as a barrier to insult and a conduit for nutrient uptake. An important component of the envelope is the cell wall or peptidoglycan (PG) layer. This essential layer is constructed from polysaccharide strands that are covalently connected along their length by cross-linked peptides to form a continuous meshwork that encapsulates the cytoplasmic membrane and protects it from osmotic rupture (Vollmer, 2008) (Figure 1). During growth and division, this tough exoskeleton is continuously remodelled by PG synthases called penicillin-binding proteins (PBPs) as well as an array of PG hydrolases capable of cleaving bonds in the PG meshwork (Vollmer et al, 2008). Tight control over these potentially lethal hydrolases must be maintained to prevent them from creating breaches in the cell wall that can lead to cell lysis. Despite the importance of such controls, the cellular mechanisms regulating PG hydrolases have long remained mysterious. Figure 1.Coordinated envelope constriction in gram-negative bacteria. Diagram of a dividing cell with an assembled cytokinetic ring apparatus (green). The box contains a close-up diagram of the division site highlighting the coordinated constriction of the envelope layers: OM, outer membrane; PG, peptidoglycan; IM, inner membrane; Z-ring, FtsZ cytoskeletal ring. The oval contains a diagram of the PG chemical structure: M, N-acetylmuramic acid; G, N-acetylglucosamine. Coloured dots represent the attached peptides. The PG structure continues in all directions to envelop the cell (red arrows). Download figure Download PowerPoint The most clearly defined function for cellular PG hydrolases is the splitting of wall material formed between daughter cells during the division process (Vollmer et al, 2008). In Escherichia coli and other gram-negative bacteria, cytokinesis proceeds via the coordinated constriction of all three envelope layers: the inner and outer membranes along with the PG layer sandwiched between them (Figure 1) (den Blaauwen et al, 2008; Uehara et al, 2009). This coordination is achieved by the divisome, a ring-shaped, multiprotein division machine organized by the bacterial tubulin protein, FtsZ (den Blaauwen et al, 2008). One of the primary functions of the divisome is to promote the synthesis of the PG layer that will eventually fortify the new daughter cell poles. This involves several divisome-associated PBPs (den Blaauwen et al, 2008). Although the septal PG produced by these synthases is initially shared by the daughter cells, it must be split shortly after it is formed to allow constriction of the outer membrane to closely follow that of the inner membrane and FtsZ ring (Figure 1). In E. coli, two sets of periplasmic factors are critical for septal PG splitting: LytC-type N-acetylmuramyl-L-alanine amidases (Pfam: Amidase_3) and LytM (lysostaphin/Pfam: Peptidase_M23) factors, both of which are widely distributed among eubacteria (Heidrich et al, 2001; Firczuk and Bochtler, 2007; Vollmer et al, 2008; Uehara et al, 2009). Mutants lacking multiple amidases or LytM factors form long chains of cells that complete inner membrane constriction and fusion, but remain connected by unsplit layers of septal PG that interfere with outer membrane invagination (Heidrich et al, 2001; Priyadarshini et al, 2007; Uehara et al, 2009). Amidases are PG hydrolases that remove the stem peptide from the glycan strands of PG and can therefore break its cross-links. E. coli encodes three factors with LytC-type amidase domains: AmiA, AmiB and AmiC, all of which are important for cell separation (Heidrich et al, 2001). The founding members of the LytM family of factors, LytM and lysostaphin, are metallo-endopeptidases that cleave pentaglycine cross-bridges in staphylococcal PG (Firczuk and Bochtler, 2007). On the basis of this activity, LytM factors in other bacteria are thought to be PG hydrolases as well, but with altered cleavage specificity because pentaglycine cross-bridges are only found among the staphylococci (Schleifer and Kandler, 1972). E. coli encodes four factors with recognizable LytM domains. Two of them, EnvC and NlpD, have major functions in cell separation (Uehara et al, 2009). The biochemical activities of the E. coli amidases and LytM factors have remained poorly defined. Amidase activity in a purified system has only been shown for AmiA (Lupoli et al, 2009). AmiC amidase activity has only been detected in crude cell extracts, whereas that of AmiB has not been reported so far (Heidrich et al, 2001). Consistent with EnvC possessing PG hydrolase activity, it was able to generate a zone of clearing in a gel-based zymogram assay (Bernhardt and de Boer, 2004). However, its activity has not been investigated in solution, nor has that of NlpD. Although EnvC, NlpD and all of the amidases except for AmiA are recruited to midcell to participate in the division process (Bernhardt and de Boer, 2003, 2004; Uehara et al, 2009) (T Dinh, unpublished results), it has remained unclear how they work together to specifically promote cell separation during cytokinesis without also promoting cell lysis during interdivisional periods of the cell cycle. Here, we show that the LytM factors, EnvC and NlpD, are not themselves PG hydrolases as previously suspected, but rather function as potent and specific activators of the PG amidases. We infer that the LytM factors have a direct and critical function in the spatiotemporal regulatory mechanism that confines cellular amidase activity to the cytokinetic ring. Results The coiled-coil domain of EnvC is a regulatory domain that is necessary and sufficient for recruitment to the divisome EnvC is a 419 amino-acid protein that contains three identifiable domains: a signal peptide (residues 1–34), a coiled-coil (CC) domain (residues 35–271) and a LytM domain (residues 318–413) (Hara et al, 2002; Ichimura et al, 2002; Bernhardt and de Boer, 2004) (Figure 2A). We hypothesized that the CC domain might serve a regulatory function controlling the putative PG hydrolase activity of EnvC. To explore this possibility, we monitored the growth and morphology of cells producing exported GFP fusions to full length, mature EnvC (35–419) [GFP-FLEnvC] or a truncated version lacking the CC domain, EnvC (278–419) [GFP-LytEnvC]. As expected from earlier results (Bernhardt and de Boer, 2004), GFP-FLEnvC was functional, did not affect growth and localized to the septum (Figure 2B; Supplementary Figure S1A and S1B). Production of GFP-LytEnvC, on the other hand, was extremely toxic and caused cell lysis after low-level induction (Figure 2B). This fusion displayed a largely peripheral (periplasmic) localization pattern and caused the formation of envelope lesions at many locations around the cell body (Figure 2C; Supplementary Figure S1C). Immunoblot analysis revealed that the lytic dose of GFP-LytEnvC was barely detectable relative to the amount of GFP-FLEnvC produced in normally growing cells supplemented with high levels of inducer (Supplementary Figure S2A and S2B). Thus, the CC domain does not control EnvC levels, but instead seems to restrict EnvC activity to the division site by mediating its recruitment to the divisome. Figure 2.The LytM domain of EnvC induces lysis. (A) Predicted domain structure of EnvC. SS, signal sequence; CC, coiled coil; LytM, LytM/lysostaphin/peptidase_M23 domain. (B) Wild-type (MG1655) cells harbouring the expression constructs pTU113 [Plac∷ssdsbA-gfp-flenvC] or pTU115 [Plac∷ssdsbA-gfp-lytenvC] integrated at the phage HK022 att site were grown in LB at 37°C to an OD600 of about 0.5. At t=0, the cultures were diluted into fresh LB with (open symbols) or without (closed symbols) 20 μM IPTG and growth was continued at 37°C. Induction of GFP-FLEnvC with much higher IPTG levels (250 μM–1 mM) also failed to affect cell growth (data not shown). Fusions encoded the signal sequence from dsbA for export and the GFP variant used was superfolder GFP (Pédelacq et al, 2006), which we have found is functional in the periplasm following Sec export (T Dinh, unpublished results). (C) Phase contrast (C1) and GFP-fluorescence (C2) micrographs of cells lysing as a result of GFP-LytEnvC production. Arrows highlight cell envelope defects, many of which correspond to regions with an enlarged periplasm as indicated by the accumulation of the exported GFP-LytEnvC fusion. Bar=4 μm. See Supplementary Figure S1C for images of cells before the onset of lysis. Download figure Download PowerPoint To determine whether the CC domain is sufficient for the recruitment of EnvC to the divisome, we studied the localization of an exported GFP-EnvC (35–277) fusion [GFP-CCEnvC]. To eliminate the possibility that GFP-CCEnvC might be recruited to the septum through EnvC–EnvC interactions rather than EnvC–divisome interactions, we studied its localization in a ΔenvC strain. Periplasmic protein localization studies in this background are complicated by the fact that unfused periplasmic GFP seems to accumulate at the septa of EnvC− cells because the delay in septal PG splitting results in an increased periplasmic volume at these sites (Bernhardt and de Boer, 2004). To account for this, we co-produced exported mCherry and GFP-CCEnvC in the ΔenvC mutant. In these cells, we observed ring or band-like accumulations of GFP-CCEnvC at nascent septa that lacked corresponding accumulations of periplasmic mCherry (Figure 3A). In contrast and as expected, when exported mCherry and unfused GFP were co-produced in ΔenvC cells, their apparent accumulations at septa were always coincident (Figure 3B and C). We conclude that the CC domain is necessary and sufficient for EnvC recruitment to the divisome. Moreover, the failure of GFP-CCEnvC to correct the EnvC− phenotype (Figure 3) indicates that the LytM domain is required for EnvC to promote proper septal PG splitting. Figure 3.The CC domain of EnvC is a septal targeting domain. (A–C) Shown are cells of TU176/pTU175 [ΔenvC/Psyn135∷ssdsbA-mCherry] containing the integrated GFP fusion constructs (A) attHKTU128 (Plac∷ ssdsbA-gfp-ccenvC) and (B, C) attHKTB263 (Plac∷ ssdsbA-gfp). Cells were grown to an OD600 of about 0.5 in M9 maltose medium without IPTG (A, B) or with 100 μM IPTG (C), and visualized with GFP (panels A1, B1 and C1), mCherry (panels A2, B2 and C2) or DIC (panels A3, B3 and C3) optics. Arrows highlight GFP-CCEnvC recruitment to nascent division sites that lack a strong periplasmic mCherry signal. Unlike the GFP-CCEnvC construct, which could be visualized in cells grown without IPTG, visualization of periplasmic GFP required the addition of 100 μM IPTG. Psyn135 is a synthetic promoter for constitutive expression. Note that the ΔenvC cells expressing periplasmic GFP-CCEnvC or GFP both display the cell division and cell separation phenotypes typical of EnvC− mutants. (D, E) Shown are cells of TB134 (attHKTB316) [ΔenvC (Plac∷flenvC-mCherry)] containing the integrated GFP fusion constructs (D) attλTU222 (Para∷ ssdsbA-gfp-ccenvC) or (E) attλTU178 (Para∷ ssdsbA-gfp). Cells were grown at 30°C in M9 maltose medium supplemented with 0.2% arabinose but without IPTG, and visualized with mCherry (panels D1 and E1), GFP (panels D2 and E2) or DIC (panels D3 and E3) optics. Bar=4 μm. Download figure Download PowerPoint To further investigate the function of protein localization in the regulation of EnvC activity, we monitored the effect of GFP-CCEnvC overproduction on the localization and function of FLEnvC–mCherry. When produced as the sole source of full-length EnvC in cells, FLEnvC–mCherry displayed a clear septal localization pattern and promoted proper cell division (Figure 3E) (Uehara et al, 2009). Overproduction of exported GFP-CCEnvC, however, both interfered with FLEnvC–mCherry recruitment to the division site and induced a dominant-negative EnvC− division defect (Figure 3D). Immunoblots indicated that FLEnvC–mCherry remained intact when GFP-CCEnvC was overproduced (Supplementary Figure S2C). We therefore conclude that GFP-CCEnvC effectively competes with FLEnvC–mCherry for a limited number of binding sites at the divisome, and that recruitment to the divisome is critical for EnvC to promote proper septal PG splitting. Interestingly, in contrast to our findings with LytEnvC, delocalized FLEnvC did not cause cell lysis in these experiments (Figure 3D). This implies that proper spatiotemporal regulation of EnvC activity involves more than just the control of its subcellular localization (see Discussion). In this regard, the differential lytic activities of delocalized FLEnvC and LytEnvC suggest that, in addition to controlling EnvC localization, the CC domain may also directly or indirectly regulate the activity of the LytM domain. Notably, although overproduction of GFP-FLEnvC from the integrated attHKTU113 construct (Plac∷ssdsbA-gfp-flenvC) failed to induce lysis at all IPTG levels tested up to 1 mM (Figure 2, data not shown), higher-level GFP-FLEnvC production from a similar construct (attλTU179) with a stronger promoter (Para) was capable of inducing lysis (Supplementary Figure S3). This indicates that the regulatory controls governing FLEnvC activity are saturable and can be overwhelmed by excess protein. EnvC does not degrade PG in solution To better define the function of the CC domain in EnvC regulation, we sought to reconstitute EnvC PG hydrolase activity in solution. FLEnvC and LytEnvC were purified (Figure 4A) and tested for their ability to degrade PG using a dye-release assay (Zhou et al, 1988). For this, purified E. coli PG was covalently labelled on its sugar moieties with the dye remazol brilliant blue (RBB). RBB-modified PG was then incubated with purified protein and hydrolytic activity was monitored as dye remaining in the supernatant after the reaction was terminated and centrifuged to pellet intact PG. Control assays readily detected lysozyme-mediated PG cleavage (Figure 4B). In contrast to earlier results using a gel-based zymogram assay (Bernhardt and de Boer, 2004), however, no PG hydrolase activity was observed for FLEnvC or LytEnvC by dye release even after overnight incubation of the reactions (Figure 4B, data not shown). Figure 4.EnvC does not degrade PG in solution. (A) Purified proteins were separated by SDS–PAGE (12.5% T) and stained with Coomassie brilliant blue. Proteins used in this study were purified with a 6xHis-SUMO tag (H-SUMO) fused to their N-termini. The tag was removed with purified 6xHis-tagged SUMO protease (H-SP). Cleaved H-SUMO tag and H-SP were depleted from the preparation using fresh Ni-NTA resin. A small amount of H-SUMO remains in the EnvC and AmiA preparations (arrow). (B) Dye-release assays for PG hydrolysis. RBB-PG was incubated with the indicated proteins (4 μM each) for 30 min at 37°C. Undigested PG was pelleted and the absorbance of the supernatant was measured at 595 nm. Longer incubation (>20 h) did not result in significant dye release by either FLEnvC or LytEnvC (data not shown). Download figure Download PowerPoint LytEnvC requires the amidases, AmiA and AmiB, to induce lysis The inactivity of both FLEnvC and LytEnvC in vitro prompted us to consider that EnvC may not be a PG hydrolase, a possibility consistent with the fact that EnvC lacks all of the zinc-chelating residues identified in the active site of LytM (Odintsov et al, 2004; Firczuk et al, 2005) (Supplementary Figure S4). Nevertheless, GFP-LytEnvC must cause PG damage in vivo, as it clearly elicited cell lysis. We therefore considered the possibility that rather than being a PG hydrolase itself, EnvC might activate one. To identify the putative EnvC-activated hydrolase(s), we selected for mutants resistant to GFP-LytEnvC production. Interestingly, two resistant mutants with transposon insertions in amiB were isolated, suggesting that EnvC activates AmiB to promote septal PG splitting. To investigate this further, we tested a panel of ami deletion mutants for resistance to GFP-LytEnvC (Figure 5). Deletion of amiB partially restored the plating efficiency of cells producing GFP-LytEnvC, whereas single amiA or amiC deletions had no effect (Figure 5, data not shown). Combining ΔamiA with ΔamiB, but not ΔamiC, fully suppressed the toxicity of LytEnvC (Figure 5). Consistent with the results on solid media, amidase defects delayed or prevented lysis in response to GFP-LytEnvC production when cells were grown in liquid medium (Supplementary Figure S5). Figure 5.Mutants with amidase defects are resistant to GFP-LytEnvC production. Cultures of the indicated strains containing the Para∷gfp-lytenvC construct pTU181 integrated at the λ att site were grown overnight at 30°C in LB. Culture densities were normalized and 10-fold serial dilutions were prepared for each. A measure of 10 μl of each dilution was spotted onto the indicated solid media and the plates were incubated overnight at 30°C. The strains used were: TB28 [WT], TU226 [amiB∷Tn4], TU227 [amiB∷Tn6], TB170 [ΔamiB], TB141 [ΔamiA], TU207 [ΔamiA ΔamiB] and TB150 [ΔamiA ΔamiC]. The amiB∷Tn4 and amiB∷Tn6 mutants were isolated in the selection for GFP-LytEnvC resistant mutants. Download figure Download PowerPoint Amidase activation in vitro Our results thus far suggested that EnvC may activate AmiA and AmiB, and that GFP-LytEnvC activates them inappropriately to induce cell lysis. To test this directly, we purified the amidases (Figure 4A) and assayed their PG hydrolase activity in the presence and absence of FLEnvC. At high concentrations (4 μM) and long incubation times (4 h to overnight), purified AmiA, AmiB or AmiC alone promoted significant levels of dye release from RBB-PG (Figure 6A). When the amidases were assayed at lower concentrations and/or using shorter reaction times (30 min), minimal dye release was observed (Figure 6B–E). However, consistent with the idea that EnvC activates AmiA and AmiB, the combination of FLEnvC and AmiA or FLEnvC and AmiB using these same conditions resulted in a level of dye release comparable to that of the lysozyme control (Figure 6B–D). FLEnvC did not lead to enhanced PG hydrolysis when it was combined with AmiC as expected from the genetic results (Figures 5 and 6E). Importantly, both end point (Figure 6C and D) and time course assays (Supplementary Figure S6) indicated that LytEnvC and FLEnvC stimulated PG hydrolysis to a similar extent when they were combined with AmiA or AmiB. Thus, the LytM domain is sufficient for enhancing PG hydrolase activity in EnvC–amidase mixtures. Figure 6.Enhanced in vitro PG hydrolysis by EnvC–AmiA and EnvC–AmiB combinations. (A) Dye-release assays measuring PG hydrolysis by the amidases in the absence of EnvC. The absorbance of supernatants from the reactions containing the indicated proteins (4 μM) were measured at 595 nm. The reactions were incubated at 37°C for 0.5, 4 or 20 h. Results shown are the average of duplicate reactions. (B) Tubes containing supernatants of dye-release reactions following the incubation of RBB-PG with the indicated proteins (4 μM each) for 30 min at 37°C. (C–E) Same as in (A) except FLEnvC or LytEnvC was combined with the amidases and reactions were all incubated for 30 min at 37°C. Numbers indicate protein concentration in μM. Amidase concentrations were held constant, whereas the amount of FLEnvC or LytEnvC varied. From left to right, a set of amidase reactions starts with no added LytM factor (0) followed by a series of LytM factor additions where the LytM factor concentration was increased by 2 × with each step through the range. Results presented are the average of three independent reactions with the error bars displaying the standard deviation. LZ, lysozyme. Download figure Download PowerPoint In the in vitro reactions, EnvC–AmiA combinations promoted a greater degree of PG hydrolysis than EnvC–AmiB reactions (Figure 6C and D). AmiB, on the other hand, seemed to have a more dominant function in causing cell lysis when GFP-LytEnvC was produced in vivo. This apparent dichotomy may be explained by the fact that AmiB accumulates at the division site in vivo (T Dinh, unpublished results), whereas AmiA does not (Bernhardt and de Boer, 2003). We suspect that uncontrolled activation of locally concentrated AmiB is more catastrophic than that of delocalized AmiA. To determine whether other LytM factors are also capable of stimulating PG hydrolysis, we purified a soluble version of NlpD (Figure 4A) and tested its effect on the in vitro activity of the amidases. Interestingly, the results perfectly complemented those with EnvC. The addition of NlpD to AmiC-containing reactions dramatically enhanced PG degradation (Figure 7). In contrast, NlpD had no effect on the activity of reactions containing AmiA or AmiB (Figure 7). Like EnvC, NlpD did not display any PG cleavage activity on its own even though it possesses two of the four LytM catalytic residues (Supplementary Figures S4 and S7). Consistent with the idea that NlpD is needed for AmiC activity in vivo, NlpD inactivation phenocopied the cell separation defect of an amiC deletion. Redundancy of the amidases required that the AmiC− phenotype be visualized in a strain lacking AmiA and AmiB, and loss of AmiC or NlpD dramatically enhanced the cell separation phenotype of this strain (Figure 8). Figure 7.Enhanced in vitro PG hydrolysis in reactions containing NlpD and AmiC. Reactions are the same as in Figure 6 except that NlpD replaces EnvC. Download figure Download PowerPoint Figure 8.Loss of NlpD phenocopies ΔamiC. Cells of (A) TB28 [WT], (B) TU207 [ΔamiA ΔamiB], (C) TU218 [ΔamiA ΔamiB ΔnlpD] and (D) TB170 [ΔamiA ΔamiB ΔamiC] were grown in LB at 37°C to an OD600 of 0.5. They were then stained with the fixable membrane dye FM1-43-FX, fixed and visualized by fluorescence microscopy as described earlier (Uehara et al, 2009). Bar=8 μm. Download figure Download PowerPoint To investigate the function of the amidases in the enhanced PG hydrolysis observed for LytM–amidase reaction mixtures, we analysed the activity of purified AmiB proteins with amino-acid substitutions in predicted active site residues. To guide the design of the mutants, we aligned the AmiB sequence with that of CwlV from Paenibacillus polymyxa and PlyPSA from Listeria monocytogenes phage PSA (Supplementary Figure S7) for which residues important for catalysis have been identified through biochemical and structural analyses (Shida et al, 2001; Korndörfer et al, 2006) (PDB 1jwq). Accordingly, we generated four AmiB mutants (H200A, E215A, H269A and E382Q). All four failed to correct the cell separation and LytEnvC-mediated lysis phenotypes of a ΔamiA ΔamiB double mutant in vivo (Figure 9A–C). Upon purification, all four mutant enzymes also failed to promote PG hydrolysis when they were mixed with EnvC (Figure 9D). Based on this, we conclude that amidase enzymatic activity is indeed necessary for the amidases to promote cell separation during division and for LytEnvC to induce lysis. Furthermore, these results provide strong support for a mechanism in which the amidases provide all the PG hydrolyzing activity in the LytM–amidase reactions. Figure 9.AmiB active site residues are required for PG hydrolysis. (A) Cultures of TU207 [ΔamiA ΔamiB] containing chromosomally integrated Para∷gfp-lytenvC and indicated Plac∷ssdsbA-amiB(23–445)-gfp expression constructs were grown overnight at 37°C in LB. Culture densities were normalized and 10-fold serial dilutions were prepared for each. A measure of 10 μl of each dilution was spotted onto the indicated solid media and the plates were incubated overnight at 37°C. (B) Cultures of TU207 [ΔamiA ΔamiB] containing the indicated Plac∷ssdsbA-amiB(23–445)-gfp expression constructs were grown at 37°C in LB to an OD600 between 0.6–0.65. Cells were stained with FM4-64FX, fixed and visualized using fluorescence microscopy. A comparison of AmiB (wt) and AmiB (H269A) is shown, but all other AmiB catalytic mutants tested displayed the same defect in cell separation activity (data not shown). (C) Immunoblots of extracts from the cultures in (B) using anti-GFP antibodies to detect the AmiB-GFP fusions. FtsZ immunoblots are shown as a control for protein loading. (D) Dye-release assays performed as in Figure 6. EnvC and AmiB proteins were present at 2 μM each in the reaction mixtures. Results are the average of duplicate reactions except for AmiB (E215A), which was assayed once. Download figure Download PowerPoint The biochemical activities of the purified proteins were defined more precisely by incubating them with unlabelled PG and analysing the cleavage products with a combination of HPLC and mass spectrometry. Again, using low levels of the amidases, significant PG cleavage was only observed in reactions with FLEnvC–AmiA, FLEnvC–AmiB and NlpD–AmiC combinations. Importantly, in each of these reactions, the only biochemical activity detected was peptide release by an N-acetylmuramyl-L-alanine amidase (Figure 10). As the odds are small that both EnvC and NlpD themselves possess this exact same activity in latent form, these results provide strong support for the following: (1) AmiA, AmiB and AmiC are indeed N-acetylmuramyl-L-alanine amidases, (2) EnvC and NlpD promote septal PG splitting by activating the amidases at the division site and (3) only specific LytM/amidase combinations yield efficient PG amidase activity. Figure 10.Analysis of PG hydrolase activity using HPLC. PG sacculi (unlabelled) were treated with buffer (A), AmiB only (4 μM) (B), FLEnvC only (4 μM) (C) or a mixture of FLEnvC and AmiB (4 μM each) (D) before mutanolysin digestion and HPLC separation of the products. Mutanolysin is an N-acetylmuraminidase that hydrolyzes the M-β-1,4-G bonds in PG. Without prior cleavage by another enzyme, mutanolysin hydrolyzes PG primarily into monomeric and dimeric disaccharide (G-M) units with attached tetrapeptides (Glauner et al, 1988). Enzymatic treatment before mutanolysin addition will alter the product composition. The numbered peaks in the chromatograms were identified using matrix-assisted laser desorption ionization (MALDI) and

ATP-binding cassette transporters are ubiquitous membrane protein complexes that move substrates across membranes. They do so using ATP-induced conformational changes in their nucleotide-binding domains to alter the conformation of the transport cavity formed by their transmembrane domains. In Escherichia coli, an ATP-binding cassette transporter-like complex composed of FtsE (nucleotide-binding domain) and FtsX (transmembrane domain) has long been known to be important for cytokinesis, but its role in the process has remained mysterious. Here we identify FtsEX as a regulator of cell-wall hydrolysis at the division site. Cell-wall material synthesized by the division machinery is shared initially by daughter cells and must be split by hydrolytic enzymes called "amidases" to drive daughter-cell separation. We recently showed that the amidases require activation at the cytokinetic ring by proteins with LytM domains, of which EnvC is the most critical. In this report, we demonstrate that FtsEX directly recruits EnvC to the septum via an interaction between EnvC and a periplasmic loop of FtsX. Importantly, we also show that FtsEX variants predicted to be ATPase defective still recruit EnvC to the septum but fail to promote cell separation. Our results thus suggest that amidase activation via EnvC in the periplasm is regulated by conformational changes in the FtsEX complex mediated by ATP hydrolysis in the cytoplasm. Since FtsE has been reported to interact with the tubulin-like FtsZ protein, our model provides a potential mechanism for coupling amidase activity with the contraction of the FtsZ cytoskeletal ring.

ABSTRACT Staphylococcus aureus is a human commensal and pathogen that is capable of forming biofilms on a variety of host tissues and implanted medical devices. Biofilm-associated infections resist antimicrobial chemotherapy and attack from the host immune system, making these infections particularly difficult to treat. In order to gain insight into environmental conditions that influence S. aureus biofilm development, we screened a library of small molecules for the ability to inhibit S. aureus biofilm formation. This led to the finding that the polyphenolic compound tannic acid inhibits S. aureus biofilm formation in multiple biofilm models without inhibiting bacterial growth. We present evidence that tannic acid inhibits S. aureus biofilm formation via a mechanism dependent upon the putative transglycosylase IsaA. Tannic acid did not inhibit biofilm formation of an isaA mutant. Overexpression of wild-type IsaA inhibited biofilm formation, whereas overexpression of a catalytically dead IsaA had no effect. Tannin-containing drinks like tea have been found to reduce methicillin-resistant S. aureus nasal colonization. We found that black tea inhibited S. aureus biofilm development and that an isaA mutant resisted this inhibition. Antibiofilm activity was eliminated from tea when milk was added to precipitate the tannic acid. Finally, we developed a rodent model for S. aureus throat colonization and found that tea consumption reduced S. aureus throat colonization via an isaA -dependent mechanism. These findings provide insight into a molecular mechanism by which commonly consumed polyphenolic compounds, such as tannins, influence S. aureus surface colonization.

Macroautophagy (hereafter autophagy) is a cellular recycling pathway essential for cell survival during nutrient deprivation that culminates in the degradation of cargo within the vacuole in yeast and the lysosome in mammals, followed by efflux of the resultant macromolecules back into the cytosol. The yeast vacuole is home to many different hydrolytic proteins and while few have established roles in autophagy, the involvement of others remains unclear. The vacuolar serine carboxypeptidase Y (Prc1) has not been previously shown to have a role in vacuolar zymogen activation and has not been directly implicated in the terminal degradation steps of autophagy. Through a combination of molecular genetic, cell biological, and biochemical approaches, we have shown that Prc1 has a functional homologue, Ybr139w, and that cells deficient in both Prc1 and Ybr139w have defects in autophagy-dependent protein synthesis, vacuolar zymogen activation, and autophagic body breakdown. Thus, we have demonstrated that Ybr139w and Prc1 have important roles in proteolytic processing in the vacuole and the terminal steps of autophagy.

Hydrolysis within the vacuole in yeast and the lysosome in mammals is required for the degradation and recycling of a multitude of substrates, many of which are delivered to the vacuole/lysosome by autophagy. In humans, defects in lysosomal hydrolysis and efflux can have devastating consequences, and contribute to a class of diseases referred to as lysosomal storage disorders. Despite the importance of these processes, many of the proteins and regulatory mechanisms involved in hydrolysis and efflux are poorly understood. In this review, we describe our current knowledge of the vacuolar/lysosomal degradation and efflux of a vast array of substrates, focusing primarily on what is known in the yeast Saccharomyces cerevisiae. We also highlight many unanswered questions, the answers to which may lead to new advances in the treatment of lysosomal storage disorders.Abbreviations: Ams1: α-mannosidase; Ape1: aminopeptidase I; Ape3: aminopeptidase Y; Ape4: aspartyl aminopeptidase; Atg: autophagy related; Cps1: carboxypeptidase S; CTNS: cystinosin, lysosomal cystine transporter; CTSA: cathepsin A; CTSD: cathepsin D; Cvt: cytoplasm-to-vacuole targeting; Dap2: dipeptidyl aminopeptidase B; GS-bimane: glutathione-S-bimane; GSH: glutathione; LDs: lipid droplets; MVB: multivesicular body; PAS: phagophore assembly site; Pep4: proteinase A; PolyP: polyphosphate; Prb1: proteinase B; Prc1: carboxypeptidase Y; V-ATPase: vacuolar-type proton-translocating ATPase; VTC: vacuolar transporter chaperone