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• W2983115969 abstract "Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Despite the crucial role of bacterial capsules in pathogenesis, it is still unknown if systemic cues such as the cell cycle can control capsule biogenesis. In this study, we show that the capsule of the synchronizable model bacterium Caulobacter crescentus is cell cycle regulated and we unearth a bacterial transglutaminase homolog, HvyA, as restriction factor that prevents capsulation in G1-phase cells. This capsule protects cells from infection by a generalized transducing Caulobacter phage (φCr30), and the loss of HvyA confers insensitivity towards φCr30. Control of capsulation during the cell cycle could serve as a simple means to prevent steric hindrance of flagellar motility or to ensure that phage-mediated genetic exchange happens before the onset of DNA replication. Moreover, the multi-layered regulatory circuitry directing HvyA expression to G1-phase is conserved during evolution, and HvyA orthologues from related Sinorhizobia can prevent capsulation in Caulobacter, indicating that alpha-proteobacteria have retained HvyA activity. https://doi.org/10.7554/eLife.03587.001 eLife digest Many bacteria have a tough outer coating known as capsule that protects them from untoward environmental conditions. This capsule also prevents viruses called bacteriophages from invading the bacterial cells, and it shields those bacteria that can infect humans from attack by our immune system. External conditions—such as a lack of nutrients and physical stresses—are known to trigger capsule formation. However, almost nothing is known about the signals from within the bacteria that control the formation of a capsule. Now, Ardissone et al. have used the capsulated bacterium called Caulobacter crescentus to show that capsule formation is regulated by the bacterial cell cycle. This cycle is a series of events and checkpoints that happen every time a cell divides to form two new cells. Ardissone et al. revealed that capsule cannot form during the first phase of the cell cycle. The bacterium only forms its capsule as this phase ends and before it copies its DNA and later divides in two. Ardissone et al. discovered that an enzyme called HvyA, which is only produced during the first phase of the cell cycle, prevents the capsule from forming. Inactivating the HvyA enzyme was also shown to make the bacteria impervious to infection by a bacteriophage. Furthermore, Ardissone et al. dissected the complicated steps involved in regulating the production of the HvyA enzyme and showed that such regulatory steps are also used by other species of bacteria. Without their capsules, bacteria can take up new genetic material from a number of sources that might help them adapt to a changing environment. Ardissone et al.'s findings suggest that by only exchanging genetic material during the first phase of the cell cycle, bacteria ensure that any useful DNA is taken up and copied along with their own DNA later in the cell cycle. Antibiotic resistance spreads between bacteria via the exchange of genetic material, making it increasingly difficult to treat bacterial infections. Interfering with the formation of the capsule during an infection could help overcome this problem by making the bacteria more vulnerable to attack either by our own immune system or by bacteriophages that can be used to treat bacterial infections. By investigating how genetic exchange and capsule formation are linked and regulated, the findings of Ardissone et al. might now open up new strategies to help combat bacterial infections. https://doi.org/10.7554/eLife.03587.002 Introduction Genetic exchange is both fundamental to the adaptation of bacterial cells faced with ever-changing environmental conditions and the cause of the alarming dissemination of antibiotic resistance determinants among the bacterial pathogens. The underlying mechanisms include direct uptake of naked DNA (transformation) by bacterial cells as well as cell- or bacteriophage-based delivery systems (respectively conjugation and generalized transduction) (Wiedenbeck and Cohan, 2011; Seitz and Blokesch, 2013). Thus, uncovering mechanisms that curb genetic exchange could provide new entry points to help intervene with the spread of antibiotic resistances. While genetic exchange can be facilitated in response to changes in the number of cells in a population (quorum sensing) or other developmental states (Seitz and Blokesch, 2013), an important but yet unresolved question is whether genetic exchange can also be regulated by systemic cues, such as those directing cell cycle progression. Recent cytological experiments provide evidence that components of the pneumococcal natural transformation (competence) machinery can be linked to cell division, at least spatially (Bergé et al., 2013), hinting that unknown mechanisms may indeed restrict genetic exchange in time or in space during the progression of the cell division cycle. A myriad of events are coordinated with progression through the eukaryotic cell cycle, but our understanding of such mechanisms and the factors that constrain them during the bacterial cell cycle are sparse. Microbial polysaccharidic capsules can also restrict bacteriophage-mediated genetic exchange. Typically, they mask bacteriophage receptor sites that are on or near the cell surface (Hyman and Abedon, 2010). Moreover, capsules are virulence factors in many Gram-negative and Gram-positive species, as they provide immune evasion by shielding or camouflaging the targets of host immune cells that are located on the surface of bacterial cells (Schneider et al., 2007; Kadioglu et al., 2008). While capsulation can be regulated by nutritional cues (Kadioglu et al., 2008; Yother, 2011), cell envelope stresses (Laubacher and Ades, 2008) or physical cues (Sledjeski and Gottesman, 1996; Tschowri et al., 2009; Loh et al., 2013), no systemic cues are currently known. As virulence regulators have recently been found to control bacterial cell cycle transcription (Fumeaux et al., 2014), capsulation might also be regulated by the cell cycle. The synchronizable and capsulated alpha-proteobacterium Caulobacter crescentus (henceforth Caulobacter) is the pre-eminent model system for cell cycle studies (Ravenscroft et al., 1991; Skerker and Laub, 2004; Curtis and Brun, 2010). It recently transpired that many of the emerging concepts of cell cycle control, and the underlying mechanisms such as those directing an asymmetric cell division (Hallez et al., 2004), are also operational in other alpha-proteobacterial lineages (Kobayashi et al., 2009; Brilli et al., 2010; Ardissone and Viollier, 2012; Pini et al., 2013; Fumeaux et al., 2014). In Caulobacter this asymmetric cell division yields a motile and piliated swarmer (SW) cell that is in a G1-arrested state and a sessile stalked (ST) cell that resides in S-phase (Figure 1A). The cellular buoyancy of the latter is higher than the former, a feature that has been exploited for synchronization of Caulobacter populations (by density gradient centrifugation) (Ely, 1991) for cell cycle studies. A vast number of transcripts are cell cycle-regulated in Caulobacter (Laub et al., 2000) and in the alpha-proteobacterium Sinorhizobium meliloti (De Nisco et al., 2014), a plant symbiont. Importantly, many transcripts of orthologous genes are cell cycle-regulated and this is in large part governed by the conserved and essential cell cycle transcriptional regulator A (CtrA). CtrA activates transcription of many late S- and G1-phase genes that are repressed by the transcriptional regulators SciP or the MucR1/2 paralogs, respectively (Fumeaux et al., 2014; Gora et al., 2010; Tan et al., 2010). In addition to acting as a transcriptional activator, CtrA functions as a negative regulator of gene expression and DNA replication initiation by binding to the conserved 5′-TTAA-(N)7-TTAA-3′ motif (CtrA box) located in many Caulobacter and Sinorhizobium promoters (Laub et al., 2000, 2002; Fumeaux et al., 2014) and the Caulobacter origin of replication (Cori) (Quon et al., 1998). Figure 1 Download asset Open asset Capsulation of Caulobacter cells is cell cycle regulated. (A) Schematic of the Caulobacter cell cycle and the regulatory interactions that determine the presence/absence of the capsule (in blue). Phosphorylated CtrA (CtrA∼P) and MucR1/2 control expression of hvyA. The antagonistic kinase/phosphatase pair, DivJ (yellow dot) and PleC (green dot), indirectly influences CtrA∼P and partitions with the stalked (ST) cell chamber or swarmer (SW) cell chamber, respectively. PleC promotes CtrA∼P accumulation in the SW cell. HvyA prevents encapsulation in SW cells. Pink denotes HvyA accumulation in the SW (G1) cell compartment. Light blue indicates the presence of the capsule in ST(S) and pre-divisional (PD) cells. (B) Schematic of cell buoyancy upon centrifugation on density gradient for WT Caulobacter cells (left). SW cells sediment in the lower band (‘heavy’, in pink) whereas ST and PD cells sediment in the upper band (‘light’, in blue). ΔpleC and ΔhvyA cells are ‘light’ due to the constitutive presence of capsule (middle). Upon transposon mutagenesis with himar1 Tn, we isolated ‘heavy’ non-capsulated mutants by cell density centrifugation (right). (C) Caulobacter loci identified by the cell density screen. The upper panel represents the CCNA_00162-CCNA_00168 locus and the lower panel the CCNA_03921-CCNA_00472 locus on the mobile genetic element of Caulobacter NA1000. The fragment deleted for each in-frame deletion is indicated (Δ). Black triangles indicate Tn insertions obtained in the ΔpleC background and white triangles indicate Tn insertions obtained in the ΔhvyA background. CCNA_00166 (hvyA) is shown in pink, and the genes hit by our buoyancy screen for ‘heavy’ mutants are in blue. The asterisks show the ORFs identified as essential by Christen et al. (2011). (D) Schematic of capsule polymerisation/export system based on the one for group 1 CPS in E. coli (Collins and Derrick, 2007). Putative functions were attributed to Caulobacter proteins based on the homology and conserved domains. CCNA_03998, CCNA_00466, and CCNA_00469 are putative glycosyltransferases; PssY, PssZ, and HfsE are polyisoprenylphosphate hexose-1-phosphotransferases; CCNA_00467 is a putative flippase; CCNA_00165 and CCNA_00470 are polymerases; CCNA_00163 and CCNA_00167 have homology to the tyrosine autokinase Wzc and its phosphatase Wzb, respectively, that regulate polymerisation and export; CCNA_00168 is the putative outer membrane lipoprotein required for translocation of the polysaccharide across the outer membrane. https://doi.org/10.7554/eLife.03587.003 Binding of CtrA to its target sites is stimulated 100-fold by phosphorylation of aspartate at position 51 (D51, CtrA∼P) (Siam and Marczynski, 2000) through a complex phosphorelay that controls both abundance and phosphorylation of CtrA as a function of the cell cycle (Biondi et al., 2006; Iniesta et al., 2006). CtrA∼P is present in G1-phase, proteolyzed during the G1→S transition to permit replication initiation and re-accumulates later in S-phase (Domian et al., 1997). G1-phase transcription is also positively dependent on PleC (Wang et al., 1993), a phosphatase that is sequestered to the SW cell pole at cell division (Wheeler and Shapiro, 1999). While PleC also regulates the cellular buoyancy properties, the molecular basis has never been determined. In this study, we use suppressor genetics to unearth the Caulobacter capsule as determinant of the buoyancy trait and we identify HvyA, a member of the poorly characterized bacterial transglutaminase-like cysteine protease (BTLCP) family, as a PleC-dependent negative regulator that is restricted to G1-phase to prevent capsulation at this time in the cell cycle. As the capsule protects Caulobacter cells from infection by the generalized transducing Caulophage φCr30 and no CRISPR/Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated)-based adaptive immunity system to protect cells from invading genetic material is encoded in the Caulobacter genome (Marks et al., 2010), HvyA is the first example of a factor restricting phage infection to a confined cell cycle phase. Results A putative capsule export machinery governs the buoyancy switch The switch in cellular buoyancy in NA1000 (WT) Caulobacter cells is cell cycle-regulated, but the underlying regulatory mechanism is elusive. We used a developmental mutant (ΔpleC) as an entry point to identify the genetic determinants conferring the change in buoyancy. Density gradient centrifugation of a WT culture yields ST and PD cells with the characteristic high buoyancy (for simplicity, we refer to these cells as ‘light’) and SW cells with the characteristic low buoyancy (‘heavy’ cells, Figure 1B). As ΔpleC cells are exclusively ‘light’, we simply sought himar1 transposon (Tn) insertions that render ΔpleC cells ‘heavy’ (Figure 1B). After backcrossing such ‘heavy’ ΔpleC::Tn mutants, we mapped the Tn insertion sites to two loci (Figure 1C). The first locus (Figure 1C, upper panel) encodes putative components of a group 1 (Wzy)-like capsular polysaccharide (CPS) export machinery, in which the saccharide precursors are first assembled on undecaprenol (Und∼P, black zigzag in Figure 1D) on the cytoplasmic membrane, flipped and assembled in the periplasm into a polymer that is then translocated across the outer membrane and anchored on the cell surface (Whitfield, 2006). Tn insertions were found in the genes encoding a putative capsular polysaccharide biosynthesis lipoprotein (CCNA_00162), a Wzc-like chain length regulator/tyrosine kinase (CCNA_00163), a putative O-antigen polymerase/ligase (CCNA_00164), a putative Wzb-like metallophosphatase (CCNA_00167), and a Wza-like outer membrane translocon (CCNA_00168), all commonly associated with capsular export systems. No Tn insertions were found in the other two genes within this cluster, CCNA_00166 and pssY. For the latter, this could be explained by a functional redundancy of pssY with the orthologs encoded by pssZ and hfsE, all encoding polyisoprenylphosphate hexose-1-phosphotransferases (Toh et al., 2008). By contrast, CCNA_00166 is predicted to encode a putative bacterial transglutaminase-like cysteine protease (BTLCP) and we describe below that an in-frame deletion of CCNA_00166 (ΔhvyA in Figure 1C) in WT cells resulted in ‘light’ cells, akin to ΔpleC cells. Consistent with the results from the Tn analysis, an in-frame deletion (Figure 1C) in pssY did not render cells ‘heavy’. By contrast, deleting CCNA_00162, CCNA_00163, CCNA_00164, or ΔCCNA_00167 gave rise to ‘heavy’ WT (and ΔpleC) cells (Figure 1C and Figure 2A, Supplementary file 1). Moreover, complementation of ΔCCNA_00162, ΔCCNA_00163, ΔCCNA_00164, ΔCCNA_00167, or CCNA_00168::Tn mutant cells with a plasmid harbouring the corresponding gene under the control of the vanillate inducible promoter (Pvan) on a medium–copy number plasmid (pMT335 (Thanbichler et al., 2007)) restored the WT buoyancy phenotype, showing that the ‘heavy’ phenotype is attributable to the loss of CCNA_00162, CCNA_00163, CCNA_00164, CCNA_00167, or CCNA_00168 function (Supplementary file 1). Figure 2 with 5 supplements see all Download asset Open asset Capsulation affects buoyancy, mucoidy, and bacteriophage sensitivity. (A) Sensitivity to bacteriophage φCr30 and buoyancy of Caulobacter WT (NA1000) and different mutant strains. Mutation in CCNA_00163 or CCNA_00470 restores sensitivity to φCr30 in ΔpleC and ΔhvyA mutant backgrounds. ΔpleC, ΔhvyA, and the double mutant ΔpleC ΔhvyA are ‘light’, whereas mutation in CCNA_00163 or CCNA_00470 renders cells ‘heavy’ (also in a ΔpleC or ΔhvyA background). (B) Mucoidy of Caulobacter WT (NA1000) and different mutant strains plated on PYE medium supplemented with 3% sucrose. WT, ΔpleC, and ΔhvyA are highly mucoid, whereas mutations in CCNA_00163 or CCNA_00470 confer a ‘rough’ non-mucoid phenotype in all three backgrounds (WT, ΔpleC, and ΔhvyA). (C) Mucoidy of Caulobacter WT (NA1000), ΔpleC, or ΔhvyA cells over-expressing hvyA under control of Pvan on a medium copy number plasmid (pMT335) or Pxyl on a low copy number plasmid (pMT375). Over-expression of hvyA confers the typical non-mucoid ‘rough’ colony phenotype on PYE agar plates supplemented with 3% sucrose, while the WT, ΔpleC, and ΔhvyA cells have a mucoid ‘smooth’ colony appearance. Sensitivity to bacteriophage φCr30 and buoyancy of Caulobacter WT (NA1000), ΔhvyA, ΔpleC, and ΔmucR1/2 strains carrying pMT335-hvyA or pMT375-hvyA are shown in Figure 2—figure supplement 1–2. RsaA extracted from the same Caulobacter strains shown in Figure 2 is displayed in Figure 2—figure supplement 3. The effect of proteinase K treatment on CCNA_00168 in Caulobacter WT, ΔhvyA, and ΔCCNA_00163 is shown in Figure 2—figure supplement 4. The effect of the ΔhvyA mutation on swarming motility of Caulobacter is shown in Figure 2—figure supplement 5. https://doi.org/10.7554/eLife.03587.004 The second cluster of genes resides on a 26-kbp mobile genetic element (MGE) that has previously been implicated in buoyancy (Marks et al., 2010) (Figure 1C, lower panel). Specifically, we recovered Tn insertions in genes predicted to encode a homolog of the putative N-acetyl-L-fucosamine transferase WbuB from E. coli that is involved in O-antigen (O26) synthesis (D'Souza et al., 2002) (CCNA_03998), a polysaccharide polymerase (CCNA_00470) and a GDP-L-fucose synthase (CCNA_00471). The three genes are near other coding sequences for polysaccharides biosynthesis proteins, including two other putative glycosyltransferases (CCNA_00466 and CCNA_00469), a Wzx-like polysaccharide flippase/translocase (CCNA_00467) and a sugar mutase homolog (CCNA_00465) (Marks et al., 2010). Consistent with a previous genome-wide Tn analysis showing that these genes cannot be disrupted (Christen et al., 2011), we were unable to engineer in-frame deletions in CCNA_00466 or CCNA_00467 in the absence of a complementing plasmid. As the entire 26-kb MGE is dispensable for viability, it appears that inactivation of either one of these four genes results in synthetic toxicity due to a polysaccharide intermediate or the sequestration of Und∼P that is also required for peptidoglycan synthesis (Yother, 2011). To confirm that CCNA_03998, CCNA_00470, and CCNA_00471 are indeed buoyancy determinants, we engineered in-frame deletions in CCNA_03998 or CCNA_00470 (CCNA_00471 was not tested) in WT or ΔpleC mutant cells and found the resulting single or double mutants to be ‘heavy’ (Figures 1C and 2A, Supplementary file 1). The association of the ‘heavy’ phenotype with a capsule synthesis and/or export defect, the resemblance of CCNA_03998 to the predicted N-acetyl-L-fucosamine transferase WbuB (D'Souza et al., 2002) and the fact that D-fucose is a known component of the extracellular polysaccharide or capsule of C. crescentus (Ravenscroft et al., 1991) prompted us to test if these mutations also affected colony mucoidy, a phenotype typically associated with the presence of capsule or exopolysaccharides. Indeed, all ‘heavy’ mutants exhibited a non-mucoid (‘rough’) colony phenotype on PYE agar plates supplemented with 3% sucrose, while the WT or ‘light’ mutants (ΔpleC) had a mucoid (‘smooth’) colony appearance (Figure 2A,B, Supplementary file 1). In support of this result, we purified capsule from WT Caulobacter, ΔCCNA_00163 (‘heavy’ and ‘rough’), ΔCCNA_00166 (ΔhvyA, ‘light’ and ‘smooth’), and ΔhvyA ΔCCNA_00163 (‘heavy’ and ‘rough’, see below) cells. As the Caulobacter capsule is primarily composed of neutral monosaccharides including fucose, mannose, galactose, and glucose (Ravenscroft et al., 1991), we used glycosyl compositional analysis as proxy to quantify capsular material from ‘heavy’ and ‘light’ strains (See ‘Materials and methods’). As shown in Table 1, the expected sugars (determined as % of total carbohydrate weight in the preparations) were abundant in preparations from the WT and the ‘light’ mutant (ΔhvyA, Table 1), whereas those from the ‘heavy’ mutants (i.e. the ΔCCNA_00163 single mutant and the ΔhvyA ΔCCNA_00163 double mutant) contained far less fucose, galactose, and mannose (up to 37-fold, 23-fold, and 5.5-fold reductions, respectively, in the ΔhvyA ΔCCNA_00163 double mutant compared to the ΔhvyA single mutant). We also observed a significant reduction in galacturonic acid in the preparations from the ‘heavy’ mutants vs WT or ‘light’ cells, raising the possibility that this saccharide is also a constituent of the NA1000 capsule (Table 1). Table 1 Glycosyl composition of per-O-trimethylsilyl (TMS) derivatives of methyl glycosides performed on purified capsular polysaccharides from WT Caulobacter (NA1000), the single mutants ΔCCNA_00166 (ΔhvyA) and ΔCCNA_00163 and the ΔhvyA ΔCCNA_00163 double mutant https://doi.org/10.7554/eLife.03587.010 NA1000ΔhvyAΔCCNA_00163ΔhvyA ΔCCNA_00163Glycosyl residueMass (μg)Weight (%)Mass (μg)Weight (%)Mass (μg)Weight (%)Mass (μg)Weight (%)Ribose0.80.40.10.10.30.30.50.8Rhamnose2.31.21.10.66.46.22.74.3Fucose19.810.326.514.70.00.00.20.4Xylose0.00.00.10.10.00.00.00.0Glucuronic Acid0.00.00.00.00.80.80.30.5Galacturonic acid28.414.932.618.15.65.42.33.7Mannose23.212.126.614.83.93.81.72.7Galactose30.115.737.020.61.41.40.50.9Glucose64.333.652.929.449.948.523.738.3N-Acetyl galactosamine2.01.10.00.02.32.21.01.7N-Acetyl glucosamine16.08.43.01.629.528.727.945.2N-Acetyl mannosamine4.12.20.00.02.22.10.71.2Σ=191.3179.9102.861.8 Mass is expressed in μg and weight % is relative to the total carbohydrate. Taken together, our results show that the loss of capsule synthesis and/or export renders cells ‘heavy’ and ‘rough’ and that the loss of capsulation is epistatic to the loss of PleC in buoyancy control. On these grounds we predicted that PleC, directly or indirectly, regulates one of the newly identified buoyancy determinants. Negative control of encapsulation by the transglutaminase homolog HvyA Since the buoyancy switch is cell cycle-regulated and since all ΔpleC cells are ‘light’ (Figure 1B), we reasoned that PleC is required to turn off capsule synthesis in G1-phase SW cells. As PleC is also required to activate motility and PilA (the structural subunit of the pilus filament) expression, when ΔpleC divides two daughter cells that are capsulated, non-piliated and non-motile are formed. By contrast division of WT yields one piliated, motile, and non-capsulated (‘heavy’) G1-phase SW progeny and one capsulated (‘light’) S-phase ST cell (Figure 1B). If PleC indeed restricts capsulation temporally, it might control expression of a negative regulator of capsulation. Interestingly, PleC is required for the accumulation of the transcript of CCNA_00166 (referred to as hvyA due to its requirement to render cells ‘heavy’) (Chen et al., 2006). The hvyA transcript peaks during the G1-phase and encodes a 272-residue protein (Figure 3A) harbouring a classical N-terminal Sec-dependent signal sequence (SS), but lacking discernible hydrophobic sequences or a lipidation signal for retention in the membrane, suggesting that it is periplasmic. The C-terminal part of HvyA features a BTLCP domain. This domain is thought to introduce intra- or inter-molecular crosslinks by transamidation, forming γ-glutamyl-ε-lysine isopeptide bonds between Gln and Lys residues, to hydrolyse amide bonds by the reverse protease reaction and/or to execute deamidation/esterification reactions of glutamine residues (Lorand and Graham, 2003; Ginalski et al., 2004). Cysteine proteases typically feature a Cys-His-Asp catalytic triad (C192, H226, and D241 for HvyA, based on sequence alignment, Figure 3—figure supplement 1) for the formation of a thioester bond intermediate by the reaction of the active site thiol (from the Cys) with Gln, followed by the transfer of the acyl group to an amine substrate (from the Lys) (Lorand and Graham, 2003). As an in-frame deletion in hvyA (ΔhvyA) phenocopied the buoyancy defect of ΔpleC cells (yielding exclusively ‘light’ mucoid cells, Figures 1b, 2a, 2b), we conclude that HvyA is required for capsule-mediated buoyancy control in Caulobacter. While expression of WT HvyA from Pvan (pMT335-hvyA) reversed the buoyancy defect of ΔhvyA and ΔpleC cells, analogous plasmids encoding the predicted catalytic mutants (C192S/A, H226Q/A, or D241A) were unable to do so, although all the HvyA variants accumulated to comparable steady-state levels as the WT protein on immunoblots (Figure 3C, Figure 3—figure supplement 2). Thus expression of catalytically active HvyA is necessary and sufficient to mitigate the buoyancy defect of ΔpleC cells. As a genetic selection for ‘heavy’ ΔhvyA::Tn mutants was answered by Tn insertions in the same genes that render ΔpleC cells ‘heavy’ (Figure 1C), we reasoned that mutations in capsule synthesis and export genes are epistatic to both the ΔhvyA and ΔpleC mutations. To confirm this notion, we engineered ΔhvyA ΔCCNA_00163, ΔhvyA ΔCCNA_00167, and ΔhvyA ΔCCNA_00470 double mutants as well as a ΔpleC ΔhvyA ΔCCNA_00167 triple mutant and found all resulting mutants to be ‘heavy’ and non-mucoid (‘rough’) on PYE sucrose plates (Figure 2A, B and Supplementary File 1). Importantly, constitutive expression of HvyA (from a Pvan- or a Pxyl-plasmid) in WT, ΔpleC, or ΔhvyA cells also renders cells ‘heavy’ and ‘rough’ (Figure 2C, Figure 2—figure supplement 1). On the basis of these findings we hypothesized that HvyA is a G1-specific negative regulator of capsulation whose expression is dependent on PleC (Figure 1A). Figure 3 with 3 supplements see all Download asset Open asset HvyA is a bacterial transglutaminase-like cysteine protease (BTLCP) homologue and its catalytic activity is required for function. (A) Schematic of HvyA domains: signal sequence (SS) and BTLCP domain (in red) are indicated. C192, H226, and D241 constitute the putative catalytic triad (C192S/A, H226Q/A, and D241A alleles are non-functional; the D241N allele is functional, consistently with some BTLCP family members having a C/H/N catalytic triad). Residues identified in the buoyancy screen for non-functional variants are indicated below (blue, non-functional; green, partially functional). (B) Schematic of the buoyancy screen for HvyA non-functional variants. The Pvan-hvyA::TAP fusion (on plasmid) was subjected to random mutagenesis, then introduced into Caulobacter cells that were subjected to multiple rounds of enrichment for ‘light’ phenotype by centrifugation on density gradient. (C) Immunoblot anti-HvyA-TAP on periplasmic proteins extracted by EGTA treatment. The HvyA alleles mutated in putative catalytic residues are expressed and exported to the periplasm like the WT protein. Immunoblot against Caulobacter β-lactamase (CCNA_02223, Bla on the lower panel) is a control for periplasmic proteins. Molecular size standards are indicated in blue on the left, with the corresponding values in kDa. (D) ΔhvyA strains harbouring hvyA catalytic mutants under control of Pvan on plasmid were tested for sensitivity to φCr30. Over-expression of the C192S or H226Q alleles does not restore sensitivity to φCr30, indicating that these alleles are non-functional. (E) Immunoblot anti-HvyA-TAP on ΔhvyA cells harbouring mutagenized Pvan-hvyA-TAP and selected for ‘light’ buoyancy. The Pvan-HvyA-TAP mutant alleles selected are still over-expressed. Molecular size standards are indicated in blue on the left, with the corresponding values in kDa. (F) Immunoblot anti-HvyA-TAP on EGTA fractions of the same clones shown in panel (E). The HvyA-TAP mutant alleles selected are exported to the periplasm like WT HvyA-TAP. Molecular size standards are indicated in blue on the left, with the corresponding values in kDa. (G) ΔhvyA strains harbouring hvyA variants under control of Pvan on pMT335 were tested for sensitivity to φCr30. The L240R, H226Y, and W178S alleles do not restore sensitivity to φCr30, whereas the P263R, R161P and D194G variants partially restore sensitivity to φCr30. The alignment of BTLCPs protein sequences from Caulobacter (HvyA), S. meliloti (SMc00998), S. fredii NGR234 (NGR_c12490), and P. fluorescens (PFL_0130) is shown in Figure 3—figure supplement 1. Immunoblots against HvyA-TAP, CtrA, and β-lactamase on whole lysates and EGTA fractions of cells expressing HvyA point mutants are shown in Figure 3—figure supplement 2–3. https://doi.org/10.7554/eLife.03587.011 Microscopic visualization of the capsule As all previous efforts to visualize the capsule directly by light or electron microscopy had been unsuccessful (Ravenscroft et al., 1991), we conducted negative stain fluorescence microscopy (FM) with fluorescein isothiocyanate (FITC)-coupled dextran to measure the zone of exclusion of FITC-dextran in capsulated (ΔhvyA, ‘light’ mucoid) and non-capsulated (ΔCCNA_00163, ‘heavy’ non-mucoid) cells (Figure 4A,B). Akin to the difference between capsulated and non-capsulated Streptococcus pneumoniae (Hathaway et al, 2012; Schaffner et al, 2014), the zone of exclusion of FITC-dextran was significantly smaller in the case of ΔCCNA_00163 compared to ΔhvyA cells, although the actual size of the cells by differential interference contrast (DIC) microscopy was comparable (Figure 4A). The increase in the exclusion radius of the dextran polymer can be explained by the presence of a capsule on ΔhvyA cells and by its absence from ΔCCNA_00163 cells. Atomic force microscopy (AFM) (Dufrêne, 2014) provided additional support for this interpretation. In these experiments, bacteria were immobilized on porous membranes, a method allowing AFM imaging of the bacteria in liquid medium. However, recording r" @default.
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• W2983115969 title "Author response: Cell cycle constraints on capsulation and bacteriophage susceptibility" @default.
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