ABSTRACT
The rare actinomycete Actinoplanes missouriensis grows as substrate mycelium and forms terminal sporangia containing a few hundred spores as dormant cells. Upon contact with water, the sporangia open up and release spores to external environments. Here, we report a cell wall hydrolase, GsmA, that is required for sporangiospore maturation in A. missouriensis. The gsmA gene is conserved among Actinoplanes species and several species of other rare actinomycetes. Transcription of gsmA is activated in the late stage of sporangium formation by the global transcriptional activator TcrA, which is involved in sporangium formation and dehiscence. GsmA is composed of an N-terminal signal peptide for the twin arginine translocation pathway, two tandem bacterial SH3-like domains, and a glucosaminidase domain. Zymographic analysis using a recombinant C-terminal glucosaminidase domain protein showed that GsmA is a hydrolase able to digest cell walls extracted from the vegetative mycelia of A. missouriensis and Streptomyces griseus. A gsmA deletion mutant (ΔgsmA) formed apparently normal sporangia, but they released chains of 2 to 20 spores under sporangium dehiscence-inducing conditions, indicating that spores did not completely mature in the mutant sporangia. From these results, we concluded that GsmA is a cell wall hydrolase for digesting peptidoglycan at septum-forming sites to separate adjacent spores during sporangiospore maturation in A. missouriensis. Unexpectedly, flagella were observed around the spore chains of the ΔgsmA mutant by transmission electron microscopy. The flagellar formation was strictly restricted to cell-cell interfaces, giving an important insight into the polarity of the flagellar biogenesis in a spherical spore.
IMPORTANCE In streptomycetes, an aerial hypha is compartmentalized by multiple septations into prespores, which become spores through a series of maturation processes. However, little is known about these maturation processes. The rare actinomycete Actinoplanes missouriensis produces sporangiospores, which are assumed to be formed also from prespores generated by the compartmentalization of intrasporangium hyphae via septation. The identification of GsmA as a cell wall hydrolase for the separation of adjacent spores sheds light on the almost unknown processes of sporangiospore formation in A. missouriensis. Furthermore, the fact that GsmA orthologues are conserved within the genus Actinoplanes but not in streptomycetes indicates that Actinoplanes has developed an original strategy for the spore maturation in a specific environment, that is, inside a sporangium.
INTRODUCTION
Peptidoglycan is a primary constituent of bacterial cell walls, and it exerts an essential function in maintaining cell shape and cytoplasmic turgor pressure. It comprises alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which make up the glycan backbone, and peptide side chains linking the parallel glycan strands together (1). Despite the rigid structure, dynamic structural modification of peptidoglycan is a universal phenomenon during life cycles of all bacterial species (2). Enzymes responsible for the peptidoglycan degradation are collectively termed cell wall lytic enzymes, which are typically grouped on the basis of the substrate specificity and cleavage products (3). Three types of glycan strand-cleaving cell wall lytic enzymes are known: N-acetyl-β-d-glucosaminidase (termed N-acetylglucosaminidase here), lysozyme, and lytic transglycosylase. The latter two enzymes are grouped as N-acetyl-β-d-muramidase (termed N-acetylmuramidase here). In addition to the glycan strand-cleaving enzymes, various amidases and peptidases, such as N-acetylmuramyl-l-alanine amidase, carboxypeptidase, and endopeptidase, are also involved in peptidoglycan remodeling (4). Thus, the tightly regulated synthesis and degradation of peptidoglycan are accomplished by coordinated activities of peptidoglycan biosynthetic enzymes and cell wall lytic enzymes.
Cell wall lytic enzymes contribute to a multitude of bacterial cellular processes, including cell elongation, sporulation, spore germination, competence, assembly of flagella and pili, and adaptation to diverse environmental conditions. In Bacillus subtilis, over 30 proteins are proven or predicted to be cell wall lytic enzymes (5). LytC and LytD, which function as N-acetylmuramyl-l-alanine amidase and N-acetylglucosaminidase, respectively, are involved in cell separation during vegetative growth. In the single gene mutants of lytC and lytD, daughter cell separation is prevented owing to the lack of hydrolysis of nascent septa, leading to an increase in the length of cell chains. Furthermore, when the lytC and lytD mutations were combined, longer cell chains were formed, indicating that these enzymes have similar roles in cell separation and mutually compensate for the lack of each other (6). It is noteworthy that most cell wall lytic enzymes in B. subtilis have cell wall (CW)-binding domains. For example, LytC has the CW-binding 2 domain, whereas three d,l-endopeptidases, LytE, CwlS, and LytF, have the peptidoglycan-anchoring LysM domain in their N-terminal regions. These three endopeptidases are highly similar to one another, and a triple mutant lacking lytE, cwlS, and lytF shows a filamentous morphology (7).
Actinomycetes are mainly soil-inhabiting Gram-positive bacteria, and many of them show a filamentous growth. Filamentous actinomycetes are characterized by a complex morphological development. In particular, morphological development of Streptomyces species has been extensively studied (8–11). In Streptomyces coelicolor A3(2), four cell wall lytic enzymes, RpfA (lysozyme), SwlA (endopeptidase/amidase), SwlB (lytic transglycosylase), and SwlC (endopeptidase), were characterized for their biochemical and physiological functions and shown to be important for spore germination, vegetative growth, heat resistance, and spore formation (12). Furthermore, a d-alanyl-d-alanine carboxypeptidase, SCO4439, which removes the terminal d-alanine from the muramyl pentapeptide, was reported to regulate the proportion of cross-linking in spore peptidoglycan by controlling the substrate pool for the transpeptidation reaction. Although sporulation proceeded normally, spores of the SCO4439 mutant showed an increase in swelling during germination, demonstrating a role of this peptidase in spore maturation (13). Recently, the composition of peptidoglycan was extensively studied in S. coelicolor A3(2), revealing that it dynamically changes over the course of morphological development and growth phase (14). While these studies highlight the importance of cell wall remodeling during life cycles of Streptomyces species, molecular insights into the peptidoglycan remodeling in rare actinomycetes, which are actinobacteria other than the genus Streptomyces, are very limited. The chemical composition of peptidoglycan in several rare actinomycetes was analyzed in detail because it is an important diagnostic feature for the taxonomy of actinomycetes (15). N-Glycolylmuramic acid was reported to be present in the glycan strands of peptidoglycan in members of the genera Mycobacterium, Corynebacterium, Gordonia, Nocardia, Actinoplanes, Catenuloplanes, Couchioplanes, Dactylosporangium, and Micromonospora (16–21).
Actinoplanes missouriensis, a well-characterized member of the genus Actinoplanes, forms branched substrate mycelium during vegetative growth and subsequently produces globose or subglobose terminal sporangia growing from the substrate mycelium through short sporangiophores on sporangium-forming humic acid-trace element (HAT) agar (22, 23). Each sporangium contains a few hundred spherical flagellated spores, and the exterior space of the spores is filled with an intrasporangial matrix substance. Sporangia open up to release the spores in response to external water through a process referred to as sporangium dehiscence (24, 25). Spores are termed zoospores after being released from sporangia because they can swim in aquatic environments. When reaching a niche suitable for vegetative growth, zoospores stop swimming and begin to germinate (26, 27). Type IV pili are used for the adhesion of zoospores to hydrophobic solid surfaces (28). On HAT agar, small sporangium-like structures are observed after 2 to 3 days of cultivation. Then, mature sporangia that can release spores under dehiscence-inducing conditions are formed after incubation for 5 to 7 days (29, 30). Previously, we revealed that an orphan response regulator, TcrA, globally controls sporangium formation, spore dormancy, and sporangium dehiscence in A. missouriensis (31). TcrA directly activates the transcription of at least 34 transcriptional units responsible for developmental processes by binding to a 21-bp direct repeat sequence (TcrA box), 5′-NNGCA(A/C)CCG-N4-GCA(A/C)CCGN-3′ (31). In the present study, we focused on a member of the TcrA regulon, AMIS_54470, to elucidate an aspect of the molecular mechanism of sporangium formation in A. missouriensis. The gene encodes a protein that is predicted to function as an N-acetylglucosaminidase. Here, we name AMIS_54470 GsmA (glucosaminidase required for spore maturation) for its function as revealed in this study. We describe the function of GsmA in the morphological development of A. missouriensis and biochemically characterize GsmA. GsmA is an N-acetylglucosaminidase for digestion of the A. missouriensis cell wall and specifically required for sporangiospore maturation.
RESULTS
GsmA is widely conserved among Actinoplanes bacteria and some other rare actinomycetes.In our previous work, we identified 34 transcriptional units as the TcrA regulon (31). gsmA, which encodes a protein of 368 amino acids, is a member of the TcrA regulon. The SignalP 5.0 server (http://www.cbs.dtu.dk/services/SignalP/) predicted that GsmA should have a signal peptide for the twin arginine translocation (TAT) pathway (the predicted cleavage site is between residues 31 and 32). A protein database search using InterPro ver. 69.0 (http://www.ebi.ac.uk/interpro/) showed that the N-terminal portion of mature GsmA harbors two bacterial SH3-like domains (accession number IPR003646; residues 35 to 105 and 127 to 198), which were suggested to mediate protein-protein interaction through the binding to the Pro-X-X-Pro motif (where X represents any amino acid) (32). In addition, a mannosyl-glycoprotein endo-β-N-acetylglucosamidase-like domain is located in the C-terminal portion (IPR002901; residues 204 to 365). The C-terminal domain of GsmA shows a 34% sequence identity to the N-acetylglucosaminidase LytG of B. subtilis (33). According to the carbohydrate-active enzyme database (http://www.cazy.org), GsmA belongs to the GH73 enzyme family, most of whose members cleave the β-1,4-glycosidic linkage between N-acetylglucosaminyl and N-acetylmuramyl moieties in the carbohydrate backbone of bacterial peptidoglycans. Taken together, these in silico analyses suggest that GsmA functions as a cell wall hydrolase.
The genome sequences with gene annotation of 23 Actinoplanes species, including A. missouriensis, have been registered in the NCBI genome database (https://www.ncbi.nlm.nih.gov/genome/). We searched for GsmA orthologues using BLAST analysis and found that highly similar proteins are conserved among all 23 Actinoplanes bacteria (see Fig. S1 in the supplemental material). GsmA orthologues were also found in other rare actinomycetes. Couchioplanes caeruleus, which forms motile spores by fragmentation of aerial hyphae, encodes two proteins showing high homology with GsmA on its genome. Partially similar genes are also found on the genomes of Catenuloplanes japonicus, which forms segmental motile spores, and members of the genera Dactylosporangium, Planomonospora, and Spirillospora, all of which produce sporangiospores. In contrast, no orthologue was found in the genus Kineosporia, which produces motile sporangiospores, the genera Kineococcus (cocci) and Angustibacter (cocci or rods), both of which have a flagellar gene cluster similar to that of A. missouriensis, and the genus Streptomyces. These results indicate that GsmA is evolutionarily conserved among the genus Actinoplanes and some other rare actinomycetes.
TcrA activates gsmA transcription during sporangium formation.We analyzed the levels of gsmA transcripts in the wild-type strain by quantitative reverse transcription PCR (qRT-PCR) analysis. The transcripts were detected at low levels in substrate hyphae cultivated on HAT agar at 30°C for 24 h (1 day) and in the mixtures of substrate hyphae and immature sporangium-like structures formed on HAT agar cultivated for 72 h (3 days) (Fig. 1A). In contrast, the transcripts were abundantly detected in the mixtures of substrate hyphae and sporangia formed on HAT agar cultivated for 144 h (6 days), indicating that the transcription of gsmA is activated in the late stage of sporangium formation (Fig. 1A). Then, we determined the transcriptional start site of gsmA by high-resolution S1 nuclease mapping using RNA samples extracted from the mixtures of substrate hyphae and sporangia cultivated on HAT agar for 144 h. The transcriptional start site was determined to be 31 nucleotides upstream from the start codon (Fig. 1B and C). As reported in our previous work (31), a TcrA box containing three direct repeat sequences is located upstream from the transcriptional start point with a spacer of 31 nucleotides (Fig. 1C); the upstream two direct repeat sequences are located at a typical position for TcrA boxes. Considering the location and sequence similarity to the consensus TcrA-binding sequence, we assume that these upstream two direct repeat sequences form the TcrA-binding site and that the third repeat sequence does not constitute an additional TcrA-binding site (Fig. 1C). In our previous study, we demonstrated that the recombinant histidine-tagged TcrA protein binds to the upstream region of gsmA by a competitive electrophoretic mobility shift assay, suggesting that TcrA directly activates the transcription of gsmA (31). Therefore, we quantified the transcript levels of gsmA in the wild-type and ΔtcrA strains cultivated on HAT agar for 144 h by qRT-PCR analysis. As expected, the level of gsmA transcript in the ΔtcrA mutant was significantly lower (<10%) than that in the wild-type strain, showing that TcrA directly activates gsmA transcription (Fig. 1D). Because the TcrA orthologues are widely conserved among rare actinomycetes (31), we searched for the TcrA box in the upstream regions from the gsmA orthologues in other rare actinomycetes described above. As a result, probable TcrA-binding sites were found in all the regions, suggesting that the TcrA orthologues directly activate the transcription of the gsmA orthologues in these rare actinomycetes (see Fig. S2 in the supplemental material).
Transcriptional analysis of gsmA. (A) Levels of gsmA transcript in the wild-type strain. The amount of gsmA transcript was examined by qRT-PCR analysis. The rpoB transcript was used as an internal standard. Data are the mean values from three biological replicates ± standard errors. (B) High-resolution S1 nuclease mapping. The 5′ terminus of the mRNA was assigned to the indicated position because fragments generated by chemical sequencing reactions migrate 1.5 nucleotides further than the fragment created by S1 nuclease digestion of the DNA-RNA hybrid. (C) Nucleotide sequence upstream of the transcriptional start point of gsmA. Two direct repeat sequences that form a TcrA box at a typical location are shaded in green. An additional repeat sequence identified by an in silico analysis is shaded in yellow (31). (D) Levels of gsmA transcript in the wild-type and ΔtcrA strains. The amount of gsmA transcript was examined by qRT-PCR using the RNA samples extracted from the cells cultivated on HAT agar for 6 days. The rpoB transcript was used as an internal standard. Data are the mean values from three biological replicates ± standard errors. The expression ratio (11.4-fold) was statistically significant (t test, P < 0.05).
The C-terminal domain of GsmA has cell wall-hydrolyzing activity.We examined the cell wall-hydrolyzing activity of GsmA, because GsmA is predicted to possess the N-acetylglucosaminidase domain in its C-terminal region. Because we attempted but failed to produce a soluble GsmA protein in its full-length form in Escherichia coli, a shorter protein containing only the C-terminal N-acetylglucosaminidase domain with an N-terminal polyhistidine tag (residues 211 to 368; His-GsmAc) was produced and purified. With the recombinant His-GsmAc protein, we performed a zymographic analysis using the cell wall extracted from A. missouriensis vegetative hyphae cultivated in peptone-yeast extract-magnesium (PYM) liquid. As a result, the cell wall was completely hydrolyzed in the presence of His-GsmAc, indicating that His-GsmAc has lytic activity toward the A. missouriensis cell wall (Fig. 2A). In this analysis, we used bovine serum albumin (BSA) as a negative control, which showed no detectable cell wall-hydrolyzing activity, as expected (Fig. 2A).
Zymographic analysis using His-GsmAc. The separating gels contained 1 mg/ml cell wall extracted from the vegetative hyphae of A. missouriensis (A) and S. griseus (B). Electrophoresis was performed in duplicate. One separating gel was stained with CBB (lanes 1 to 3), and the other gel was stained with alkaline methylene blue after renaturation (lanes 4 and 5). Lane 1, protein molecular size standards (Bio-Rad); lanes 2 and 4, BSA; lanes 3 and 5, His-GsmAc.
As described in the introduction, N-glycolylmuramic acid instead of MurNAc is present in the glycan strand of peptidoglycan in Actinoplanes species (20). To examine the substrate specificity of His-GsmAc, we conducted another zymographic analysis using the cell wall extracted from the Streptomyces griseus vegetative hyphae, whose glycan strands contain MurNAc instead of N-glycolylmuramic acid. Because GsmA orthologues are conserved among rare actinomycetes, such as members of the genera Actinoplanes, Catenuloplanes, Couchioplanes, and Dactylosporangium, whose peptidoglycan contains N-glycolylmuramic acid instead of MurNAc, we suspected that GsmA should cleave the glycosidic bond specifically between GlcNAc and N-glycolylmuramic acid. Contrary to our expectation, however, His-GsmAc showed a hydrolytic activity toward the S. griseus cell wall, too, indicating that N-glycolylmuramic acid in the A. missouriensis peptidoglycan is not an essential moiety for the cleavage by His-GsmAc (Fig. 2B).
GsmA is required for sporangiospore maturation.Considering that the transcription of gsmA is activated by TcrA at the late stage of sporangium formation (Fig. 1A), we postulated that GsmA should function as a cell wall hydrolase that plays a role in morphological development. To investigate the physiological role of GsmA, we generated a gsmA null mutant (ΔgsmA) and compared its phenotype with that of the wild-type strain. No difference was observed between the wild-type and ΔgsmA strains by macroscopic observation of mycelia and sporangia on yeast extract-beef extract-NZ amine-maltose monohydrate (YBNM) and HAT agars (data not shown). To examine sporangium formation in detail, we observed the mycelia of the wild-type and ΔgsmA strains grown on HAT agar at 30°C for 7 days by scanning electron microscopy (SEM). However, both strains produced normal globose or subglobose sporangia with short sporangiophores (data not shown). Next, we observed sporangium dehiscence using the sporangia harvested from the mycelia grown on HAT agar at 30°C for 7 days. Sporangium dehiscence can be induced by incubating the sporangia in 25 mM histidine solution (our unpublished observation). In the dehiscence-inducing solution, sporangia of the wild-type strain normally dehisced and released a large number of zoospores into the surrounding environment (Fig. 3A to C). Sporangia of the ΔgsmA mutant strain also dehisced under the same condition, but to our surprise, most cells released from the sporangia were arranged in long chains, in which up to 20 sporulating cells were connected in tandem, indicating that sporulation did not proceed properly in the ΔgsmA mutant sporangia (Fig. 3D to G). It should be noted that almost all spores released from the wild-type sporangia were observed as free-swimming zoospores, and no spore chains were found (Fig. 3C). The ΔgsmA mutant also released short spore chains of 2 to 5 spores that moved awkwardly, and a small number of free-swimming zoospores were also observed (Fig. 3F and G). In a gene complementation test, introduction of the wild-type gsmA gene with its own promoter into the ΔgsmA mutant resulted in the formation of normal sporangiospores that were released from the sporangia as free-swimming zoospores under the dehiscence-inducing condition (Fig. 3H to J). Finally, we quantified the state of released zoospores, i.e., free or connected, by the optical microscopic observation of more than 1,300 zoospores of the wild-type and ΔgsmA mutant strains, both of which contained the empty vector pTYM19-Apra on the chromosome. As a result, almost all spores (96%) released from the wild-type sporangia were observed as free single zoospores (Fig. 3K). Only a small portion of zoospores were observed in spore chains of double spores (1.5%) and triple spores (2.7%). However, we cannot eliminate the possibility that these spore chains came from the aggregation of free single zoospores. In contrast, many spores (67%) released from the ΔgsmA mutant sporangia were arranged in chains, although free single zoospores (33%) were also observed (Fig. 3L). Similarly, the ΔgsmA mutant harboring the gsmA complementation plasmid was examined. In this strain, almost all spores (98%) released from the sporangia were observed as free single zoospores as in the wild-type strain (Fig. 3M).
Observation of sporangia and zoospores by optical microscopy and quantification of single and tandemly connected zoospores. Sporangia produced on HAT agar after 7 days of cultivation were harvested and suspended in 25 mM histidine solution to induce sporangium dehiscence. (A to J) Microscopic images of the wild-type strain (A to C), ΔgsmA mutant (D to G), and ΔgsmA mutant harboring the gsmA complementation plasmid (H to J). Panels A, D, and H were taken immediately after the suspension. Panels B, E, and I were taken 15 min after the suspension. Panels C, F, G, and J were taken 30 min after the suspension. Sporangium membrane just after the suspension reflects the light from the microscope (A, D, and H) and then gradually becomes transparent before the spore release (B, E, and I). Bars, 10 μm (A to F and H to I) and 4 μm (G). (K to M) Proportions of single and tandemly connected zoospores in the wild-type strain (K) and ΔgsmA mutant (L), both of which contained pTYM19-Apra, and ΔgsmA mutant harboring the gsmA complementation plasmid (M). The total number (n) of the observed zoospores of each strain is shown. Some zoospores are observed as single free zoospores, and others are observed as components of spore chains with 2, 3, 4, 5, and >5 spores.
Next, we observed the spores and spore chains released from sporangia by SEM. While separate spores were observed in the wild-type strain, cells of the spore chains of the ΔgsmA mutant were obviously connected with each other at their surface layers (Fig. 4). Furthermore, the tandem arrangement of the spores in spore chains indicated that sporangiospore maturation was arrested in the ΔgsmA mutant (Fig. 3G and Fig. 4B).
Observation of zoospores by SEM. The zoospores or spore chains released from the sporangia were dropped on an agar piece and dried before coating. (A) Zoospores of the wild-type strain. (B) A spore chain of the ΔgsmA mutant. Bars, 1 μm.
Because a small number of free zoospores of the ΔgsmA mutant swam actively (Fig. 3F and G), we supposed that they would be flagellated. To examine this, we analyzed the free zoospores of the wild-type and ΔgsmA strains by transmission electron microscopy (TEM). As expected, the swimming zoospores of the ΔgsmA mutant were flagellated similarly to the wild-type zoospores (Fig. 5A and B). Furthermore, the spore chains of the ΔgsmA mutant were analyzed with TEM, because short spore chains of 2 to 5 spores moved awkwardly in the observation by optical microscopy (Fig. 3F and G). As a result, flagella were successfully observed around the spore chains. Unexpectedly, however, we found that the flagellation was strictly restricted to the interfaces between the connected spores (Fig. 5C and D), suggesting that flagella are assembled at the cell-cell boundary regions during sporangiospore maturation in A. missouriensis (see Discussion).
Observation of zoospores by TEM. (A) A zoospore of the wild-type strain. (B) A zoospore of the ΔgsmA mutant. (C) A spore chain of the ΔgsmA mutant. (D) An enlarged view of a portion of panel C. The samples were negatively stained with 1% (wt/vol) phosphotungstic acid. Arrowheads indicate flagellar filaments in panels A and B. All filaments observed around zoospores and spore chains are flagella. Bars, 1 μm.
DISCUSSION
In this study, we revealed that GsmA plays a pivotal role in morphological differentiation in A. missouriensis. GsmA is predominantly produced in the late stage of sporangium formation and presumably secreted into the intrasporangial matrix space through the TAT pathway, where it functions as a cell wall hydrolase to separate sporangiospores along sporulating hyphae. Cell chain formation due to the lack of cell wall lytic enzymes has been observed in a wide variety of bacterial species. In E. coli, a mutant strain with deletions of seven genes encoding amidases, endopeptidases, and lytic transglycosylases grew in chains comprising up to 100 cells per chain (34). In Lactococcus lactis, loss of the N-acetylmuramidase AcmA resulted in the growth in long chains (35, 36). A similar long-chain formation was also observed in a mutant lacking the N-acetylglucosaminidase LytB of Streptococcus pneumoniae (37). Furthermore, the cell wall lytic enzyme p60 in Listeria monocytogenes and the muramidase 2 in Enterococcus hirae were reported to have an essential role in cell separation (38, 39). To the best of our knowledge, however, no cell wall lytic enzyme required for septation along spore chains has been identified so far in spore-forming actinomycetes. Therefore, GsmA is a novel enzyme that hydrolyzes peptidoglycan at septum-forming sites to separate mature spores in the spore-forming actinomycetes.
The glycan strands of bacterial peptidoglycan invariably become modified before or immediately after insertion into the cell wall. The N-deacetylation of the GlcNAc or MurNAc residues and O-acetylation of the MurNAc residue were reported to reduce the hydrolytic activity of lysozyme toward peptidoglycan, suggesting that bacteria have evolved the peptidoglycan modifications as a means of resistance to ubiquitously existing cell wall lytic enzymes (40, 41). As described in the introduction, the glycan strands of wall peptidoglycans in the genus Actinoplanes contain N-glycolylmuramic acid instead of MurNAc. Previously, a hydroxylase-encoding gene, namH, was reported to be required for the glycolyl modification of the cytoplasmic precursor UDP-MurNAc-pentapeptide in Mycobacterium smegmatis (42). While the glycan strands of peptidoglycan in the M. smegmatis wild-type strain contain N-glycolylmuramic acid, the namH null mutant (ΔnamH) strain is devoid of N-glycolylated precursors and instead synthesizes only N-acetylated ones. Furthermore, the ΔnamH mutant exhibited an increased susceptibility to β-lactam antibiotics and lysozyme, suggesting that the N-glycolylated peptidoglycan is more resistant to these antibacterial substances than the N-acetylated one. In A. missouriensis, the AMIS_70000 gene product shares high homology with NamH (52% identity in amino acid sequence), supporting the notion that the N-glycolylated peptidoglycan is produced in this bacterium. Considering the results of our zymographic analysis (Fig. 2), we concluded that GsmA is a unique cell wall hydrolase that cleaves not only the lysozyme-sensitive N-acetylated peptidoglycan but also the antibacterial substance-resistant N-glycolylated peptidoglycan.
In our previous studies, we observed that flagella were assembled in a limited area of the zoospore surface in A. missouriensis (28, 29). Although the molecular mechanism that assembles flagella in these restricted regions remains elusive, observation of the ΔgsmA mutant spore chains by TEM gave an important mechanistic insight, leading to the prediction that flagellar biogenesis proceeds at the sporulating cell-cell interfaces prior to completion of the sporangiospore separation (Fig. 5C and D). This model enables the flagella to be assembled in the restricted space around the zoospores. Because flagellar filaments were not observed at the tip of spore chains, they seem to be synthesized only at the cell pole near the root of spore chains in each spore. Furthermore, our recent study revealed that the A. missouriensis zoospores possess both flagella and pili on their surfaces (28). The spatiotemporal regulation of the flagellar and pilus biogenesis would enable the pili to be assembled at locations on the zoospore surface different from that for the flagellar assembly; the pilus biogenesis also may proceed at the cell-cell interface opposite that for the flagellar assembly in sporulating cells. To prove this working model, exact areas of the flagellation and piliation around the zoospore surface need to be determined.
Based on the phenotypic changes observed in the ΔgsmA mutant, we assume that other cell wall lytic enzymes in addition to GsmA are inevitably involved in the sporangiospore formation in A. missouriensis. According to our previously obtained transcriptomic data during sporangium formation (29), a gene candidate is AMIS_65730, encoding a putative N-acetylmuramyl-l-alanine amidase, whose transcription is highly induced at the early stage of sporangium formation (42 times higher at day 3 than at day 1 on HAT agar). Another gene candidate is AMIS_13350, encoding a putative d-alanyl-d-alanine carboxypeptidase, whose transcription is highly induced during sporangium formation (104 and 456 times higher at days 3 and 6, respectively, than at day 1 on HAT agar), suggesting that the gene product is also involved in the sporangiospore formation through the modification of peptidoglycan structures, although clear phenotypic change was not observed between an isogenic AMIS_13350 null mutant and the wild-type strain (our unpublished data). It is worth noting that the AMIS_65730 gene product contains four copies of the Pro-X-X-Pro motif, which is a probable interaction site for the SH3-like domain (32). In B. subtilis, LytD was suggested to interact with other cell wall lytic enzymes or cell surface proteins through its SH3-like domain, forming a cell wall-modifying protein complex (43). Therefore, we assume that GsmA interacts with the AMIS_65730 gene product and other extracellular enzymes through the tandem SH3-like domains in its N-terminal region to produce a cell wall-modifying protein complex during sporangium formation/maturation. Because the AMIS_65730 homologues are widely conserved among actinobacteria, including streptomycetes (approximately 40% amino acid sequence identity in streptomycetes), the gene products may be a core component of the probable cell wall-modifying protein complex. Considering that GsmA orthologues are highly conserved among Actinoplanes but not in streptomycetes, members of the genus Actinoplanes may have evolved GsmA and its orthologues as an original element of the cell wall-modifying protein complex to form mature spores in the peculiar environment, specifically, inside a sporangium.
MATERIALS AND METHODS
Bacterial strains, plasmids, media, and primers.A. missouriensis 431T (NBRC 102363T) and S. griseus IFO 13350 (NBRC 13350) were obtained from the National Institute of Technology and Evaluation (NITE, Chiba, Japan) and the Institute of Fermentation (Osaka, Japan), respectively (23, 44). A. missouriensis was grown at 30°C on YBNM or HAT agar for solid culture and in PYM broth for liquid culture as previously described (30). S. griseus was grown at 30°C on yeast extract-magnesium-peptone-glucose (YMPD) agar for solid culture and in YMPD broth for liquid culture as previously described (45). Modified ISP4 or mannitol-soy flour (MS) agar medium (28) was used for transformation of A. missouriensis by conjugation with E. coli ET12567(pUZ8002). Modified ISP4 agar was used for the construction of the ΔgsmA mutant, and MS agar was used to construct the strains for gene complementation testing. E. coli ET12567(pUZ8002) was obtained from the John Innes Centre (Norwich, United Kingdom) and used as the donor in intergeneric conjugation. E. coli JM109, pUC19, and pColdII were purchased from TaKaRa Biochemicals (Shiga, Japan) (46). E. coli BL21(DE3) was purchased from Merck Millipore (Darmstadt, Germany). The media and growth conditions for E. coli were the same as those described by Maniatis et al. (47). Apramycin (50 μg/ml), spectinomycin (50 μg/ml), and ampicillin (50 μg/ml) were added when necessary. Primers used in this study are listed in Table S1 in the supplemental material.
RNA extraction.A. missouriensis cells for RNA extraction were prepared as previously described (31). Cells were disrupted by grinding with a mortar and pestle, and the cell lysate was mixed with the lysis/binding solution from the RNAqueous total RNA isolation kit (Thermo Fisher Scientific, MA). After the debris was removed by centrifugation at 21,000 × g for 5 min, total RNA was extracted according to the manufacturer’s instructions. The total RNA was treated with DNase I to eliminate contaminating genomic DNA and purified by phenol-chloroform extraction and ethanol precipitation.
qRT-PCR.Total RNA samples (1 μg each) were used for reverse transcription reactions for the first-strand cDNA synthesis using the ThermoScript RT-PCR system (Thermo Fisher Scientific) according to the manufacturer’s instructions. Following RNase H treatment, the synthesized cDNA libraries were used as the templates for PCR. Quantitative PCRs were performed with the SYBR premix Ex Taq II reaction mixture (TaKaRa Biochemicals) using the AriaMx real-time PCR system (Agilent Technologies, CA) under the following conditions: 5 min at 95°C, followed by 40 cycles of 5 s at 95°C and 10 s at 60°C. The rpoB gene was used as an internal standard. All reactions were performed in biological triplicate, and the data were normalized using the average for the internal standard.
S1 nuclease mapping.S1 nuclease mapping was performed using the method described by Bibb et al. (48) and Kelemen et al. (49). A hybridization probe was prepared using PCR and labeled at both 5′ ends with [γ-32P]ATP (220 TBq/mM) using T4 polynucleotide kinase. Labeling at one side of the 5′ ends was eliminated by digestion with EcoRI. For hybridization, 40 μg of total RNA was used. Protected fragments were analyzed on 6% polyacrylamide DNA sequencing gels according to the method of Maxam and Gilbert (50).
Production and purification of recombinant His-GsmAc protein.A 499-bp DNA fragment containing the 3′ portion of the gsmA coding sequence was amplified by PCR. The fragment was digested with EcoRI and HindIII and cloned into pUC19 digested with the same restriction enzymes, generating pUC19-gsmAc. Plasmid pUC19-gsmAc was sequenced to confirm that no PCR-derived error was introduced. The fragment was digested with NdeI and HindIII and cloned into pColdII digested with the same restriction enzymes, generating pColdII-gsmAc. Plasmid pColdII-gsmAc was introduced into E. coli BL21(DE3). The transformant was cultivated in LB broth (50 ml) at 37°C for 2.5 h and at 15°C for 30 min. Then, isopropyl-β-d-1-thiogalactopyranoside was added to the culture to a final concentration of 1 mM. After a further cultivation at 15°C for 24 h, cells were collected by centrifugation at 3,000 × g for 10 min and suspended in 5 ml of lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0), followed by disruption by sonication. After removal of cell debris by centrifugation at 10,000 × g for 30 min, GsmAc-His was purified from the cell extract using Ni-nitrilotriacetic acid Superflow resin (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. GsmAc-His was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 400 mM imidazole, 10% glycerol, pH 8.0). The quality of the purified protein was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Extraction of crude cell wall.Cell wall components were extracted from the vegetative hyphae of A. missouriensis and S. griseus as described by Fukushima and Sekiguchi (51). The wild-type strain was cultivated in PYM or YMPD broth for 72 h, and the vegetative hyphae were collected by centrifugation at 10,000 × g for 10 min. After removal of the supernatant, the pellet was suspended in 30 ml of 4 M LiCl solution, followed by boiling for 15 min. After cooling down to room temperature, the samples were disrupted by sonication, and the lysate was centrifuged at 10,000 × g for 10 min. After removal of the supernatant, the precipitated crude cell wall was suspended in 20 ml of distilled water, further dissolved by sonication for 15 min, and centrifuged at 10,000 × g for 10 min. After removal of supernatant, the precipitated cell wall was suspended in 40 ml of 4% (wt/vol) sodium dodecyl sulfate solution and boiled for 15 min. After centrifugation at 10,000 × g for 10 min, the pellet was suspended in 40 ml of 1 M NaCl solution and centrifuged at 10,000 × g for 10 min. The washing procedure using 1 M NaCl solution was repeated five times in total. Then, the cell wall pellet was suspended in 40 ml of distilled water and centrifuged at 10,000 × g for 10 min. The washing procedure using distilled water was repeated three times in total. Finally, the crude cell wall was suspended in distilled water. The concentration of the cell wall was calculated by drying 1 ml of the cell wall suspension, followed by the measurement of the dry weight.
Zymography.A zymographic analysis using the A. missouriensis or S. griseus crude cell wall was performed as described by Fukushima and Sekiguchi (51). The cell wall was added to the separating gel solution at a concentration of 1 mg/ml. SDS-PAGE was performed in duplicate using His-GsmAc and BSA (Merck Millipore). The protein samples were mixed with the SDS-PAGE sample buffer (final concentration, 31 mM Tris-HCl [pH 6.8], 0.003% bromophenol blue, 10% glycerol, 1% SDS) and boiled for 5 min. One gel was stained with Coomassie brilliant blue (CBB), and the other was used for the following zymographic assay. The separating gel was placed in 50 ml of distilled water and gently shaken for 5 min. Then, the gel was transferred to 50 ml of the renaturing buffer (50 mM Na2HPO4, 1% [vol/vol] Triton X-100, pH 7.0) and incubated at 37°C for 6 h. The renatured gel was stained with 50 ml of the staining solution (0.01% [wt/vol] methylene blue, 0.01% [wt/vol] KOH) until the entire gel became blue. The stained gel was put into 50 ml distilled water and gently shaken until a destained band appeared in the gel.
Construction of the ΔgsmA mutant.To construct a ΔgsmA mutant, the upstream and downstream regions of gsmA were amplified by PCR. The amplified DNA fragments were digested with EcoRI and XbaI (for the upstream region) and XbaI and HindIII (for the downstream region) and cloned into pUC19 digested with the same restriction enzymes, generating pUC19-ΔgsmA-u and pUC19-ΔgsmA-d. Plasmids pUC19-ΔgsmA-u and pUC19-ΔgsmA-d were sequenced to confirm that no PCR-derived error was introduced. The fragments were digested with EcoRI and XbaI (for the upstream region) and XbaI and HindIII (for the downstream region) and cloned together between the EcoRI and HindIII sites of pK19mobsacB (52), whose kanamycin resistance gene had been replaced with the apramycin resistance gene aac(3)IV (30), generating pK19mobsacB-ΔgsmA. Plasmid pK19mobsacB-ΔgsmA was introduced into A. missouriensis by conjugation as described previously (29). Apramycin-resistant colonies resulting from a single crossover recombination were isolated. One of them was cultivated in PYM liquid medium at 30°C for 36 h, and the mycelia suspended in 0.75% NaCl solution were spread onto Czapek-Dox broth agar medium (Becton Dickinson, NJ) containing extra sucrose (final concentration, 5%). After incubation at 30°C for 5 days, the sucrose-resistant colonies were inoculated onto YBNM agar with or without apramycin to confirm that they were sensitive to apramycin. The apramycin-sensitive and sucrose-resistant colonies resulting from the second crossover recombination were isolated as candidates for the ΔgsmA mutant. The disruption of gsmA was confirmed by PCR (data not shown).
Construction of the recombinant strain for complementation test.A 2.0-kbp DNA fragment containing the promoter and coding sequences of gsmA was amplified by PCR. The amplified fragment was digested with EcoRI and HindIII and cloned between the EcoRI and HindIII sites of pTYM19-Apra (31), resulting in pTYM19-Apra-gsmA. Plasmid pTYM19-Apra-gsmA was sequenced to confirm that no PCR-derived error was introduced, and it was introduced into the ΔgsmA mutant by conjugation as described previously (29). Plasmid pTYM19-Apra was also introduced into the wild-type and ΔgsmA mutant strains for the vector control strains. Apramycin-resistant colonies were obtained.
Microscopic observations.To prepare sporangia for the observations by microscopy, the wild-type and ΔgsmA mutant strains were grown on HAT agar for 7 days. The dehiscence of the sporangia was analyzed via optical microscopy. Sporangia and substrate hyphae formed on HAT agar were harvested with a spatula and mixed with 25 mM histidine solution in a 2.0-ml plastic tube. Just after mixing or following incubation with rotation for 15 or 30 min, the sporangia were observed with a BH-2 light microscope (Olympus, Tokyo, Japan). For the observation of zoospores by SEM, 25 mM NH4HCO3 solution was poured onto sporangia formed on HAT agar to induce sporangium dehiscence. Following incubation at 30°C for 1 h, the zoospores released from sporangia were dropped on an agar piece and dried. SEM was performed with an S-4800 scanning electron microscope (Hitachi, Tokyo, Japan) as described previously (53). The observation by TEM was performed as previously described (29). The samples were negatively stained with 1% (wt/vol) phosphotungstic acid (pH 7.0) and observed with a JEM-1010 electron microscope (JEOL, Tokyo, Japan).
Quantification of free zoospores, spore chains, and component zoospores in each spore chain.To prepare sporangia, the wild-type and ΔgsmA mutant strains, both of which contained pTYM19-Apra on the chromosome, and the ΔgsmA mutant harboring the gsmA complementation plasmid were grown on HAT agar for 7 days. Sporangia and substrate hyphae formed on HAT agar were harvested and mixed with 25 mM histidine solution to induce sporangium dehiscence. After incubation with rotation for 1 h at room temperature, glycerol (final concentration 50%) was added to the solution to repress zoospore motility. The microscopic images of zoospores of each strain were recorded with the BH-2 light microscope (Olympus), and the free zoospores and spore chains, as well as the component zoospores in each spore chain, were counted by visual inspection.
ACKNOWLEDGMENTS
This research was supported in part by Grants-in-Aid for Scientific Research (A) (26252010), (B) (18H02122), and (C) (17K07711) and Grants-in-Aid for Scientific Research on Innovative Areas (19H05685 and 19H05679) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
FOOTNOTES
- Received 6 August 2019.
- Accepted 23 September 2019.
- Accepted manuscript posted online 30 September 2019.
Supplemental material for this article may be found at https://doi.org/10.1128/JB.00519-19.
- Copyright © 2019 American Society for Microbiology.
