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Characterization of zoospore type IV pili in Actinoplanes missouriensis 1

2

Tomohiro Kimura,1 Takeaki Tezuka,1,2,* Daisuke Nakane,3 Takayuki Nishizaka,3 3

Shin-Ichi Aizawa,4 Yasuo Ohnishi1,2,* 4

5

1Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The 6

University of Tokyo, Bunkyo-ku, Tokyo, Japan. 7

2Collaborative Research Institute for Innovative Microbiology, The University of Tokyo, 8

Bunkyo-ku, Tokyo, Japan. 9

3Department of Physics, Gakushuin University, Toshima-ku, Tokyo, Japan. 10

4Department of Life Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima, 11

Japan. 12

13

*Address correspondence to Takeaki Tezuka, [email protected]; Yasuo 14

Ohnishi, [email protected] 15

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Running Head: Zoospore pili of a rare actinomycete 17

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Keywords: adhesion; gene regulation; rare actinomycete; type IV pili; zoospore 19

JB Accepted Manuscript Posted Online 29 April 2019J. Bacteriol. doi:10.1128/JB.00746-18Copyright © 2019 American Society for Microbiology. All Rights Reserved.

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ABSTRACT 20

The rare actinomycete Actinoplanes missouriensis produces terminal sporangia 21

containing a few hundred flagellated spores. After release from the sporangia, the spores 22

swim rapidly in aquatic environments as zoospores. The zoospores stop swimming and 23

begin to germinate in niches for vegetative growth. Here, we report the characterization 24

and functional analysis of zoospore type IV pili in A. missouriensis. The pilus gene (pil) 25

cluster, consisting of three apparently FliA-dependent transcriptional units, is activated 26

during sporangium formation similar to the flagellar gene cluster, indicating that the 27

zoospore has not only flagella but also pili. With a new method in which zoospores 28

were fixed with glutaraldehyde to prevent pilus retraction, zoospore pili were observed 29

relatively easily using transmission electron microscopy: 6 ± 3 pili per zoospore (n = 37 30

piliated zoospores) and 0.62 ± 0.35 m in length (n = 206), via observation of 31

fliC-deleted, non-flagellated zoospores. No pili were observed in the zoospores of a 32

prepilin-encoding pilA deletion (pilA) mutant. In addition, the deletion of pilT, which 33

encodes an ATPase predicted to be involved in pilus retraction, substantially reduced the 34

frequency of pilus retraction. Several adhesion experiments using wild-type and pilA 35

zoospores indicated that the zoospore pili are required for the sufficient adhesion of 36

zoospores to hydrophobic solid surfaces. Many zoospore-forming rare actinomycetes 37

conserve the pil cluster, which indicates that the zoospore pili yield an evolutionary 38

benefit in the adhesion of zoospores to hydrophobic materials as footholds for 39

germination in their mycelial growth. 40

41

IMPORTANCE 42

Bacterial zoospores are interesting cells in that their physiological state changes 43

dynamically: they are dormant in sporangia, exert temporary mobility after awakening, 44

and finally stop swimming to germinate in niches for vegetative growth. However, the 45

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cellular biology of a zoospore remains largely unknown. This study describes 46

unprecedented zoospore type IV pili in the rare actinomycete Actinoplanes 47

missouriensis. Similar to usual bacterial type IV pili, zoospore pili appeared to be 48

retractable. Our findings that the zoospore pili have a functional role in the adhesion of 49

zoospores to hydrophobic solid surfaces and that the zoospores use both pili and flagella 50

properly according to their different purposes provide an important insight into the 51

cellular biology of the zoospore. 52

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INTRODUCTION 53

Pili, also referred to as fimbriae, are hair-like non-flagellar appendages that are 54

located over cell surfaces in a wide range of bacteria. These dynamic extracellular 55

organelles range between 5 and 9 nm in diameter and can reach several micrometers in 56

length. There are five known types of pili in bacteria: chaperone-usher pili, type IV pili, 57

conjugative type IV secretion pili, curli fibers, and type V pili (1). Type IV pili serve 58

diverse functions including motility along solid surfaces, adhesion to host cells, 59

microcolony or biofilm formation, electron transfer, and DNA uptake. For example, the 60

Gram-negative -proteobacterium Myxococcus xanthus relies on type IV pili for social 61

motility and for fruiting body and biofilm formation (2). The human pathogen Neisseria 62

gonorrhoeae moves on surfaces by attaching and retracting type IV pili (3). Among 63

Gram-positive bacteria, on the other hand, mutants lacking type IV pili have been 64

shown to be deficient in twitching and gliding motility in Clostridium difficile and 65

Clostridium perfringens, respectively (4, 5). These functions are dependent on the three 66

basic activities of type IV pili: (i) extension—lengthening the pilus through the 67

polymerization of filament-constituting pilin subunits; (ii) adhesion—the ability of pilus 68

subunits to bind to target surfaces or specific biomolecules; and (iii) 69

retraction—shortening the pilus through pilin depolymerization (3, 6). 70

The type IV pilus system is similar to the type II secretion system, which 71

translocate folded proteins from periplasm into the extracellular environment in 72

Gram-negative bacteria (1, 7). In the type IV pilus system, the substrates for 73

translocation are the pilin subunits. The prepilin protein encoded by pilA has the 74

following three characteristic structures: (i) a signal peptide; (ii) a recognition site for a 75

prepilin peptidase, GFxxxE (x, any amino acid); and (iii) an N-terminal 76

transmembrane-like -helix (8). In the first step of type IV pilus biogenesis, prepilin 77

subunits are inserted into the plasma membrane by the Sec machinery. Then, the signal 78

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peptide is cleaved off in the membrane by the prepilin peptidase PilD (9, 10). During 79

the elongation of pilus filaments, the mature pilin subunits are extracted from the 80

membrane and incorporated into the base of growing filaments by the type IV pilus 81

biogenesis machinery, which is composed of several subcomplexes (11). The motor 82

subcomplex is composed of the membrane protein PilC and the cytoplasmic ATPases 83

PilB and PilT, which are responsible for pilus elongation and retraction, respectively (3, 84

12). In Gram-negative bacteria, the alignment subcomplex, which is composed of PilM, 85

PilN, PilO, and PilP, bridges the motor subcomplex and the outer membrane secretin 86

subcomplex, which is a gated pore for pilus assembly and disassembly (13, 14). The 87

final structural component of the type IV pilus system is the helical pilus filament, 88

which is composed of a major pilin subunit. In some bacteria, minor pilin subunits and 89

adhesins are also components of the filament (15). 90

Actinomycetes are high GC Gram-positive, mainly soil-inhabiting bacteria. 91

Many of them show filamentous growth and are often characterized by complex 92

morphological development (16-19). Streptomyces is the most representative genus 93

isolated with very high frequency, and actinomycetes (especially, filamentous 94

actinomycetes) other than genus Streptomyces are often called rare actinomycetes. 95

Members of the genus Actinoplanes are rare actinomycetes with the ability for 96

remarkable morphological development. They form a substrate mycelium from a 97

germinating spore and subsequently produce terminal sporangia growing from the 98

substrate mycelium through short sporangiophores (20). Terminal sporangia contain 99

flagellated spores and open up to release the spores in response to water. This process is 100

referred to as sporangium dehiscence (21, 22). Spores are termed zoospores after release 101

from the sporangia, because they can swim in aquatic environments and show 102

chemotactic properties toward various substances. When reaching a niche suitable for 103

vegetative growth, zoospores stop swimming and begin to germinate (23, 24). While 104

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they have such an interesting life cycle, molecular biological studies on their 105

morphological development have been very limited. 106

An extensively characterized species Actinoplanes missouriensis produces 107

terminal sporangia in a round shape when cultivated on HAT (humic acid-trace element) 108

agar. Each sporangium contains a few hundred spherical flagellated spores (25). 109

Recently, we revealed that the transcriptional regulator TcrA globally controls 110

sporangium formation, spore dormancy, and sporangium dehiscence in A. missouriensis 111

(26). TcrA is predominantly produced during sporangium formation and activates the 112

transcription of genes responsible for the developmental processes by binding to the 113

21-bp direct repeat sequence 5′-nnGCA(A/C)CCG-n4-GCA(A/C)CCGn-3′ (TcrA box; n, 114

any nucleotide). Based on comparative RNA sequencing (RNA-Seq) analysis between 115

wild-type and isogenic tcrA null (tcrA) mutant strains, we listed a total of 263 genes 116

whose transcription is down-regulated in the tcrA mutant (26). We found a gene 117

cluster consisting of nine genes that encode putative type IV pilus system components 118

in this TcrA-dependent gene’s list, which suggests that the zoospores of A. missouriensis 119

are piliated (26). To the best of our knowledge, pili are unprecedented in the cellular 120

biological studies of a zoospore. In this study, we genetically analyzed the pilus gene 121

cluster and functionally characterized A. missouriensis zoospore pili. 122

123

RESULTS 124

In silico analysis of the A. missouriensis type IV pilus gene cluster. Among the 263 125

genes whose transcript levels were down-regulated (over 4.0-fold) in the tcrA mutant, 126

we found nine consecutive genes, AMIS_8980 to AMIS_9060, that constitute a putative 127

pilus gene (pil) cluster. AMIS_9030 is annotated as pilA, which encodes a structural 128

component of pilus fiber, and the deduced amino acid sequences of five other gene 129

products are homologous to the type IV pilus system components in C. difficile (Table 130

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1). Thus, this gene cluster encodes sufficient proteins for assembling a functional type 131

IV pilus of Gram-positive bacteria: PilA (prepilin), PilB (ATPase for pilus elongation), 132

PilC (integral membrane protein required for pilus elongation), PilD (prepilin peptidase), 133

PilM and PilO (components of the alignment subcomplex), and PilT (ATPase for pilus 134

retraction). One of the remaining two genes, AMIS_9000, is predicted to encode a minor 135

pilin subunit (see Discussion). The function of the other gene product, AMIS_8980, is 136

unknown because this protein shows no significant homology with any characterized 137

proteins (Table 1). 138

The genome sequences of 16 Actinoplanes species including A. missouriensis 139

have been registered in the NCBI genome database with gene annotation 140

(https://www.ncbi.nlm.nih.gov/genome/). We searched for homologous genes of the pil 141

cluster using the A. missouriensis pil genes as queries, and found that all 16 142

Actinoplanes species have a very similar pilus gene cluster. The gene organization of the 143

pil cluster in Actinoplanes lutulentus is the same as that in A. missouriensis (Fig. 1). The 144

pil clusters of the other 14 Actinoplanes species have the same gene organization as the 145

A. missouriensis pil cluster except that all of them lack an AMIS_8980 ortholog. 146

Furthermore, gene clusters showing high homology with the A. missouriensis pil cluster 147

were found in other zoospore-forming rare actinomycetes. Couchioplanes caeruleus and 148

Catenuloplanes japonicus, both of which produce segmental motile spore, have a gene 149

cluster that is quite similar to the A. missouriensis pil cluster; they also lack an 150

AMIS_8980 ortholog (Fig. 1). Dactylosporangium aurantiacum, which produces motile 151

sporangiospore, also has a gene cluster showing high homology with the A. 152

missouriensis pil cluster; it has an additional gene between pilM and AMIS_9000 153

orthologs. Partially homologous gene clusters are also encoded on the actinobacteria 154

genomes of the members of the genera Planomonospora, Kineosporia, and 155

Spirillospora, all of which produce motile sporangiospore, and genera Kineococcus 156

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(motile cocci) and Angustibacter (non-motile cocci to rods). In contrast, no pil cluster 157

was found in Streptomyces species. These results indicate that the type IV pilus system 158

is evolutionarily conserved among zoospore-producing rare actinomycetes. 159

160

Transcriptional analysis of the pil cluster. Previously, we performed an RNA-Seq 161

analysis to obtain transcriptional profiles during sporangium formation using the total 162

RNA extracted from wild-type cells cultivated for 1, 3, 6, and 40 days on HAT agar (27). 163

On this agar plate, small sporangium-like structures were observed after 2 or 3 days of 164

cultivation. Then, mature sporangia that can release spores under dehiscence-inducing 165

conditions were formed after 5–7 days of incubation. Based on the results, the 166

transcriptional profile of the pil cluster is presented in Fig. 2. All genes of the pil cluster 167

were scarcely transcribed on day 1. However, transcription was significantly activated 168

on day 3. The transcript levels of the pilA- and pilT-containing operons increased 169

substantially with the analyzed time course just like group iv genes in the flagellar gene 170

cluster (27), while the transcripts of pilB were detected at nearly equal levels on days 3, 171

6, and 40 as for group iii genes in the flagellar gene cluster (27). In this way, both the 172

flagellar and pil gene clusters are actively transcribed during sporangium formation and 173

seem to be controlled by common regulatory mechanisms. This transcriptional profile 174

of pil cluster indicates that components for the pilus biogenesis are produced during 175

sporangium formation. Pilus structures are presumably assembled in the process of 176

sporangium formation. 177

RNA-Seq analysis also clarified the transcriptional units of the pil genes; the pil 178

cluster consists of three major transcriptional units, as shown in Fig. 2. While pilB is 179

transcribed as a monocistronic transcript, the other genes are transcribed as 180

polycistronic operons. We determined transcriptional start points in the pil cluster (Fig. 181

2). The transcriptional start site of pilB was determined to be the first nucleotide of the 182

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translational start codon using the 5-rapid amplification of cDNA ends (RACE) 183

procedure, whereas the transcriptional start points upstream from the pilT- and 184

pilA-containing operons were determined using high-resolution S1 nuclease mapping. 185

The transcriptional start points of the pilT-containing operon were 28 and 29 186

nucleotides upstream from the start codon of pilT, and those of the pilA-containing 187

operon were 229 and 231 nucleotides upstream from the start codon of pilA (Fig. S1 in 188

the supplemental material). Alignment of the promoter regions upstream from the 189

transcriptional start sites revealed that all of the pil genes shared similar promoter 190

elements that were in accord with the putative FliA-recognizing promoter, 191

5-CTCA-(n15-17)-GCCGA(A/T)-3 (26) (Fig. 3). This result suggests that a FliA-family 192

sigma factor(s) initiates the transcription of all genes in the pil cluster (see Discussion). 193

194

Observation of zoospore pili. To examine whether zoospores are piliated, a 195

transmission electron microscopic (TEM) analysis was performed to observe zoospores 196

released from sporangia of the wild-type strain by using a negative staining method for 197

flagellar observation (21, 27). At first, we were unable to observe any pili, while many 198

flagella were clearly observed. After repeated experiments, we obtained only one 199

picture that shows the presence of pilus filaments extending from the surface of a 200

zoospore (Fig. 4A–C). The pilus filaments are thinner (diameter, approximately 5 nm) 201

than flagellar filaments (approximately 13 nm) and appear to be somewhat linear 202

compared with the gently curved flagellar filaments (Fig. 4A–C). The extremely low 203

probability of pilus observation prompted us to examine a new method for the 204

observation of zoospore pili. Based on the assumption that zoospores should withdraw 205

their pili in response to physical contact with the grids for TEM observation, we fixed 206

zoospores with glutaraldehyde before putting them on the grids. This new method 207

drastically increased the frequency of pilus observation (up to 20% of the observed 208

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zoospores), and although the images were rather less clear (Fig. 4D and E), we used this 209

method hereafter in this study unless otherwise mentioned. 210

To clearly show that the observed thin filaments around the cell surfaces are 211

distinct from flagellar filaments, we observed zoospores of the flagellin-encoding fliC 212

deletion (fliC) mutant constructed in our previous study (27). TEM observation of the 213

non-motile fliC mutant zoospores revealed that they were not flagellated but piliated 214

(Fig. 5A and B). Furthermore, we constructed a pilA deletion (pilA) mutant to show 215

that the observed thin filaments are produced by the type IV pilus system encoded by 216

the pil cluster. The pilA mutant grew and formed sporangia normally, and the 217

sporangia released zoospores by normal dehiscence. We did not observe any pilus 218

structures on the pilA mutant zoospores with TEM analysis (Fig. 5C), confirming that 219

pilA encodes the prepilin subunit. The piliation of the zoospores was restored by the 220

introduction of the pilA gene with its own promoter region into the pilA mutant (Fig. 221

5D and E; this picture was taken by using the conventional method without 222

glutaraldehyde fixation). Next, we generated a pilT deletion (pilT) mutant to reduce 223

the frequency of the pilus retraction of the zoospores. The pilT mutant also formed 224

sporangia normally and the sporangia released zoospores under dehiscence-inducing 225

conditions. As expected, the pilT deletion improved the frequency of the pilus 226

observation; without glutaraldehyde treatment, we were able to observe piliated 227

zoospores with much higher frequency (more than 50%) in our TEM analysis (Fig. 5F 228

and G), indicating that the pilT gene product is responsible for the pilus retraction. 229

We counted the number of pili extending from each zoospore of the fliC mutant 230

in the electron microscopic images. Zoospores with five pili were most abundant; the 231

average (± standard deviation) was 6 ± 3 pili per zoospore (n = 37 piliated zoospores; 232

Fig. 5H). It should be noted that some zoospores were observed to be non-piliated 233

presumably owing to the retraction of pili and limitation of the method, and these 234

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apparently non-piliated zoospores were excluded from the calculation. The largest 235

number of pili observed on a zoospore was 13. In addition, we measured the length of 236

each pilus in the images using ImageJ (http://rsb.info.nih.gov/ij/) (28). The average (± 237

standard deviation) pilus length was 0.62 ± 0.35 m (n = 206; Fig. 5I). The length of the 238

longest pilus observed in the microscopic images was 2.49 m. Furthermore, we also 239

counted the number of pili of the pilT mutant zoospores in the TEM images without 240

glutaraldehyde fixation. Because the frequency of the pilus observation was improved 241

in the pilT mutant zoospores, non-piliated zoospores were included in the calculation. 242

The average number of pili (± standard deviation) was 3 ± 3 pili per zoospore (n = 21) 243

and the average (± standard deviation) pilus length was 0.45 ± 0.22 m (n = 69) (Fig. 244

5J). It should be noted that the pilT zoospores have both flagella and pili (see 245

Discussion). Interestingly, the pilus filaments appeared to extend from only a restricted 246

area of the zoospore surface (Fig. 5A; see Discussion). 247

248

Zoospore pili are required for adhesion to hydrophobic solid surfaces. As 249

mentioned in the introduction section, bacterial type IV pili have been reported to serve 250

fundamental functions in diverse cellular processes. In A. missouriensis, we 251

hypothesized that the zoospore pili have an important function in adhesion to solid 252

surfaces upon the cessation of swimming behavior before the onset of germination. 253

Thus, we analyzed the ability of zoospores to adhere to the surface of a plastic dish. The 254

average proportion (± standard deviation) of the zoospores that adhered to the dish 255

surface was 40.6% ± 2.5% in the wild-type strain (Fig. 6A). In the pilA mutant, 256

however, it was reduced to only 3.9% ± 1.0%, indicating that zoospore pili are required 257

for sufficient adhesion to the surface of the plastic dish under the test conditions (Fig. 258

6A). The proportion of the pilA mutant zoospores that adhered to the dish surface was 259

partially restored to 20.2% ± 1.7% by the introduction of the pilA gene with its own 260

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promoter region (Fig. 6A). 261

We also attempted to examine the ability of zoospores to adhere to the 262

hydrophilic glass surface, but the proportion of the wild-type zoospores that adhered to 263

the glass surface was only 7.2% ± 1.3% under the same conditions for the plastic dish 264

(Fig. 6B), suggesting that zoospores can adhere predominantly to hydrophobic solid 265

surfaces. We speculate that a small proportion of zoospores adhered to the glass surface 266

through flagella. To analyze the hypothesis that zoospores predominantly adhere to 267

hydrophobic solid surfaces, we further examined whether zoospores adhere to the 268

surface of a plastic dish treated with 1% (wt/vol) bovine serum albumin (BSA) solution. 269

As expected, only 0.2% ± 0.1% zoospores adhered to the BSA-treated dish surfaces 270

under the test conditions, indicating that the zoospore pili possess a much higher affinity 271

to hydrophobic solid surfaces than hydrophilic ones (Fig. 6B). 272

273

DISCUSSION 274

In this study, we successfully identified and characterized unprecedented 275

zoospore type IV pili of A. missouriensis. We also demonstrated the adhesion property 276

of the A. missouriensis zoospore to hydrophobic surfaces. The zoospore type IV pili 277

were indicated to play a pivotal role in the adhesion property of the zoospore. This 278

function is expected for bacterial type IV pili. However, when we consider the unique 279

features of the zoospore, our findings become more important. Zoospores of 280

filamentous actinomycetes are highly differentiated cells that aim for the rapid 281

expansion of their habitat; they can swim far away to seek niches and settle themselves 282

in the niches to grow as mycelia. Flagella and pili are required for swimming and solid 283

surface adhesion (i.e., the initiation of colonization), respectively, both of which are 284

very important in the biology of the zoospore. Therefore, to understand the physiology 285

of the zoospore, we should pay attention not only to flagella but also to pili. 286

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While type IV pilus filaments are composed of repeating units of the major pilin, 287

other proteins such as minor pilins and adhesins can be incorporated into the filament in 288

some cases. For example, the pilus filament of Pseudomonas aeruginosa is composed 289

of major (PilA) and several minor (PilE, PilV, PilW, PilX, and FimU) pilin subunits and 290

adhesion molecules such as PilY1 (29). One of the minor pilins is an initiator pilin that 291

forms the template upon which the polymerization of major pilins begins. The initiator 292

pilins lack a conserved Glu residue at position 6 in the recognition site for the prepilin 293

peptidase because this Glu residue forms a salt bridge with the N-terminus of the 294

previously incorporated pilin subunit and hence is not required for the first pilin subunit. 295

Furthermore, the initiator pilins are larger in size than their cognate major pilins (15). In 296

the pil cluster of A. missouriensis, AMIS_9000 encodes a protein consistent with these 297

two features, suggesting that the gene product is the initiator pilin located at the tip of 298

the pilus filament. For AMIS_8980, the precise function of the gene product remains 299

elusive. Considering that homologs of AMIS_8980 are not found in the type IV pilus 300

gene clusters of most Actinoplanes species and many other rare actinomycetes, we 301

postulate that the gene product is not required for the pilus biogenesis. 302

Putative FliA-recognizing promoters were identified in the upstream regions 303

from all three transcriptional start points of the pil genes (Fig. 3). In a previous study, 304

we revealed that TcrA activates transcription of the genes involved in sporangium 305

formation, spore dormancy, sporangium dehiscence, flagellar biogenesis, and 306

chemotaxis (26). Considering that three FliA-family sigma factors, FliA1, FliA2, and 307

FliA3, are under transcriptional control of TcrA, we predict that one or more of these 308

sigma factors are responsible for transcription in the pil cluster, leading to lower 309

transcript levels of the pil genes in the tcrA mutant compared with the wild-type strain 310

(26). Detailed functional analysis of the FliA-family sigma factors is in progress and 311

will be published elsewhere. 312

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In the adhesion test, the proportion of adhesive zoospores of the pilA 313

complemented strain was not fully restored to that of the wild-type strain (Fig. 6A). We 314

speculate that the transcription level of pilA in the pilA complemented strain is lower 315

than that in the wild-type strain and that this explains the partial restoration of adhesion 316

activity. The transcriptional profile of the pil cluster indicates that pilA is transcribed not 317

only from its own promoter but also as a read-through from the pilT-containing operon 318

(Fig. 2). However, the pilA gene is transcribed only from its own promoter in the pilA 319

complemented strain, which probably results in an insufficient transcript level of pilA. 320

In the pilT zoospore, the frequency of the pilus retraction was greatly reduced; 321

we could observe piliated zoospores of the pilT mutant without glutaraldehyde 322

treatment with high frequency (more than 50%). This enabled us to count the number of 323

pili and their length in the flagellated zoospores, because it was really difficult to 324

observe pili on the flagellated wild-type zoospore with glutaraldehyde fixation, which 325

reduced clearness of the TEM images. The numbers of pili per pilated zoospore and the 326

average length of observed pili were not so varied between non-flagellated (fliC) and 327

flagellated (pilT) zoospores. Apparently, this result suggests no link between 328

flagellation and piliation. However, it may be somewhat strange that only the frequency 329

of pilus observation was greatly improved and hyper piliation (in number and/or length) 330

was not induced by the deletion of pilT. Further investigation of the possible link 331

between flagellation and piliation, including their specific location (see below), is our 332

future research subject. 333

We previously reported that flagellar formation was observed in a restricted area 334

of the zoospore surface in A. missouriensis (27). In this study, pilus filaments were also 335

often observed in a restricted area of the zoospore surfaces. Interestingly, some TEM 336

images indicate that flagella and pili are extended from areas that are opposite each 337

other (Fig. 4D and E). The molecular mechanism and physiological role of the specific 338

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localization of flagella and pili remain elusive. However, we think that possible 339

different localization of flagella and pili is very reasonable, because pili would be an 340

obstacle for flagellar rotation if the pili and flagella extended from the same region. Our 341

ongoing studies on the zoospore’s swimming behavior are also important for 342

understanding the zoospore biology. We believe that our multilateral analyses of the A. 343

missouriensis zoospore are revealing the molecular mechanisms of the species’ 344

characteristic survival strategy. 345

346

MATERIALS AND METHODS 347

Bacterial strains, plasmids, media, and primers. A. missouriensis 431T (NBRC 348

102363T) was obtained from the National Institute of Technology and Evaluation (NITE, 349

Chiba, Japan). A. missouriensis was grown on YBNM or HAT agar at 30°C for a solid 350

culture and in PYM broth at 30°C for a liquid culture, as previously described (30). MS 351

(2% soy flour and 2% mannitol) or modified ISP4 (ISP medium 4 [Difco] supplemented 352

with 0.05% yeast extract [Difco] and 0.1% tryptone [Difco]) agar medium was used for 353

transformation by conjugation with E. coli ET12567 (pUZ8002). MS and modified 354

ISP4 agar media were supplemented with MgCl2·6H2O at a final concentration of 40 355

mM. MS agar was used for the construction of the recombinant strain for the 356

complementation test, and modified ISP4 agar was used for the construction of the 357

pilA mutant. E. coli ET12567 (pUZ8002) was obtained from the John Innes Centre 358

(Norwich, UK) and used as the donor in intergeneric conjugation. E. coli JM109 and 359

pUC19 were purchased from Takara Biochemicals (Shiga, Japan). The media and 360

growth conditions for E. coli were as described by Maniatis et al. (31). Apramycin 361

(50 µg/mL), spectinomycin (50 µg/mL), and ampicillin (50 µg/mL) were added when 362

necessary. The primers used in this study are listed in Table S1 in the supplemental 363

material. 364

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365

RNA extraction. A. missouriensis cells for RNA extraction were prepared as previously 366

described (26). Cells were disrupted by rubbing with a mortar and pestle, and the cell 367

lysate was mixed with the lysis/binding solution of the RNAqueousTM Total RNA 368

Isolation Kit (Thermo Fisher Scientific, MA, USA). After the debris was removed by 369

centrifugation at 21,000 × g for 5 min, total RNAs were extracted according to the 370

manufacturer’s instructions. The total RNAs were treated with DNase I to eliminate 371

contaminating genomic DNA and purified by phenol-chloroform extraction and ethanol 372

precipitation. 373

374

S1 nuclease mapping. S1 nuclease mapping was performed using a method described 375

by Bibb et al. (32) and Kelemen et al. (33). Hybridization probes were prepared using 376

PCR and labeled at both 5-ends with [-32P]-ATP (220 TBq/mmol) using T4 377

polynucleotide kinase. Labeling at one side of the 5-ends was eliminated by restriction 378

enzyme digestion. For hybridization, 40 g of total RNA was used. Protected fragments 379

were analyzed on 6% polyacrylamide DNA sequencing gels according to the method of 380

Maxam and Gilbert (34). 381

382

5-RACE. Mapping of the 5-end was carried out using a Full RACE Core Set (Takara 383

Biochemicals) according to the manufacturer’s instructions. The PCR products were 384

cloned into pUC19 and sequenced by FASMAC (Kanagawa, Japan). 385

386

TEM observation. Zoospores were released from the sporangia by pouring 10 mL of 387

25 mM NH4HCO3 onto one HAT plate and incubating the plate at 30°C for 1 h. After 388

being collected from the plate, zoospores were observed by TEM according to the 389

method reported previously (21, 27). In this method, the zoospore samples were 390

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negatively stained with 1% (wt/vol) phosphotungstic acid (pH 7.0) and observed with 391

the electron microscope JEM-1010 (Jeol, Tokyo, Japan) using the formvar-coated 392

copper grids. As depicted in Fig. 4D and E and Fig. 5A-C, we used a new method as 393

follows. The zoospore samples were incubated in 1% (wt/vol) glutaraldehyde solution 394

for a few minutes at room temperature. These samples were centrifuged, and the 395

supernatants were removed. The zoospore pellets were resuspended in 25 mM 396

NH4HCO3. The samples were negatively stained with 2% (wt/vol) ammonium 397

molybdate and observed with the electron microscope JEM-1400 using the 398

carbon-coated copper grids, as described previously (35). 399

400

Construction of the pilA and pilT mutant strains. For construction of pilA and 401

pilT mutant strains, 2.5-kbp upstream and downstream regions of pilA and pilT were 402

amplified by PCR. The amplified DNA fragments for pilA were cloned into pUC19 403

digested with XbaI and HindIII using an In-Fusion HD cloning kit (Takara 404

Biochemicals) according to the manufacturer’s instructions, generating pUC19-pilA. 405

The amplified upstream and downstream fragments for pilT were digested with EcoRI 406

plus XbaI and XbaI plus HindIII, respectively, and cloned together into pUC19 digested 407

with EcoRI and HindIII, generating pUC19-pilT. Plasmids pUC19-pilA and 408

pUC19-pilT were sequenced to confirm that no PCR-derived error was introduced. 409

Then pUC19-pilA and pUC19-pilT were digested with XbaI plus HindIII and EcoRI 410

plus HindIII, respectively, and the insert fragments were cloned into pK19mobsacB 411

digested with the same restriction enzymes, whose kanamycin resistance gene had been 412

replaced with the apramycin resistance gene aac(3)IV (30), generating 413

pK19mobsacB-pilA and pK19mobsacB-pilT. Plasmids pK19mobsacB-pilA and 414

pK19mobsacB-pilT were introduced into A. missouriensis by conjugation as described 415

previously (27). Apramycin-resistant colonies resulting from a single crossover 416

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recombination were isolated. One of them was cultivated in PYM liquid medium for 2 417

days, and the mycelia suspended in 0.75% NaCl solution were spread onto Czapek-Dox 418

Broth agar medium (BD, NJ, USA) containing extra sucrose (final concentration; 5%). 419

After incubation at 30°C for 5–7 days, the sucrose-resistant colonies were inoculated 420

onto YBNM agar with or without apramycin to confirm that they were sensitive to 421

apramycin. The apramycin-sensitive and sucrose-resistant colonies resulting from the 422

second crossover recombination were isolated as candidates for the pilA and pilT 423

mutant strains. The disruption of pilA and pilT was confirmed by PCR (data not shown). 424

425

Construction of the recombinant strain for complementation test. A 0.9-kbp DNA 426

fragment containing the promoter and coding sequences of pilA was amplified by PCR. 427

The amplified fragment was cloned into pTYM19-Apra (26) digested with EcoRI and 428

HindIII using the In-Fusion HD cloning kit, resulting in pTYM19-Apra-pilA. Plasmid 429

pTYM19-Apra-pilA was sequenced to confirm that there was no PCR-derived error and 430

was then introduced into the pilA mutant by conjugation as described previously (27). 431

Apramycin-resistant colonies were obtained. 432

433

Zoospore adhesion test. A cover glass was put on a plastic (polystyrene) dish (IWAKI 434

#1000-035, AGC, Shizuoka, Japan) using two narrow double-sided tapes that were 435

arranged at the both side edges as parallel lines to seal the edges, and a 436

zoospore-containing solution (approximately 104 cells/l) was poured into the space 437

between the dish and cover glass. After 10 min, the whole zoospores were photographed 438

with a high-speed camera by scanning the microscopic fields along the vertical direction. 439

Then, to remove zoospores that did not adhere to the solid surfaces, 25 mM NH4HCO3 440

was poured into the chamber from one side, and the overflowed solution was absorbed 441

with a paper filter on the other side. Then the images of the zoospores that adhered to 442

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the dish surface were recorded. From the microscopic images, the adhesion ratios 443

(proportions of adhesive zoospores to whole zoospores) were calculated. The numbers 444

of whole zoospores in the solution were counted using the Color_Footprint plugin for 445

ImageJ (36). A glass dish (IWAKI #3970-035, AGC) was used for the adhesion test to 446

the hydrophilic glass surface. For the BSA coating, the plastic dish was treated with 1% 447

(wt/vol) BSA for a few minutes at room temperature and washed with 25 mM 448

NH4HCO3 twice. To increase the viscosity of the solution, zoospores were suspended in 449

the 25 mM NH4HCO3 solution containing 10% (wt/vol) polyethylene glycol (PEG6000, 450

Sigma-Aldrich, MO, USA) or 0.1% (wt/vol) methylcellulose (M0512-100G, 451

Sigma-Aldrich). 452

453

Optical Microscopy. Cells were visualized under a phase-contrast microscope (IX73, 454

Olympus, Tokyo, Japan) equipped with an objective lens (LUCPLFLN 20×PH, 455

Olympus), a CMOS camera (DMK33UX174, Imaging Source, Bremen, Germany), and 456

an optical table (HAX-0605, JVI, Shizuoka, Japan). For high-speed imaging, a 457

lab-recorder system (LRH1540, Digimo, Tokyo, Japan) was used at a speed of 200 458

frames per second. The cell images were captured as 8-bit images and converted into 459

TIF files without compression. All data were analyzed using ImageJ and its plugins. 460

461

ACKNOWLEDGMENTS 462

This research was supported in part by Grants-in-Aid for Scientific Research (A) 463

(26252010 to Y.O.), (B) (18H02122 to Y.O.), and (C) (17K07711 to T.T.), Grants-in-Aid 464

for Young Scientists (A) (16H06230 to D.N.) and (B) (15K18669 to T.T.), and 465

Grant-in-Aid for JSPS Research Fellow (15J07768 to T.K.) from Japan Society for the 466

Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science, 467

and Technology of Japan (MEXT). 468

469

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Burrows LL. 2015. Pseudomonas aeruginosa minor pilins prime type IVa pilus 571

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30. Mouri Y, Konishi K, Fujita A, Tezuka T, Ohnishi Y. 2017. Regulation of sporangium 575

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31. Maniatis T, Fritsch EF, Sambrook J. 1982. Molecular Cloning: A Laboratory Manual. 579

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32. Bibb MJ, Janssen GR, Ward JM. 1985. Cloning and analysis of the promoter region 582

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33. Kelemen GH, Brian P, Flärdh K, Chamberlin L, Chater KF, Buttner MJ. 1998. 586

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spore pigment in Streptomyces coelicolor A3(2). J Bacteriol 180:2515-2521. 588

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34. Maxam AM, Gilbert W. 1980. Sequencing end-labeled DNA with base-specific 590

chemical cleavages. Methods Enzymol 65:499-560. 591

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35. Nakane D, Nishizaka T. 2017. Asymmetric distribution of type IV pili triggered by 593

directional light in unicellular cyanobacteria. Proc Natl Acad Sci U S A 594

114:6593-6598. 595

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bacteria. Proc Natl Acad Sci U S A 103:13618-13623. 598

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Figure legends 599

Fig. 1. Gene organization of the pil clusters in A. missouriensis (A), A. lutulentus (B), 600

and C. japonicus (C). Arrows indicate the locations of the open reading frames, 601

including their length and direction. Gene identifiers (IDs) and names are shown above 602

and below the arrows, respectively. Gene names are not shown when the functions of 603

the gene products are unknown. 604

605

Fig. 2. Transcriptional profile of the pil cluster during sporangium formation. 606

Distributions of the mapped RNA-Seq read counts in the 1-, 3-, 6-, and 40-day cultures 607

are denoted by different colored lines. All genes of the cluster were hardly transcribed 608

on day 1. Bold arrows indicate open reading frames. An open arrowhead indicates a 609

transfer RNA gene. The read counts mapped to the transfer RNA gene were eliminated 610

from the profile. The transcriptional start points are shown by bent arrows. The three 611

major transcriptional units are shown by light blue arrows. 612

613

Fig. 3. Sequence alignment of promoter regions upstream from the transcriptional start 614

points in the pil cluster. The first genes downstream of each transcriptional start site are 615

shown on the left side. The putative FliA-recognizing promoter element is shown below 616

the alignment. Conserved promoter elements are shaded. The transcriptional start points 617

are shown with bent arrows. n, any nucleotide; W, A or T. 618

619

Fig. 4. Observation of the wild-type zoospores by TEM. (B), (C), and (E) are enlarged 620

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views of a portion of panels (A), (B), and (D), respectively. Arrowheads indicate the 621

pilus filaments. The thick filaments are flagella. A conventional method for flagellar 622

observation (21, 27) was used for (A), (B), and (C), while a new method was used for 623

(D) and (E). Scale bars = 500 nm. 624

625

Fig. 5. Observation of the fliC (A, B), pilA (C), pilA-complemented pilA/pilA+ (D, 626

E), and pilT (F, G) zoospores by TEM, and numbers and lengths of pili in the fliC (H, 627

I) and pilT (J) mutants. The pilA/pilA+ strain harbors the pilA complementation 628

plasmid on the chromosome. (B), (E), and (G) are enlarged views of a portion of panels 629

(A), (D), and (F), respectively. Arrowheads indicate pilus filaments. The thick filaments 630

in panels (C) to (G) are flagella. Scale bars = 500 nm. (H) Distribution of pilus number 631

per piliated fliC mutant zoospore (n = 37). (I) Distribution of length of fliC zoospore 632

pili (n = 206). (J) Distribution of pilus number per piliated or apparently non-piliated 633

pilT mutant zoospore (n = 21) and distribution of length of pilT zoospore pili (n = 634

69). 635

636

Fig. 6. Zoospore adhesion to solid surfaces. (A) Proportion of adhesive zoospores to the 637

hydrophobic plastic (polystyrene) surface. Data are the mean values from three 638

biological replicates ± standard deviations. Microscopic images of the adhesive 639

zoospores are shown below the graph. Scale bars = 10 m. (B) Proportion of adhesive 640

zoospores of the wild-type strain to the hydrophilic glass surface and BSA-coated 641

hydrophilic plastic surface. 642

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Table 1. Genes in the pil cluster in A. missouriensis643

Gene ID 644

(AMIS no.) 645

8980 646

8990 647

9000 648

9010 649

9020 650

9030 651

9040 652

9050 653

9060 654

Length 655

(aa) 656

104 657

204 658

232 659

341 660

259 661

158 662

410 663

372 664

563 665

Gene 666

product 667

- 668

PilOb 669

- 670

PilM 671

PilD 672

PilA 673

PilC 674

PilT 675

PilB 676

Putative function 677

678

Unknowna 679

Component of alignment subcomplex 680

Unknowna 681

Component of alignment subcomplex 682

Prepilin peptidase 683

Prepilin 684

Component of motor subcomplex 685

ATPase for pilus retraction 686

ATPase for pilus elongation 687

Homologue in 688

C. difficile 689

- 690

- 691

- 692

CD630_32930 693

CD630_35040 694

CD630_32940 695

CD630_35110 696

CD630_35050 697

CD630_32960 698

Identity/ 699

similarity (%) 700

- 701

- 702

- 703

13/36 704

28/55 705

33/55 706

26/56 707

47/70 708

37/62709

a These proteins exhibit no significant sequence homology to any characterized proteins. 710

b The gene product is homologous to PilO in Neiserria meningiditis (17% and 45% in identity and similarity, respectively). There is 711

no PilO homologue in C. difficile R20291. 712

713

714

715

716

717

718

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719 a These proteins exhibit no significant sequence homology to any characterized proteins. 720

b The gene product is homologous to PilO in Neiserria meningiditis (17% and 45% in identity and similarity, respectively). There is 721

no PilO homologue in C. difficile R20291. 722

723

724

1

Table 1. Genes in the pil cluster in A. missouriensis

Gene ID

(AMIS no.)

8980

8990

9000

9010

9020

9030

9040

9050

9060

Length

(aa)

104

204

232

341

259

158

410

372

563

Gene

product

-

PilOb

-

PilM

PilD

PilA

PilC

PilT

PilB

Putative function

Unknowna

Component of alignment subcomplex

Unknowna

Component of alignment subcomplex

Prepilin peptidase

Prepilin

Component of motor subcomplex

ATPase for pilus retraction

ATPase for pilus elongation

Homologue in

C. difficile

-

-

-

CD630_32930

CD630_35040

CD630_32940

CD630_35110

CD630_35050

CD630_32960

Identity/

similarity (%)

-

-

-

13/36

28/55

33/55

26/56

47/70

37/62

a These proteins exhibit no significant sequence homology to any characterized proteins.

b The gene product is homologous to PilO in Neiserria meningiditis (17% and 45% in identity and similarity, respectively). There is

no PilO homologue in C. difficile R20291.

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725

726

Fig. 1. Gene organization of the pil clusters in A. missouriensis (A), A. lutulentus (B), 727

and C. japonicus (C). Arrows indicate the locations of the open reading frames, 728

including their length and direction. Gene identifiers (IDs) and names are shown above 729

and below the arrows, respectively. Gene names are not shown when the functions of 730

the gene products are unknown. 731

732

733

734

735

736

737 738

Fig. 2. Transcriptional profile of the pil cluster during sporangium formation. 739

Distributions of the mapped RNA-Seq read counts in the 1-, 3-, 6-, and 40-day cultures 740

are denoted by different colored lines. All genes of the cluster were hardly transcribed 741

on day 1. Bold arrows indicate open reading frames. An open arrowhead indicates a 742

transfer RNA gene. The read counts mapped to the transfer RNA gene were eliminated 743

from the profile. The transcriptional start points are shown by bent arrows. The three 744

major transcriptional units are shown by light blue arrows. 745

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746

Fig. 3. Sequence alignment of promoter regions upstream from the transcriptional start 747

points in the pil cluster. The first genes downstream of each transcriptional start site are 748

shown on the left side. The putative FliA-recognizing promoter element is shown below 749

the alignment. Conserved promoter elements are shaded. The transcriptional start points 750

are shown with bent arrows. n, any nucleotide; W, A or T. 751

752

753

754

755

756

Fig. 4. Observation of the wild-type zoospores by TEM. (B), (C), and (E) are enlarged 757

views of a portion of panels (A), (B), and (D), respectively. Arrowheads indicate the 758

pilus filaments. The thick filaments are flagella. A conventional method for flagellar 759

observation (21, 27) was used for (A), (B), and (C), while a new method was used for 760

(D) and (E). Scale bars = 500 nm. 761

762

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763

Fig. 5. Observation of the fliC (A, B), pilA (C), pilA-complemented pilA/pilA+ (D, 764

E), and pilT (F, G) zoospores by TEM, and numbers and lengths of pili in the fliC (H, 765

I) and pilT (J) mutants. The pilA/pilA+ strain harbors the pilA complementation 766

plasmid on the chromosome. (B), (E), and (G) are enlarged views of a portion of panels 767

(A), (D), and (F), respectively. Arrowheads indicate pilus filaments. The thick filaments 768

in panels (C) to (G) are flagella. Scale bars = 500 nm. (H) Distribution of pilus number 769

per piliated fliC mutant zoospore (n = 37). (I) Distribution of length of fliC zoospore 770

pili (n = 206). (J) Distribution of pilus number per piliated or apparently non-piliated 771

pilT mutant zoospore (n = 21) and distribution of length of pilT zoospore pili (n = 772

69). 773

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774

775

776

Fig. 6. Zoospore adhesion to solid surfaces. (A) Proportion of adhesive zoospores to the 777

hydrophobic plastic (polystyrene) surface. Data are the mean values from three 778

biological replicates ± standard deviations. Microscopic images of the adhesive 779

zoospores are shown below the graph. Scale bars = 10 m. (B) Proportion of adhesive 780

zoospores of the wild-type strain to the hydrophilic glass surface and BSA-coated 781

hydrophilic plastic surface. 782

783

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500 bp(A) Actinoplanes missouriensis

8980 8990 9000 9010 9020 9030 9040 9050 9060

pilO pilM pilD pilA pilC pilT pilB

(B) Actinoplanes lutulentus

8180 8175 8170 8165 8160 8155 8150 8145 8140


(C) Catunuloplanes japonicus

20500 20495 20490 20485 20480 20475 20470 20465


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180,000

Cov

erag

e

0

Day 3Day 6 Day 40

Day 1

500 bp

8980 9000pilO pilM pilD pilA pilC pilT pilB 9070

Transcriptional unit

tRNA-Ala

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pilA

pilB

pilT

sFliA-recognizing promoter `q`^ JJJJJEå NRJNTFJJJJ d``d^t

qd^^d^^^d`dd``^d^q`^ dq`^ d^qd`qdd^ `^^ddqq d``d^ ` qq`q^`d^`^d^

``q^q`d``dd^`dqq``dd ` ^`^ `ddddqd^` q`^d`^^ q``d dqd`d^`dq``d^

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A

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B

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C

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D E

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A B C

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F GD E

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0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14

(H)

Fre

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Number of pili per zoospore<

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Fre

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Length (mm)0 0.5 1 1.5 2 2.5

1

2

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4

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Number of pili per zoospore

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5

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15

20

25

30

35

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2

3

4

5

6

7

8

9

10

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40

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