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Gas vesicles in actinomycetes Keulen, Geertje van; Hopwood, David A.; Dijkhuizen, Lubbert; Sawers, R. Gary

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DOI: 10.1016/j.tim.2005.06.006

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Citation for published version (APA): Keulen, G. V., Hopwood, D. A., Dijkhuizen, L., & Sawers, R. G. (2005). Gas vesicles in actinomycetes: old buoys in novel habitats? Trends in Microbiology, 13(8), 350 - 354. https://doi.org/10.1016/j.tim.2005.06.006

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Genome Analysis Gas vesicles in actinomycetes: old buoys in novel habitats?

Geertje van Keulen1,2, David A. Hopwood1, Lubbert Dijkhuizen2 and R. Gary Sawers1

1Department of Molecular Microbiology, John Innes Centre, Norwich Research Park Norwich, UK 2Microbial Physiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands

Gas vesicles are gas-filled prokaryotic organelles that experiments indicated that 13 gvp genes are essential for function as flotation devices. This enables planktonic wild-type gas vesicle formation in halophilic [3], and halophilic archaea to position them- whereas the findings of a more recent study suggest that selves within the water column to make optimal use of eight genes, gvpFGJKLM-gvpAO, suffice [4]. GvpA repre- light and nutrients. Few terrestrial microbes are known sents the major envelope component. Two sets of to contain gas vesicles. Genome sequences that have paralogous proteins, the GvpA paralogues GvpJ and become available recently for many from non- GvpM and the paralogous GvpF and GvpL proteins, are planktonic habitats reveal gas vesicle gene clusters in also found in archaeal gas vesicles [2]. The function of members of the actinomycete genera Streptomyces, GvpG, -K and -O is still unclear; however, GvpC provides Frankia and Rhodococcus, which typically live in soils rigidity to the vesicle but is not essential for vesicle and sediments. Remarkably, there is an additional level of integrity. Moreover, GvpC has been suggested to shape gas complexity in cluster number and gene content. Here, we vesicles [5–8]. discuss whether putative gas vesicle proteins in these Intriguingly, orthologues of the eight essential gvp actinomycetes might actually be involved in flotation or genes occur in duplicate or even triplicate in the genomes whether they might fulfil other cellular functions. of the saprophytic soil bacteria Streptomyces coelicolor and Streptomyces avermitilis (Figure 1) [4,9,10]. These organisms are developmentally complex, mycelial Introduction members of the actinomycetes, which are high-GC Gas vesicles are gas-filled organelles that have been Gram-positive bacteria with a remarkable capacity to studied extensively in cyanobacteria and halophilic produce a vast array of secondary metabolites, including archaea (for a review see [1]). The gas vesicle envelope many antibiotics. The unexpected discovery of gvp gene consists of an amphipathic membrane with a clusters in the genomes of several actinomycetes raises ribbed ultrastructure, and comprises minimally seven interesting questions concerning their functions. proteins [2]. These gas vesicle proteins (Gvps) are encoded by a cluster of gvp genes (Figure 1). Early gene disruption gvp gene clusters in Streptomyces species

Corresponding author: van Keulen, G. ([email protected]). The gvp clusters of S. coelicolor and S. avermitilis have a Available online 29 June 2005 similar gene order: gvpO, A, F, G, J, L, S, K (Figure 1). In www.sciencedirect.com Update TRENDS in Microbiology Vol.13 No.8 August 2005 351

Gas vesicle genes in other actinomycetes Archaea Several other soil actinomycetes also contain gvp clusters Halobacterium sp. NRC-1 pNRC200 MLKJIHGFEDACNO (Figure 1). Whether these organisms actually synthesize Halobacteriumsp. NRC-1 pNRC100 MLKJIHGFEDACNO gas vesicles remains to be established; nevertheless, the Halobacterium mediterranei c-vac MLKJIHGFEDACNO filamentous erythromycin-producer Saccharopolyspora Natronobacterium vacuolatum EDACNOFGH erythraea (P. Leadlay, personal communication) and the Methanosarcina barkeri AAAFGJKLM filamentous balhimycin-producer Amycolatopsis balhimy- Cyanobacteria cina (W. Wohlleben and T. Weber, personal communi- Anabaena flos-aquae AACNJKF cation) both have at least one gvp cluster. The unicellular Nostoc sp.ATCC29413 ABCNJKFG animal pathogen Rhodococcus equi (http://www.sanger.ac. uk/Projects/R_equi/) and the filamentous PCB-degrader, Pseudoanabaena sp.PCC6901 ACNJ Rhodococcus sp. RHA1 (http://www.rhodococcus.ca/), both Microcystis aeruginosa AAACNJXKFGVW contain a gvp cluster. Perhaps most surprising is the Trichodesmium erythraeum AAAJKJGF discovery of gvp genes in Frankia spp., which are Actinomycetes filamentous N2-fixing actinomycetes that live symbioti- Streptomyces coelicolor gvp1 OAFGYZJLSK cally in actinorhizal root nodules of shrubs and trees Streptomyces coelicolor gvp2 OAFGYZJLSK (P. Normand, personal communication; http://genome.jgi- Streptomyces avermitilis gvp1 OAFG JLSK psf.org/mic_home.html). Interestingly, Frankia sp. Ccl3, Streptomyces avermitilis gvp2 OAFGYZJLSK with the smallest genome and only a limited host-range, Streptomyces avermitilis gvp3 OAFGYZJLSK also contains the smallest gvp gene cluster, comprising only gvpAF (Figure 1). It is noteworthy that the sequenced Streptomyces scabies gvp1 OAFGYZJLSK mycobacteria and corynebacteria, with smaller genomes Streptomyces scabies gvp2 OAFGYZJSK than most other actinomycetes, lack gvp genes (http:// Streptomyces peucetius gvp1 OAFGJLK www.ncbi.nlm.nih.gov/; http://www.sanger.ac.uk/Projects/ Streptomyces peucetius gvp2 YZOAFJLSK Pathogens/). Streptomyces diversea TM gvp1 OAFGYZJLSK Streptomyces diversa TM gvp2 YZOAFGJLSK Saccharopolyspora erythraea YZOAFGJLSK Actinomycete GvpA proteins have a C-terminal extension Frankia alni OJLSKAFYZ An amino acid sequence alignment of various putative Frankiasp.EAN1pec OJLSKAFYZ GvpA proteins revealed a long C-terminal extension on Frankiasp.CcI3 AF actinomycete GvpAs but not on other GvpAs (Figure 2). Rhodococcus sp.RHA1gvp1 YZAFGOJLSK This extension almost doubles the length of actinomycete Rhodococcus sp.RHA1 gvp2 YZAFOJ GvpA proteins and does not resemble other known Rhodococcus equi YZAFGOJLSK sequences. Moreover, this additional domain has extra- Other bacteria ordinarily high levels of glutamate and arginine, and to a Bacillus megaterium APQBRNFGLSKJTU lesser extent proline, which are often present in long, alternating acidic and basic tracts. Epitope display Rhodobacter sphaeroides AKGFJ experiments imply that the C terminus of GvpA might Ancylobacter aquaticus ACN be situated on the cytosolic side of the gas vesicle in

Figure 1. Organization of gvp gene clusters. Each letter identifies a gvp gene. Halobacterium sp. NRC-1 [11,12] and the unusual proper- Orthologous genes, and paralogues in the same cluster, are in identical colours. ties of actinomycete C-terminal GvpA could hint towards Transcription direction is indicated by arrows. alternative functions for gas vesicles in actinomycetes. The N-terminal half of actinomycete GvpAs aligns well four of these five clusters, two novel genes – gvpY and Z –lie with the full-length GvpAs of non-actinomycetes. This between gvpG and J,whereas,ingvp1 of S. avermitilis,these enabled construction of an unrooted phylogenetic tree are replaced by a single open reading frame (ORF). None of (Figure 3). Cyanobacterial, archaeal and actinomycete the clusters encodes GvpC, which is required for gas vesicles GvpAs each form a distinct clade. GvpA of Streptomyces to resist higher pressures, particularly in deep lakes [5]. and Frankia spp. formed separate subclades within the Recently, genome-sequencing projects have revealed actinomycete clade. GvpA2 of S. coelicolor and GvpA3 of pairs of gvp clusters in three other Streptomyces species: S. avermitilis formed a deep-branching group within the the plant pathogen S. scabies (http://www.sanger.ac.uk/ Streptomyces sub-clade. Surprisingly, GvpA of Sac. Projects/S_scabies); the adriamycin-producer S. peucetius erythraea displayed particularly deep-branching, group- (J.K. Sohng, personal communication); and ‘S. diversae’, ing most closely with GvpA of rhodococci as a third a derivative of S. venezuelae engineered for enhanced subclade. secondary metabolism (M.J. Bibb, personal communi- The separate actinomycete GvpA branch indicates that cation). Of these, gvp1 in S. scabies and ‘S. diversae’ these gvp genes were not acquired by lateral gene transfer. resembles the S. coelicolor and S. avermitilis clusters, Furthermore, a global comparison of the genomes of whereas the others differ in the presence and position of S. coelicolor and Frankia alni (P. Normand, personal gvpYZ or in lacking gvpL (S. scabies gvp2), gvpS communication) showed significant overall synteny (S. peucetius gvp1)orgvpG (S. peucetius gvp2). including gvp of F. alni and gvp1 of S. coelicolor. This www.sciencedirect.com 352 Update TRENDS in Microbiology Vol.13 No.8 August 2005

N Cyanobacteria C 71Ð72 aa

N B. megaterium/Rb. sphaeroides/A. aquaticus/T. pendens C 66Ð86 aa

N Archaea C 76Ð79 aa

N Actinomycetes C 124Ð170 aa

SCO6500 R L D LE AG P RK D --- PG LP D LV G E MT ESG A RG KS KG AL SG AA ET IS D AF KQ A ------R DD GGS E R E ------TSS R P R A RK AA ------PS RRK EE Q E - 144 SAV1889 R L D LE SG PH KS --- PG LP D LV G D IT ESG A RG KS KG AL SG AA ET IS D AF KQS ------R EE GQS E ------S R P R A RK ST ------AS RRK EE Q E - 141 SCABGVPA1 R LD LE AG P RK D --- PG LP D LV G E LT ESG A RG KS KG AL SG AA ET IS D AF KQS ------R EE SQS E ------S R P R A RR TT ------S A RKK EE Q E - 141 SP529 R L D LE AGN RK D --- PG LP D LV G E IT ESG A RG KS KG ALA G AA QT IS D AF GGG ------H DEE K E ------SG P R A RR SS ------GS RRK EE Q E - 140 SCABGVPA2 R LD LE SG RK AP --- SQ LT D IV GNT V ESG A KG KS KG AL TG AV E AV T DS LH KG --- RG DD -- DD R ES ------G R E RQ R V RS E R ----- G AS RR SS RE REE --- 152 SAV598 R L D LE SG RK AP --- AQ LT D LV G E VT EGG A KG KS KG AL TG AV E AV T ES FHQG --- H EED R EEE R E PAA ------E RKR P R PT RR P ------A RRR EE ---- 149 SP8137 R L D LE SG RK AP --- TQ LT D IV G DT V ESG AHG KS KG AL GG AM E AV T ES LTGGG KS KR DE S DEE A EE K E ------PV E RRR PA RR A ------T RR E R E RGG E 156 SMD10157 R L D LE AG RK AP --- AQ LP E VV GG ML ED G AHG KS KG AL SG AV E AV T ES LSG KRR G DDEE P EEE Q EE P EEEE A E APVP RRRR PAA RR S ------A RR E KE ---- 161 SMD00942 R L D LE SG RK AP --- TQ LT D IV G E VT ESG A KG KS KG AL TG AV E AF T DS LQS K ---- R EE S EEE P RKR P ------A RK STSG RTS ------AT RR EE ---- 145 RR3717 R L E IGS - E P KG - LS D LV G ---- G AT EGG A KS KT KG AL E AA G D K LG D LL GGG EE - E P E R E R VS RK SS R ------G D K ------124 RE R L E IS ETQ P KG - LP D MV Q ---- D MTTG VA EG KT KG AI E AA G E K VL D FL GGSQ D - E R E PQ RR GHSGGG ------KD R ------132 SE R L D LH A KGG K D - LP E LM QG MSQG VTQGG AHG KT KG VL D AA K E K I DE F RT EE P E R E RQ PA RRR ES RG R ------E RG ES ------144 FKA5660 R L D LQ P R EQ VA G LP G LM H E VT EGT A RH KTSG AL GG L K DT A EE VV G AL RG APV EE RR G R D LPV G R E AP ------A E RR G ------G KK G ------143 FKE6294 R L D IQ P K EQ VGG LP G LM R E VT EGSS RH R V KG AL GG I R DT A ED VA E AL RGSSSS DQNG RG MPA E R VAP ------A E RR G ------G R EG ------143 FKC4060 R L D LAP KE Q VP G LP G LM H E VT DGT A RQ KS KG AL EG L K DT A EE AV GS L RGGSS EE H A RR D LPA G RS AP ------G D RR S ------G R EG ------143 SCO0650 R L D LE H D V RS K --- T VP E MF GS PMA KT VG R AG A RR T A RS LT D K V R D VL T P EH EH EEE P EE A ED R P R AG A E RG RSTQ R P RS R PAA R P R DEDD R P RS R P RRR T EEED R 170 SAV2355 R L D LE RD SGST --- T VP E LI GGG AV KS MG KRK V RK AA ES VG DT V RK AV GGG R DEDEE P DEDEEE QQ E ------E R P RKRR AP ------A RSG AS RRR PV E A - 156

Figure 2. Domain organization of GvpA showing extraordinary length and content of the C termini of actinomycete GvpA proteins. Amino acid (aa) numbers indicate the full length of proteins.

10% 100 CAE11898 Microcystis aeruginosa PCC7806 GvpAI 15 CAE11900 Microcystis aeruginosa PCC7806 GvpAIII 84 CAE11899 Microcystis aeruginosa PCC7806 GvpAII 44 AAA82497.1 Anabaena flos-aquae Cyanobacteria 35 CAA40898 Pseudoanabaena sp. PCC6901 27 79 AVAR03001663 Anabaena variabilis ATCC29413 ASL2254 Anabaena sp. PCC7120 76 TERY02003711Trichodesmium erythraeum IMS101 39 AAF16863M100 Ancylobacter aquaticus α-proteo β 75 RSPH030013202.4.1Rhodobacter sphaeroides -proteo Thiocapsa pendens GvpA Chromatiales, γ-proteo 12 O68680 Bacillus megaterium low GC Gram+ Archaea 23 METH02001626 Methanosarcina barkeri 99 VNG6241G Halobacteriumsp.NRC-1 GvpA2 100 VNG6029G Halobacteriumsp.NRC-1 GvpA1 45 CAA39503 Haloferax mediterranei c-vac 49 CAA69881 Natronobacterium vacuolatum DSM8800 SMD10157 Streptomyces diversaTM GvpA2 94 SMD00942 Streptomyces diversa TM GvpA1 46 70 SAV598 Streptomyces avermitilis GvpA1 28 Streptomyces scabies GvpA2 100 SP8137 Streptomyces peucetius GvpA2 60 Streptomyces scabies GvpA1 95 100 47 SAV1889 Streptomyces avermitilis GvpA2 SCO6500 Streptomyces coelicolor GvpA SP529 Streptomyces peucetius GvpA1 48 100 SAV2355 Streptomyces avermitilis GvpA3 SCO0650 S.coelicolor GvpA2 FKE6294 Frankia sp. EAN1pec 75 100 Streptomyces 59 FKC4060 Frankia sp. CcI3 FKA5660 Frankia alni Other actinomycetes 78 Saccharopolyspora erythraea 93 Rhodococcus equi 3717 Rhodococcus sp. RHA1

Figure 3. Phylogenetic relationship of full-length GvpA of non-actinomycetes and the N termini of actinomycete GvpA. The phylogenetic tree was constructed using the Treecon v 1.3b program [23] inferred from alignments created by the ClustalX program [24]. Phylogenetic distances were determined by neighbour-joining analysis; numbers on branching points are bootstrap values with 100 replicates. www.sciencedirect.com Update TRENDS in Microbiology Vol.13 No.8 August 2005 353 indicates that gvp sequences must have been present in a actinomycetes, and in particular to determine whether the common actinomycete ancestor. long C-terminal extension confers roles other than in flotation. Are gas vesicle proteins required for flotation in It is notable that the actinomycetes that have gvp gene actinomycetes? clusters generally have large genomes. Whether there is a The habitats of saprophytic actinomycetes range from correlation between genome size and the presence of gvp soils to sediments; more recently, actinomycetes have also genes remains to be determined. It seems more likely that been discovered in marine environments [13]. the absence of gvp genes from pathogenic mycobacteria Recently, it was shown that the non-motile S. coelicolor and corynebacteria reflects their roles as specialized forms floating colonies in standing liquid cultures [14],a parasites rather than free-living saprophytes. It is condition that resembles flooded, water-logged soils. unlikely that filamentous growth is the significant factor These environments readily become anoxic; therefore, in actinomycetes with gvp genes because the Thermo- flotation and subsequent sporulation at the air–water bifida fusca genome does not have gvp genes (http:// interface provides an excellent escape from the unfavour- genome.jgi-psf.org/mic_home.html). able, oxygen-poor environment. Surprisingly, however, Does the different composition of gvp clusters in the S. coelicolor mutants lacking both gvp clusters still floated various actinomycetes perhaps suggest different functions and reached the air interface in standing liquid cultures for these multiprotein assemblies in these strains? And (G. van Keulen, et al., unpublished). This indicates that why do streptomycetes have more than one gvp gene gas vesicle proteins, including the intriguing C-terminal cluster? These are exciting questions that need to be extension on GvpA in actinomycetes, are not essential for addressed to understand the role of the gas vesicle flotation and suggests the existence of other, unknown proteins in the complex biology of these microbes. mechanism(s) of buoyancy in addition to other roles for the Gvp proteins: what could they be? Acknowledgements This work is supported by a Marie Curie Postdoctoral Fellowship to Involvement of actinomycete Gvp proteins in other G.V.K. (MEIF-CT-2004–506056), by the BBSRC (EGH16080), and by EU cellular processes grants to L.D. (COMBIGTOP, LSHB-CT-2003–503491 and ACTINOGEN, IP005224). We gratefully acknowledge P. Normand (Lyon) and Genoscope Transcript profiling has indicated that gvp1 and neigh- (Evry), J.K. Sohng (Asan), P. Leadlay (Cambridge), M. Bibb (Norwich) and bouring genes are transiently induced in expression in Diversa (San Diego), W. Wohlleben and T. Weber (Tu¨ bingen), and A. Ward shaken liquid cultures of S. coelicolor after osmotic and (Newcastle) for making unpublished (gvp) sequences and other data temperature upshifts [15]. Recent evidence indicates that available. osmoadaptation is also important in erection of aerial hyphae and secondary metabolite production [16–18]. References Whether the properties conferred by the C-terminal 1 Walsby, A.E. (1994) Gas vesicles. Microbiol. Rev. 58, 94–144 extension on GvpA have a role in actinomycete stress 2 Shukla, H.D. and DasSarma, S. (2004) Complexity of gas vesicle response or in differentiation remains to be established. biogenesis in Halobacterium sp. strain NRC-1: Identification of five new proteins. J. Bacteriol. 186, 3182–3186 One possibility is that the arginine-, glutamate- and 3 DasSarma, S. et al. 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