Archaeal Flagella, Bacterial Flagella and Type IV Pili

J Mol Microbiol Biotechnol 2006;11:167–191 DOI: 10.1159/000094053

Archaeal Flagella, Bacterial Flagella and Type IV Pili: A Comparison of Genes and Posttranslational Modifications Sandy Y.M. Ng Bonnie Chaban Ken F. Jarrell Department of Microbiology and Immunology, Queen’s University, Kingston, Canada

Key Words Archaeal flagella Bacterial flagella Type IV pili Genes and posttranslational modifications, a comparison Flagellar gene families Preflagellin/prepilin signal peptide processing Flagellin/pilin subunit glycosylation

Abstract The archaeal flagellum is a unique motility organelle. While superficially similar to the bacterial flagellum, several similarities have been reported between the archaeal flagellum and the bacterial type IV pilus system. These include the multiflagellin nature of the flagellar filament, N-terminal sequence similarities between archaeal flagellins and bacterial type IV pilins, as well as the presence of homologous proteins in the two systems. Recent advances in archaeal flagella research add to the growing list of similarities. First, the preflagellin peptidase that is responsible for processing the N-terminal signal peptide in preflagellins has been identified. The preflagellin peptidase is a membrane-bound enzyme topologically similar to its counterpart in the type IV pilus system (prepilin peptidase); the two enzymes are demonstrated to utilize the same catalytic mechanism. Second, it has been suggested that the archaeal flagellum and the bacterial type IV pilus share a similar mode of assembly. While bacterial flagellins and type IV pilins can be modified with O -linked glycans, N -linked glycans have recently been reported on archaeal flagellins. This mode of glycosylation,

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as well as the observation that the archaeal flagellum lacks a central channel, are both consistent with the proposed assembly model. On the other hand, the failure to identify other genes involved in archaeal flagellation by homology searches likely implies a novel aspect of the archaeal flagellar system. These interesting features remain to be deciphered through continued research. Such knowledge would be invaluable to motility and protein export studies in the Archaea. Copyright © 2006 S. Karger AG, Basel

Introduction

Motility, including both swimming and surface-mediated translocation, is a widespread feature among prokaryotes; different species attain this through various physical mechanisms. Among these, flagella and type IV pili are extremely effective motility organelles commonly found on prokaryotic cells. While bacterial flagella and type IV pili systems are relatively well-studied, the archaeal flagellar system is also receiving growing attention. Significant evidence exists to suggest that the archaeal flagellar system is distinct from the bacterial flagellar system and instead shares more common features with the bacterial type IV pilus system. Similarities include the multiflagellin nature of the archaeal flagellar filament, presence of homologous proteins, N-terminal

Ken F. Jarrell Department of Microbiology and Immunology, Queen’s University Kingston, Ont. K7L 3N6 (Canada) Tel. +1 613 533 2456, Fax +1 613 533 6796 E-Mail [email protected]

sequence similarities between archaeal flagellins and bacterial type IV pilins, and likely also some parallels in the mechanism of assembly [for detailed reviews, see Bardy et al., 2003; Thomas et al., 2001a, b]. Recent progress in the study of the archaeal flagellar system has unveiled posttranslational modification processes on archaeal flagellins, including signal peptide processing and glycosylation. Also, the explosion of available genomic data has allowed for more comprehensive and meaningful analyses of the archaeal flagella gene families. This review focuses on the assembly and composition of these three motility systems. Comparisons of gene sequences as well as the molecular biology and posttranslational modifications of the archaeal flagellar system to both the bacterial flagellum and type IV pilus systems will be made based on the most recent data. Unique features of the archaeal flagellar system will also be highlighted.

Ultrastructure

Examination of many archaea by electron microscopy revealed the presence of thin filaments that were identified as flagella in keeping with the observed motility of many of these strains. Knockouts in the fla gene clusters lead to a disappearance of these structures linking the genes to the structure in different archaea [Jarrell et al., 1996; Patenge et al., 2001]. The archaeal motility structure is unusual in many respects in ultrastructure compared to the bacterial flagellum or type IV pilus. One consistent feature of the archaeal flagellum is its thin diameter of 10–13 nm [Thomas et al., 2001a], which places it between the diameters usually reported for type IV pili (6 nm) [Nudleman and Kaiser, 2004] and bacterial flagella (approx. 20 nm) [Jones and Aizawa, 1991]. Archaeal flagella are composed of a filament with an attached hook [Bardy et al., 2002] like that of bacterial flagella and unlike that of type IV pili. Hooks in archaea seem to be more variable in length than that observed in bacteria [Bardy and Jarrell, 2002; Bardy et al., 2002; Cruden et al., 1989]. However, convincing reproducible archaeal basal bodies equivalent to the well-defined rings (MS, P, L and C) and rod of bacterial flagella [Macnab, 1999; Minamino and Namba, 2004] have not yet been routinely observed. Instead, a knoblike structure is often observed in flagella preparations from Methanococcus species [Bardy et al., 2002; Kalmokoff et al., 1988] and extreme halophiles [Jaschke et al., 1994; Kupper et al., 1994]. Possibly because of the unusual wall structure in many archaea, specifi168

J Mol Microbiol Biotechnol 2006;11:167–191

cally the lack of murein, the flagella of archaea may be necessarily anchored to a sub-cytoplasmic membrane layer, such as the polar cap in halophiles [Kupper et al., 1994; Speranskii et al., 1996]. In Halobacterium salinarum (formerly H. halobium; H. salinarium), a plateau-like intracellular structure below the cytoplasmic membrane, termed the ‘discoid lamellar structure’, has been reported at the cell pole where flagella are located/inserted [reviewed in Metlina, 2004]. Specific anchoring structures have not been reported in type IV pili. Recently, however, it has been shown for the bundle-forming pilus (bfp) of Escherichia coli, through crosslinking and yeast two-hybrid studies, that 10 of the 14 proteins encoded by the bfp operon interact, suggesting that they form an oligomeric complex [Crowther et al., 2004; Hwang et al., 2003]. The nature of the protein composition of the three motility structures also varies. The shaft of the type IV pilus is composed of a major pilin and likely several minor pseudopilins (made as preproteins with prepilin-like signal peptides and processed by the prepilin peptidase), which may also be involved in forming a base [Mattick, 2002]. This seems analogous to the archaeal flagellum in which the filament is almost universally composed of multiple flagellins, often in very different stoichiometries ranging from major to very minor [Bardy et al., 2004]. Bacterial flagella, on the other hand, are usually composed of thousands of copies of a single type of flagellin protein [Minamino and Namba, 2004], although there are many cases of multiple flagellins in bacteria as well [Limberger, 2004; Wilson and Beveridge, 1993]. Both bacterial and archaeal flagella appear to consist of protofilaments – longitudinal arrays of flagellin subunits [Fedorov et al., 1994; Yonekura et al., 2003]. It has been suggested that in archaea, helicity of the flagella is determined by the presence of several flagellins with different structures instead of the ability of a single flagellin to exist in two conformations as in bacteria [Pyatibratov et al., 2002]. A significant similarity between bacterial and archaeal flagella is the observation that both are rotating structures with switches [Aizawa, 1996; Marwan et al., 1991], while the mechanism for translocation movement determined by type IV pili is an extension and retraction of the pilus [Merz et al., 2000; Skerker and Berg, 2001]. However, orthologs of bacterial flagella motor components were not identified in any of the completely sequenced archaeal genomes. Recent data from reverse engineering and mathematical modeling have demonstrated that the archaeal flagellar switching mechanism and sensory control is fundamentally different from that in bacteria. All the published models on the mechanism on Ng/Chaban/Jarrell

Aeropyrum pernix B2

G

B2

G

I

H

J

Sulfolobus tokodaii B2

G

F

I

H

J

Archaeoglobus fulgidus B1-2

B1-1

D/E

G

F

I

H

J A1

Halobacterium salinarum J

K

I

H

G

F

C/D/E

D

F

G

H

htplX

B1

A2 B2

B3

Methanococcus voltae A

B1

B2

C

B3

D

E

I

J

Methanosarcina acetivorans J

I

F

H

G

D/E

B

B

2kb

B

D/E G

F

H

I

J

Pyrococcus abyssi B1

B2

C

B3

D/E

F

G

I

H

J

Thermoplasma volcanium B

C

D/E

F

G

H

I

J

B2

1 kb

Fig. 1. Flagella gene families of selected archaea. Similar colors represent homologues shared among families.

The genes are transcribed in the direction of the respective arrows. The white, unlabeled arrows in Aeropyrum pernix and Sulfolobus tokodaii have no sequence similarity to genes in universal databases. The B flagellin genes of H. salinarum are adjacent to the accessory genes, while the A flagellin genes are located elsewhere on the chromosome.

motor switching in E. coli were tested against H. salinarum and none provided a satisfactory fit with observed experimental results. A different model was proposed that quantitatively reproduce all experimental observations thus far, suggesting the existence of a novel and yet unknown switching mechanism in the domain Archaea [Nutsch et al., 2005].

Flagellar Gene Families

Thus far, only one major gene cluster (comprising up to 13 genes) involved in flagellation has been identified in archaea (fig. 1, table 1). No genes homologous to ones involved in flagella structure or assembly have been detected in the complete genomes of any archaeon [Faguy and Jarrell, 1999]. There are no homologues to rod, rings, basal body components, mot proteins, hook, hook-associated proteins, switch proteins, specific flagella export system proteins or even to bacterial flagellins. The arArchaeal Flagella, Bacterial Flagella and Type IV Pili

chaeal flagellins are instead similar in amino acid sequence at the N-terminus to type IV pilins [Faguy et al., 1994]. This system, in turn, shares homology with type II protein secretion systems [Peabody et al., 2003]. Archaeal flagellins also share with type IV pilins unusual, short signal peptides and their processing by a homologous signal peptidase. In Pseudomonas aeruginosa, it has been estimated that about 35 genes are involved in type IV pilus assembly and function [Mattick, 2002]. However, a much smaller number of genes are required for the assembly of functional bfp (14 genes) [Donnenberg et al., 1997]. Among archaeal flagella and type IV pili systems, only a few direct homologues have been identified [Jarrell et al., 1996; Peabody et al., 2003; Thomas et al., 2001a]. These are the flagellins/pilins and pseudopilins themselves, usually multiple in each case; the associated processing signal peptidase (PilD in P. aeruginosa type IV pili system and FlaK/PibD in archaeal flagella systems); conserved ATPase (PilT/PilB/TadA in type IV pili systems and FlaI in archaeal flagella), and a conserved memJ Mol Microbiol Biotechnol 2006;11:167–191

169

Table 1. Distribution of fla gene clusters in archaea Organism

Flagellin

Fla genes C

D

E

F

G

H

I

J

K

Crenarchaeota Aeropyrum pernix

14601712 14601713

–

–

–

–

14601709 14601708

14601707

14601706 14601914 14600901

14601705

–

Pyrobaculum aerophilum

–

–

–

–

–

–

–

18313579 18312170 18313110

18313580

–

Sulfolobus acidocaldarius

70606948

–

–

–

70606945

70606946

70606944

70606943 70608025 70607238

70606942

70605985

Sulfolobus solfataricus

15899081

–

–

–

15899078

15897078

15899077

15899076

15899075

15897089

Sulfolobus tokodaii

15922853

–

–

–

15922856

15922855

15922857

15922858 15920739 1521686

15922859

15922589

Archaeoglobus fulgidus

11498659 11498660

–

11498656

11498657

11498655

11498654

11498653

11498541

Ferroplasma acidarmanus

–

–

–

–

–

–

–

–

Haloarcula marismortui

55376132 55380089 55378891

55378884

55378883

55378882

55378881 55378048

55378880 55378047

55379184

Halobacterium salinarum

15790079 15790080 15790081 15790120 15790121 15790252

–

–

15790074

16554480

16554479

15790072 15789975 15789521

15790071

15791095

Methanocaldococcus jannaschii

15669081 15669082 15669083

15669084

15669086

15669087

15669088

15669089

15669090 1591924 1591480

15669091 1591923

15669092

Methanococcoides burtonii

68185311 68185312 68185254

68186333

68186334

68186335 68185307

68186336 68185306

68186337 68185305

68184794

Methanococcus maripaludis

45359229 45359230 45359231

45359232

45359233

45359234

45359235

45359236

45359237

45359238

45359239

45358118

Methanopyrus kandleri

–

–

–

–

–

–

–

20094144

19887149

20094140

Methanosarcina acetivorans

20091879 20091880 20091895

–

20091878 20091896

20091876 20091898

20091877 20091897

20091875 20091899

20091874 20091900 20089132 20091914 20092769

20091873 20091901

20089408

Methanosarcina barkeri

68080180

–

68080179

68080177

68080178

68080176

68080175 68081047

68080174

68133328

Euryarchaeota

170

11498658 –

–

55378885

15790076

15790075

15669085

68186332 68185854


Ng/Chaban/Jarrell

Table 1 (continued) Organism

Flagellin

Fla genes C

D

E

21226423 21226519

F

G

H

I

J

K

21226421 21226517

21226422 21226518

21226420 21227041 21226516

21226419 21226515 21227618 21227040 21228400

21226418 21227039 21226514

21227780

Methanosarcina mazei

21226424 21226425 21226520

–

Methanothermobacter thermoautotrophicus

–

–

–

–

–

–

–

2622833

2622834

2621485

Nanoarchaeum equitans

–

–

–

–

–

–

–

41614965 41615211

–

–

Natronomonas pharaonis

76801687 76801688 76801689

76801721

76801690

76801721

76801691

76801692 76801693

76801722

76801723

76801724

76801289

Picrophilus torridus

–

–

–

–

–

–

–

–

–

–

Pyrococcus abyssi

14521692 14521693 14521694

14521691

14521690

14521689

14521688

14521687

14521686 14521589

14521685 14521587

14521786

Pyrococcus furiosus

18976709 18976710

18976708

18976707

18976706

18976705

18976704

18976703 18977366

18976702 18977364

18976843

Pyrococcus horikoshii

14590447 14590448 14590449 14590450 14590451

14590452

14590453

33359301

14590455

14590456

14590457 14590539

14590458 14590540 14590541

14590360

Thermococcus kodakarensis

57639973 57639974 57639975 57639976 57639977

57639978

57639979

57639980

57639981

57639982

57639983 57639935 57641788

57639984

57639988

Thermoplasma acidophilum

16082620 16081658

16081659

16081660

16081661

16082646

16081662

16081664 16081951

–

Thermoplasma volcanium

13542257 13541438

13541439

13541440

13541441

13541442

13541443

16081663 16081953 16081950 13541444 13541846 13541843

13541445 13541844

–

GI numbers are for proteins from genome sequences at NCBI Genomes (http://www.ncbi.nlm.nih.gov/genomes/static/a.html).

brane protein (PilC/TadB in type IV pili systems and FlaJ in archaeal flagella). Archaeal Flagellins Archaeal flagellins are members of COG 1681 (Clusters of Orthologous Groups of proteins, http://www.ncbi. nlm.nih.gov/COG/). Among the 26 sequenced archaea genomes (available at the NCBI website http://www.ncbi. Archaeal Flagella, Bacterial Flagella and Type IV Pili

nlm.nih.gov/), flagellin genes can be readily detected in 20. This includes three species of Methanosarcina in which flagella and/or motility have never been reported. However, another species of Methanosarcina, M. baltica, can be flagellated [Singh et al., 2005] and other members of the family Methanosarcinaceae such as Methanolobus, Methanococcoides and Methanosalsum have been reported as motile. In addition to the archaea with completely J Mol Microbiol Biotechnol 2006;11:167–191

171

sequenced genomes, flagellins have been sequenced in Methanococcus voltae, Methanococcus thermolithotrophicus, Methanococcus vannielii and Natrialba magadii [Bayley et al., 1998; Kalmokoff and Jarrell, 1991; Nagahisa et al., 1999; Nutsch et al., 2003a; Serganova et al., 2002; Thomas and Jarrell, 2001]. Flagellins have not been reported in the completely sequenced genomes of Pyrobaculum aerophilum, Methanothermobacter thermoautotrophicus, Methanopyrus kandleri, Ferroplasma acidarmanus, Picrophilus torridus and Nanoarchaeum equitans. Of these, M. kandleri [Kurr et al., 1991] and P. aerophilum [Volkl et al., 1993] have been reported to be motile. N. equitans has never been reported to be motile although it does have some sort of appendage [Waters et al., 2003]. Cells of M. thermoautotrophicus are not motile or flagellated [Balch et al., 1979]. Motility/flagellation has not been reported in F. acidarmanus although Ferroplasma acidophilum is not flagellated [Golyshina et al., 2000]. Archaea, as a rule, have multiple flagellin genes, anywhere from 2 to 6, usually located in tandem on the chromosome. Sulfolobus species may be the only exception with a single identified flagellin gene. Interestingly however, two apparent flagellin bands of 31 and 33 kDa were found by SDS-PAGE analysis of flagella preparations of Sulfolobus shibatae [Faguy et al., 1996]. Multiple proteins with prepilin signal peptides are found in the type IV pilus system as well [Alm and Mattick, 1997]: usually a major structural pilin and several pseudopilins which may be minor structural components of the pilus. On the other hand, most bacteria make the flagellum filament from a single type of flagellin subunit, although multiple flagellin-containing bacterial species are not uncommon [Wilson and Beveridge, 1993]. One feature of type IV pilin primary sequence is the presence of two conserved cysteines that form a disulfide bridge essential for pilus assembly [Craig et al., 2004]. The distance between the cysteines can vary considerably. Cysteines are found only rarely in bacterial flagellins and this is true of archaeal flagellins as well. Only the two Aeropyrum pernix flagellins, M. voltae FlaA and M. vannielii FlaB2 have two cysteines, while one of the two A. fulgidus flagellins has three. Eight other archaeal flagellins have one cysteine while the vast majority has none. Type IV pilins and archaeal flagellins also share homology with components of the type II secretion system. These components, dubbed pseudopilins, share significant amino acid sequence similarity over the first 30 positions when compared to type IV pilins. This suggested that the pseudopilins might form a pseudopilus between 172


the two membranes in Gram-negative bacteria and drive, like a piston, the secreted proteins through the outer membrane [Mattick and Alm, 1995; Sandkvist, 2001]. In the past few years, it has been shown that overexpressed pseudopilins of type II secretion systems can indeed form pilus-like structures [Durand et al., 2003; Kohler et al., 2004; Vignon et al., 2003]. Preflagellin Peptidase It is believed that all archaeal flagellins are made with short signal peptides that are cleaved by the preflagellin peptidase prior to incorporation of the flagellins into the filament. This unusual enzyme, distinct from a signal peptidase I equivalent, is a member of COG 1989 and should be present in the genome of at least all flagellated archaea. Since it has been shown to also cleave flagellinlike signal peptides from other proteins apparently unrelated to flagellation in at least Sulfolobus species [Albers et al., 2003], it is possible that this enzyme could also be found in non-flagellated archaea. So far, archaeal genes have been biochemically shown to encode this enzymatic function in the case of flaK in Methanococcus maripaludis [Bardy and Jarrell, 2002] and M. voltae [Bardy and Jarrell, 2003] as well as pibD in Sulfolobus solfataricus [Albers et al., 2003]. In the sequenced genomes of flagellated archaeal species, a preflagellin peptidase can be identified in all cases thus far except in A. pernix, two Thermoplasma species, and in P. acidophilum, which is flagellated but has no annotated flagellins or preflagellin peptidase. Since it appears all archaeal flagellins have a short signal peptide which is cleaved prior to incorporation into the filament and this processing is essential for flagellation (at least in M. voltae) [Bardy and Jarrell, 2003], then such an enzymatic activity must be present in these organisms. A preflagellin peptidase has also been detected in the three Methanosarcina species that have flagellin genes even though they have no flagella. In cases where complete genome sequences are not available, the preflagellin peptidase gene and/or activity has also been found in M. voltae and M. thermolithotrophicus [Bardy and Jarrell, 2003, Correia and Jarrell, 2000]. Among the clearly non-flagellated archaea, a preflagellin peptidase has also been observed in M. thermoautotrophicus. As well, a preflagellin peptidase is present in M. kandleri, which is motile and flagellated although no flagellin genes have been identified yet. Assuming that the preflagellin peptidase equivalents are transcribed and the gene products active, then they apparently must be processing non-flagellin proteins with preflagellin-like signal peptides in these cases. Ng/Chaban/Jarrell

Rarely, as in Methanocaldococcus jannaschii, the preflagellin peptidase gene is located at the end of the gene cluster that also contains the flagellin genes. It is likely cotranscribed with the flagellins. More commonly, the gene for the preflagellin peptidase is located at a distance from the flagellin genes surrounded by apparently unrelated genes. In S. solfataricus, it was noted that while pibD is not within an apparent operon structure, remnants of an insertional element are present upstream of the gene, suggesting the possibility of a previous gene rearrangement [Albers et al., 2003]. All of the abovementioned preflagellin peptidases have the two conserved aspartic acid residues shown to be essential for activity in M. voltae [Bardy and Jarrell, 2003]. Several archaea have more than one member of COG 1989 but, at least in some cases, the additional preflagellin peptidase homologues lack the essential aspartic acid residues so whether these gene products are biochemically competent to perform the cleavage reaction is presently unknown. In contrast, M. acetivorans appears to have as many as four and M. jannaschii three homologues that have the conserved catalytic aspartic acids. In the case of the Halobacterium NRC1 (Halobacterium NRC-1 now classified as a strain of H. salinarum [Gruber et al., 2004]), a preflagellin peptidase homologue was found but apparently annotated with an incorrect start that begins after the conserved aspartic acid residues (compare NCBI sequence with TIGR annotation of the sequence with an alternative upstream start site at www. tigr.org). Bacteria can have multiple paralogues of the prepilin peptidase: seven have been identified in E. coli [Peabody et al., 2003] while only one (pilD) is found in the complete sequence of P. aeruginosa [Peabody et al., 2003]. The presence of the prepilin peptidase has been identified in a wide diversity of bacteria and it has been speculated that it might have different functions with related or unrelated substrate specificities in some of these organisms [Peabody et al., 2003]. The same enzyme (PilD/XcpA) cleaves pseudopilins involved in type II secretion as well as type IV prepilins in P. aeruginosa [Alm and Mattick, 1997, Peabody et al., 2003]. Bacterial flagellins are not made with signal peptides and so this system does not possess a preflagellin peptidase equivalent. FlaHIJ Among the genes known or suspected to be involved in flagellation in archaea, few have homologues outside of the archaea. Exceptions are the preflagellin peptidase, as well as flaI and flaJ. FlaI possesses a Walker box A (inArchaeal Flagella, Bacterial Flagella and Type IV Pili

volved in nucleotide binding) and is a homologue of the ATPase found in the type IV pilin system involved in pilus extension and retraction (PilB/PilT in P. aeruginosa [Bayley and Jarrell, 1998] and TadA in Actinobacillus actinomycetemcomitans [Kachlany et al., 2001]). These are members of COG 630. Homologous proteins are widespread ATPases known to be involved in protein export systems as well [Peabody et al., 2003]. PilT is a member of the AAA family of motor proteins and has recently been shown to exist as a hexamer [Forest et al., 2004]. Recent evidence in the bfp system indicates that the PilT equivalent (BfpF) also forms homocomplexes predicted to be homohexamers [Crowther et al., 2004]. It has been suggested that PilT exists as a hexameric ATPase surrounding the base of the pilus and providing an axial power stroke like the F1 ATPase [Kaiser, 2000]. A single PilT complex has been suggested to be responsible for the retraction of a single pilus [Maier et al., 2004]. A multimeric nature for FlaI has not yet been demonstrated but would not be unexpected. The assembly of bacterial flagella also requires an ATPase, FliI, a protein which has significant sequence similarity to the -catalytic subunit of the proton-translocating F0F1 ATPase, although with a flagellum-specific N-terminal extension [Gonzalez-Pedrajo et al., 2002]. Recently, FliI has been shown to likely exist as a homohexamer and to form ring structures with sixfold symmetry and having an internal cavity of about 2.5–3.0 nm [Claret et al., 2003]. It is postulated that FliI is located near the integral membrane components of the type III export system in the patch of membrane within the flagellum MS ring. ATP hydrolysis would result in the export of the substrates into the flagellar channel [Claret et al., 2003]. FlaJ (COG 1955) is a conserved polytopic cytoplasmic membrane protein with homologues also found in type IV pili systems (PilC in P. aeruginosa and TadB in A. actinomycetemcomitans [Kachlany et al., 2001; Peabody et al., 2003]). The PilC homologues may interact with the corresponding nucleotide-binding protein [Alm and Mattick, 1997] and suggest that, in the archaeal flagella system, FlaJ may interact with FlaI. Peabody et al. [2003] have suggested that the bacterial genes (pilC) arose by one internal gene duplication event while the larger archaeal equivalent (flaJ) arose from a second internal gene duplication. In archaeal flagellar systems, flaI and flaJ are always coupled with flaH (COG 2874). Despite also containing a Walker box A motif [Thomas and Jarrell, 2001], FlaH has no strong similarity to any bacterial protein and none to ones involved in type IV pili or type II secretion. FlaJ Mol Microbiol Biotechnol 2006;11:167–191

173

HIJ are found in the immediate vicinity (almost always downstream) of flagellins in all cases where flagellins are found, usually after a series of other genes suspected to be involved in flagella synthesis. In many cases, there are two copies of flaHIJ, located at different loci on the chromosome. Since these genes are likely also involved in protein export (type II secretion involvement), it may be that the set located next to the flagellins is directly and specifically involved in flagellin export while the other set is involved in export of other proteins. The requirement for the FlaI and FlaJ located in the proximity of the flagellins for the production of flagella has been shown in methanogens and halophiles and presumed to be true of all archaea [Patenge et al., 2001; Thomas et al., 2002]. In the case of H. salinarum, an in-frame deletion was created in flaI, which led to non-flagellated cells [Patenge et al., 2001]. Since H. salinarum has multiple copies of flaI, this suggests that the flaI located next to the flagellin genes is critical for flagellation and is not compensated for by other flaI-like genes located elsewhere on the chromosome. In type IV pili systems, mutants in pilT are unable to retract their pili and hence have a hyperpiliated phenotype while mutants in pilB, unable to polymerize pilin, are non-piliated [Mattick, 2002; Whitchurch et al., 1990; Wolfgang et al., 2000]. FlaCDEFG In many flagellated archaea, such as methanococci, halophiles and pyrococci, there are a number of genes located between the flagellins and the conserved flaHIJ genes. It has been shown that the whole region from flaB1 to the end of flaJ is cotranscribed in methanococci [Thomas et al., 2002]. In H. salinarum, the orientation of the neighboring flagella-associated genes is such that they are transcribed in the direction opposite to the flagellins. As more archaea have had their genomes sequenced, it is now apparent that genes with at least weak similarity to some of flaC, D, E, F and flaG are found in the same location between flagellins and flaHIJ in many flagellated archaea. Most of the genes flaC, D, E, F, and G are found in all flagellated euryarchaeota especially methanococci, halophiles, pyrococci and Thermoplasma, and in a similar order. It is more difficult to find strong hits to these proteins in the crenarchaeotes. While the flagellins and flaHIJ are easily detected in the flagellated crenarchaeotes, only weak hits to flaF and flaG are found and convincing hits to flaC, D and E are not found. In some cases, the number of orfs found between the flagellins and the conserved flaHIJ genes simply do not leave room for all of flaC, D, E, F and G. The role of these genes 174


in archaeal flagellation is an area of active research. There is significant identity (42.5% identity over 127 amino acid overlap) over the entire length of FlaE compared to FlaD in M. voltae. Because of the similarity of flaD and flaE, some genes in other archaea give significant hits to both in homology searches. Furthermore, it has been shown [Thomas and Jarrell, 2001] that two proteins are made from flaD, one a full-length version of approximately 52 kDa and a second smaller C-terminal version of about 15 kDa. Both versions are detected in immunoblots of E. coli overexpressing the flaD gene and both versions are also detected in immunoblots of M. voltae membranes. The smaller protein is apparently derived from an internal translational start preceded by a ribosome-binding site. The truncated version of FlaD is almost identical in size and similar in sequence and pI to FlaE. Similar findings concerning FlaD were also observed for all other methanococci examined (M. jannaschii, M. thermolithotrophicus and M. maripaludis). A 16-kDa C-terminal portion of FlaD was detected in a 2D gel study of M. jannaschii indicating that the truncated form of FlaD is also made in the hyperthermophile [Mukhopadhyay et al., 2000]. Interestingly, several archaea (i.e. H. salinarum NRC1 and Methanococcoides burtonii) have one large gene with significant similarity to all of flaC, D and E. In the case of H. salinarum NRC1, the protein FlaCDE is 504 amino acids long. It aligns with the M. voltae FlaC (188 amino acids) over residues 30–233 and then the halophile protein aligns with M. voltae FlaD (362 amino acids) over the remainder of the protein. Because of the strong similarity between FlaD and FlaE, the halophile protein also has strong similarity to FlaE (135 amino acids) from amino acid 380 to the end of the protein. If this one large protein is fulfilling the roles of FlaC, D and E, it may indicate that in those archaea which have the three separate proteins that these proteins may interact directly. This may be analogous to an observation that has been made in the bacterial flagella system, where fusions of the switch protein genes fliF and fliG were found which were still functional [Francis et al., 1992]. These fusions indicated a specific interaction of these two proteins presumably in a 1: 1 ratio in wild-type cells, interactions subsequently demonstrated in coprecipitation studies [Tang et al., 1996]. The archaeal flaCDEFG genes appear to have no significant homology to any bacterial genes and may be archaeal-specific genes involved in flagellation in this domain. In support of this hypothesis, none of these genes have yet been found in any archaea lacking flagellin genes. Ng/Chaban/Jarrell

Bacterial Flagella Rings and Type IV Pili Secretins Type IV pili systems possess a multimeric outer membrane protein (PilQ) which acts as a gated pore to allow passage of the pilus through the outer membrane [Mattick, 2002]. Corresponding proteins are found in type II secretion systems. The proteins are part of a large family of so-called secretins [Bitter, 2003], which, in the case of PilQ, forms a dodecameric donut-shaped complex whose internal hole diameter closely matches that of the external diameter of the corresponding pilus [Bitter et al., 1998]. In bacterial flagella systems, the same structural problem is faced, namely how to get the large filament through the outer membrane. It is accomplished by the presence of the L ring which acts as a bushing for the filament through the outer membrane [Minamino and Namba, 2004]. The archaeal flagella system appears to lack a corresponding protein, as would be expected since a second membrane has only rarely been detected in archaea [Nather and Rachel, 2003], obviating the need for a protein-mediated hole for the flagellum. Indeed, in the best-studied cases of archaeal flagella, namely Methanococcus and Halobacterium species, the outer surface is simply an S-layer of proteins or glycoproteins overlaying a cytoplasmic membrane. The sole requirement for the filament here is to emerge through the S-layer, an event which has been shown in bacteria to require only a localized perturbation of the S-layer and no apparent specialized mechanism or structure [Sleytr, 1978]. Archaeal Flagellin Glycosylation Genes Recently, for the first time, genes involved in the glycosylation of archaeal flagellins have been identified. In M. voltae, the glycosylation found on the flagellins is a unique trisaccharide that is N-linked to several positions on all 4 flagellins, as well as to the S-layer protein [Voisin et al., 2005]. Two genes critical for glycosylation have been identified thus far. One is a homologue of the oligosaccharyltransferase STT3 subunit. Homologues of this gene are found among the pgl genes in such bacteria as Campylobacter (pglB) and in eukaryotes (stt3p) where they are critical in N-linked glycosylation systems [Burda and Aebi, 1999; Szymanski et al., 2003]. The STT3 homologues are responsible for the transfer of the completed glycan to the protein target in a reaction that occurs on the periplasmic side of the cytoplasmic membrane in bacteria [Szymanski et al., 2003]. BLAST searches using the M. voltae gene sequence reveals the presence of genes with high similarity to the M. voltae STT3 gene in most archaea, including non-flagellated ones. The second gene demonstrated to be necessary for glycosylation in M. Archaeal Flagella, Bacterial Flagella and Type IV Pili

voltae is one that has strong similarity to glycosyltransferases in many bacteria such as Thermus and Pseudomonas. In M. voltae, this gene product appears to be the one that transfers the third, terminal sugar in the glycan structure attached to the flagellins and S-layer proteins [Chaban et al., 2006]. Genes with significant similarity to this gene are also found in a variety of archaea, again including non-flagellated ones. As with the STT3, this glycosyltransferase and homologues are likely involved in general glycan synthesis, not specific to flagellins, and may play a role in posttranslational modification of S-layer proteins and possibly other cell surface proteins. In the related M. maripaludis, in-frame deletions in the corresponding homologous genes were generated and effects similar to that in the M. voltae system were observed [D. VanDyke, B. Chaban and K.F. Jarrell, unpubl. data]. Chemotaxis Genes It is clear that the bacterial flagellum, the type IV pilus and the archaeal flagellum can all have an associated chemotaxis system [Szurmant and Ordal, 2004]. Chemotaxis systems have been well studied in E. coli, Bacillus subtilis and many other bacteria [Armitage, 1999; Szurmant and Ordal, 2004]. At the heart of the system is a classic two-component regulation system best studied in bacteria in relation to flagella. Changes in the concentration of attractants or repellents are detected by methyl-accepting chemotaxis proteins usually located in the cytoplasmic membrane. The signal is transduced to CheA via CheW and then to CheY. CheA is a histidine kinase which can autophosphorylate and transfer the phosphate to a conserved aspartate residue on CheY. CheY-P binds to the flagellar switch protein, FliM, and causes a change in flagella rotation. Other proteins are involved in adaptation to the signal and removal of the phosphate from CheY-P. Many archaea have been shown to be chemotactic and examination of their complete genomes reveals often a seemingly complete set of bacterial-like chemotaxis genes. These include proteins involved in signal recognition and transduction (methyl-accepting chemotaxis proteins or MCPs, CheD), excitation (CheA, CheW, CheY), adaptation (CheR, CheB) and signal removal (CheC, CheX) [Szurmant and Ordal, 2004]. Notably, the cells contain one or more genes annotated as CheY although in bacteria this response regulator delivers its signal via interaction with the flagellar switch protein FliM located at the base of the flagellum and so far no homologue to FliM has yet been reported in archaea [Faguy and Jarrell, 1999; Nutsch et al., 2003b]. To date, how CheY delivers its signal to the archaeal flagellum is a mystery. J Mol Microbiol Biotechnol 2006;11:167–191

175

4

Fig. 2. A sequence LOGO of archaeal fla-

gellins aligned at their cleavage site (without gaps). The default settings of the WebLogo site (http://weblogo.berkeley.edu/ logo.cgi) were used. The signal peptide cleavage site is indicated by the arrow.

2 1 0 N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

Bits

3

At the genetic level, chemotaxis in archaea has been best studied in H. salinarum where deletion analysis of the che operon has led to insights into functions of the various proteins in archaea and contributed to knowledge about the switch [Kokoeva et al., 2002; Nutsch et al., 2003b, 2005; Rudolph and Oesterhelt, 1996]. Interestingly, H. salinarum cells alternate between forward and reverse swimming as determined by CW and CCW rotation of their right-handed flagella, in contrast to most bacteria, like E. coli, which alternate between forward swimming (CCW rotation of left-handed flagella) and tumbling (CW rotation of left-handed flagella). The type IV pili system also has an associated chemotaxis system, although the signals to which this translocation system responds are as yet undefined. A complex chemosensory system tied to type IV pili twitching has recently been studied in P. aeruginosa [Darzins, 1994; Huang et al., 2003; Whitchurch et al., 2004] and similar systems detected in many other bacteria, including Myxococcus xanthus [Ward and Zusman, 1999; Winther-Larsen and Koomey, 2002]. In P. aeruginosa, the system includes previously identified proteins PilGHIJK as well as novel proteins ChpA, ChpB, ChpC and possibly ChpD and ChpE. Together, this system has putative MCPs, CheR, CheB and CheW homologues in addition to multiple CheY modules. An unusually large and complex protein, ChpA, belongs to the CheA-like histidine kinase family and contains nine potential sites of phosphorylation, as well as a CheY-like receiver domain. The Chp chemosensory system may influence twitching motility by affecting pilus assembly, site of pilus assembly or by affecting pilus extension and/or retraction. Presumably the connection between the Chp system and the type IV pilus is via the CheY homologues, PilG and/or PilH, which may interact with the pilus motor [Whitchurch et al., 2004]. 176


C

An obvious, but not exclusive, way that this could occur is through binding of the CheY homologues directly to the ATPases responsible for pilus extension (PilB) or retraction (PilT/PilU), although proof of this interaction is currently lacking. Notable, however, is that many bacteria with type IV pili, such as Neisseria gonorrhoeae, do not have a detectable associated chemotaxis system orthologous to the Chp system [Whitchurch et al., 2004].

Preflagellin/Prepilin Signal Peptide Processing

Archaeal flagellins share several hallmark features with type IV pilins that are absent in the bacterial flagellar system. Bacterial flagellins are not synthesized as preproteins; they are exported via a type III secretion system through the flagellar filament. On the other hand, archaeal flagellins and bacterial type IV pilins are synthesized as preproteins, with the signal peptide being removed by a membrane-bound signal peptidase. Proteins of the type IV pilin superfamily are all small proteins (100–240 amino acids). Similarly, from amino acid sequences deduced from cloned flagellin genes, archaeal flagellins fall in the size range of 193–580 amino acids, with majority of them being approximately 190–220 amino acids [Thomas et al., 2001a]. Unlike other classes of signal peptides, both the type IV pilin and the archaeal preflagellin signal peptides are atypical in that cleavage occurs before the hydrophobic region. Signal Peptide Amino Acid Sequence A recent genomic survey in Archaea revealed the highly conserved nature of the amino acids surrounding the cleavage site of archaeal flagellins [Bardy et al., 2003]. The signal peptide almost always ends with two charged amiNg/Chaban/Jarrell

no acid residues (lysine or arginine) followed by a glycine at the –1 position. Alanine is also found in the -1 position in a few cases, and in the B3 flagellin of Thermoplasma acidophilum (GenBank ID CAC12527.1), the –1 position is occupied by a serine residue. At the +3 position, glycine is found exclusively in all the available flagellin sequences (fig. 2). Site-directed mutagenesis data in M. voltae indicated that the –1 glycine, –2 basic amino acid and +3 glycine are crucial for proper preflagellin processing [Thomas et al., 2001b]. In type IV pili, site-directed mutagenesis has revealed that the glycine residue at –1 position relative to the cleavage site is also essential for prepilin processing. Mutation to any other amino acid resulted in loss of function, except a change to alanine which resulted in partial cleavage. It was suggested that an amino acid with a small side group is probably required at this position to allow access for the peptidase to mediate the cleavage event. A variety of amino acids are found at the +1 position of the archaeal flagellin. This is consistent with the finding that a wide variety of amino acids substitutions are tolerated in the +1 position of the prepilin and still allowed processing. Like that in archaeal flagellins, the –2 position in type IV pilins is almost always lysine. However, in the case of P. aeruginosa, changing the lysine to either threonine or asparagine had no effect on cleavage of pilin or assembly of pili. The N-terminal 40–50 amino acids of the mature archaeal flagellins are extremely conserved and hydrophobic [Bardy et al., 2003]. While sequence analyses revealed no noticeable similarities between the archaeal and bacterial flagellins, sequence similarities at the mature Nterminus were observed when archaeal flagellin sequences were compared to that of type IV pilins in universal databases [Faguy et al., 1994]. The conservation of the Nterminal region throughout all archaeal flagellins would suggest that it has an important role in either the assembly and/or function of the flagella. In type IV pilins, the Nterminal 53 residue stretch mediates pilus subunit assembly through the formation of a hydrophobic helical bundle in the filament core [Craig et al., 2004]. Likewise, secondary structure prediction of archaeal flagellin sequence suggests that the N-terminal hydrophobic conserved regions could form helices [Faguy et al., 1994]. Preflagellin/Prepilin Peptidase The prepilin peptidase, PilD in P. aeruginosa, is a bifunctional enzyme that catalyses the cleavage and Nmethylation of prepilin subunits. An in vitro assay was developed for the archaeal system based on the in vitro prepilin peptidase assay. Preflagellin peptidase activity Archaeal Flagella, Bacterial Flagella and Type IV Pili

was detected in crude membrane fractions of M. voltae [Correia and Jarrell, 2000]. Recently, through comparative genomic analyses and utilization of the COG database, archaeal PilD homologues were identified in M. maripaludis (FlaK), M. voltae (FlaK) and S. solfataricus (PibD). Positive enzymatic activities of these proteins were directly demonstrated through in vitro assays utilizing E. coli membranes containing the heterologously expressed enzymes in question [Albers et al., 2003; Bardy and Jarrell, 2002, 2003]. Furthermore, in M. voltae, insertional inactivation of flaK resulted in mutants that were non-flagellated and non-motile, demonstrating the essential role of this enzyme in flagellar biogenesis. The flagellins of the flaK– cells were unprocessed, as determined by immunoblotting [Bardy and Jarrell, 2003]. Similar results were observed in a M. maripaludis mutant carrying an in-frame deletion of flaK [D. VanDyke, B. Chaban and K.F. Jarrell, unpubl. data]). A notable dissimilarity between the prepilin peptidase and the archaeal preflagellin peptidase is that while PilD also catalyses methylation of the mature N-terminal amino acid, so far there is no evidence to suggest that the +1 amino acids of mature archaeal flagellins are modified. The methylation of type IV pilins is not required for secretion and its biological function remains unknown [Pepe and Lory, 1998]. In terms of signal peptide removal, bacterial type IV prepilin peptidases and archaeal preflagellin peptidases share a common mechanism of action. Although conserved cysteines were initially implicated in catalysis in bacterial type IV prepilin peptidases, there are proteins belonging to the same family in which cysteine residues are absent [Hu et al., 1995]. In addition, an alignment of type IV prepilin peptidase sequences revealed the absence of any conserved histidine residue – a necessary component in the cysteine protease mechanism of action. A systematic site-directed mutagenesis approach was employed against all the highly conserved potential catalytic residues, including serine, aspartic acid, lysine and glutamic acid, in two type IV prepilin peptidase family members (VcpD with type 4a pilin target proteins and TcpJ with type 4b pilin target proteins). It was revealed that only two conserved aspartic acid residues are required for protease activity (Asp147 and Asp212 in VcpD of Vibrio cholerae and Asp125 and Asp189 in TcpJ). It is now clear that bacterial type IV prepilin peptidases belong to a novel family of aspartic acid proteases [LaPointe and Taylor, 2000]. Alignment of archaeal enzymes reveals the conservation of these two residues (Asp18 and Asp79; M. voltae numbering), as well as three additional J Mol Microbiol Biotechnol 2006;11:167–191

177

Fig. 3. Secondary structures of (a) M. maripaludis FlaK, (b) M. voltae FlaK, and (c) S. solfataricus PibD, as predicted by TMHMM [Krogh et al., 2001]. Arrows indicate the aspartic acid residues proven or implicated (from sequence alignment) to be important in activity. This panel was generated using TOPO transmembrane protein display software (S.J. Johns and R.C. Speth) available online at http://www.sacs.ucsf.edu/TOPO/.

aspartic acid residues that are highly conserved (Asp 186, 190 and 224; M. voltae numbering). Site-directed mutagenesis was employed targeting all five conserved aspartic residues. It was revealed that Asp18 and Asp79 are of essential nature in the enzymatic activity of M. voltae FlaK. Mutations to either alanine or asparagine resulted in a loss of enzymatic activity. The conservative substitution of D79E still produced a functional preflagellin peptidase, whereas the D18E substitution resulted in an unstable protein that could not be detected by immunoblotting. On the other hand, D186A, D190A and D224A substitutions still resulted in enzymatic activity of FlaK, indicating the non-essential nature of these aspartic acid residues [Bardy and Jarrell, 2003]. Archaeal preflagellin peptidases have a similar topology to type IV prepilin peptidases. PilD is an integral cytoplasmic membrane protein with five to six predicted transmembrane helices [Strom and Lory, 1993]. Using TMHMM, FlaK of M. maripaludis and M. voltae, as well as PibD of S. solfataricus, are predicted to have 4, 5 and 5 transmembrane helices, respectively. Most aspartic acid proteases are bilobed molecules in which the active site cleft is located between the lobes with an active site aspartic acid contributed from each lobe. This feature is conserved in type IV prepilin peptidases [LaPointe and Taylor, 2000]. Based on data from site-directed mutagenesis, amino acid alignment and topology, FlaK of M. voltae and M. maripaludis are found to be consistent with this model (fig. 3a, b). An interesting deviation is the case of PibD of S. solfataricus, where the two aspartic acid 178


residues that align with the catalytically active ones in M. voltae are predicted to be located in a periplasmic loop and a transmembrane helix respectively (fig. 3c). Further studies are required to establish whether these residues are truly catalytic, and if so how the unusual predicted positioning of the residues function in the catalytic mechanism of the enzyme. In terms of substrate specificity, the prepilin peptidase is a dedicated enzyme responsible for processing the prepilins and a few other proteins that are part of a proteinexport machinery. These proteins are termed PddABCD (PilD-dependent proteins) [Strom and Lory, 1993]. Likewise, it is believed that the archaeal preflagellin peptidase is a dedicated enzyme towards the processing of flagellins. Like prepilin peptidases, archaeal preflagellin peptidases have stringent requirements towards amino acids surrounding the cleavage site. It is very possible that the preflagellins might be the only substrates of FlaK in methanogens, since methanogens do not utilize sugars as substrates (and hence do not contain sugar-binding proteins). An interesting disparity is PibD in S. solfataricus, for which broader substrate specificity is implicated based on data from a recent genomic survey. Apart from preflagellin, PibD processing of GlcS (glucose-binding protein) was also demonstrated in vitro. Site-directed mutagenesis on the GlcS signal peptide revealed that a myriad of substitutions around the cleavage site still permitted processing. The allowed substitutions were consistent with the signal peptide sequences of a list of proposed PibD substrates. From the observation that homologues of Ng/Chaban/Jarrell

Table 2. N-terminal amino acid alignment of all available archaeal flagellin sequences

% A. fulgidus

FlaB1 FlaB2

MGMRFLKNEKG MRVGSRKLRRDEKG

FTGLEAAIVLIAFVTVAAVFSYVLLGAGFFATQK FTGLEAAIVLIAFVVVAAVFSYVMLGAGFYTTQK

A. pernix

FlaB1 FlaB2

MRRRRG MRRRRG

IVGIEAAIVLIAFVIVAAALAFVALNMGLFTTQK IVGIEAAIVLIAFVIVAAALAFVALNMGLFTTQK

H. marismortui

FlaA FlaA FlaB

MFEKIANENERG MFERITNPEDRG

QVGIGTLIVFIAMVLVAAIAAGVLINTAGFLQSS MVLVAAIAAGVLINTAGFLQSS QVGIGTLIVFIAMVLVAAIAAGVLINTAGFLQSS

H. salinarum

FlgA1 FlgA2 FlgB1 FlgB2 FlgB3

MFEFITDEDERG MFEFITDEDERG MFEFITDEDERG MFEFITDEDERG MFEFITDEDERG

QVGIGTLIVFIAMVLVAAIAAGVLINTAGFLQSK QVGIGTLIVFIAMVLVAAIAAGVLINTAGFLQSK QVGIGTLIVFIAMVLVAAIAAGVLINTAGYLQSK QVGIGTLIVFIAMVLVAAIAAGVLINTAGYLQSK QVGIGTLIVFIAMVLVAAIAAGVLINTAGYLQSK

H. salinarum NRC-1

FlaA1 FlaA2 FlaA1a FlaB1 FlaB2 FlaB3

MESMRRG MFEKIANENERG MFEFITDEDERG MFEFITDEDERG MFEFITIDEDERG MFEFITIDEDERG

QVGIGTLVVFMAMILVAAMAASTLVDIGGMLQSR QVGIGTLIVFIAMVLVAAIAAGVLINTAGFLQSK QVGIGTLIVFIAMVLVAAIAAGVLINTAGFLQSK QVGIGTLIVFIAMVLVAAIAAGVLINTAGYLQSK QVGIGTLIVFIAMVLVAAIAAGVLINTAGYLQSK QVGIGTLIVFIAMVLVAAIAAGVLINTAGYLQSK

H. volcanii

FlaA1 FlaB2

MFENINEDRG MFNNITDDDRG

QVGIGTLIVFIAMVLVAAIAAGVLVNTAGFLQAT QVGIGTLIVFIAMVLVAAIAAGVLVNTAGFLQAT

Mcc. burtonii

Fla Fla Fla

MKANKHLMMNNDRA MNQWNKIIKDDKA MFKKFFNDDKA

QAGIGTLIIFIAMVLVAAVAAAVLIQTSGVLQER FTGLEAAIVLVAFVVVAAVFSYVMLGAGFYTTQK FTGLEAAIVLVAFVVVAAVFSYVMLGAGFYTTQK

M. jannaschii

FlaB1 FlaB2 FlaB3

MKVFEFLKGKRG MKVFEFLKGKRG MLLDYIKSRRG

AMGIGTLIIFIAMVLVAAVAAAVLINTSGFLQQK AMGIGTLIIFIAMVLVAAVAAAVLINTSGFLQQK AIGIGTLIIFIALVLVAAVAAAVIINTAANLQHK

M. maripaludis

FlaB1 FlaB2 FlaB3

MKIKEFLKTKKG MVKKEFMKNKKG MVKKFMKNKKG

ASGIGTLIVFIAMVLVAAVAASVLINTSGFLQQK ASGIGTLIVFIAMVLVAAVAASVLINTSGYLQQK AVGIGTLIIFIAMVLVAAIAASVIINTAGKLQHK

M. thermolithotrophicus

FlaB1 FlaB2 FlaB3 FlaB4

MKIAQFIKDKKG MKIAQFIKDKKG MKIFEFLKNKKG MLKKFFKNRRG

ASGIGTLIVFIAMVLVAAVAASVLINTSGFLQQK ASGIGTLIVFIAMVLVAAVAASVLINTSGFLQQK ASGIGTLIVFIAMVLVAAVAASVLINTSGFLQQK AVGIGTLIIFIALVLVAAVAASVIINTAGKLQHK

M. vannielii

FlaB1 FlaB2 FlaB3

MSVKNFMNNKKG MKITEFLNNKKG MMKKFLMDKKG

DSGIGTLIVFIAMVLVAAVAASVLINTSGFLQQK ASGIGTLIVFIAMVLVAAVAASVLINTSGFLQQK AVGIGTLIIFIAMVLVAAVAASVIINTAGNLQHK

M. voltae

FlaA FlaB1 FlaB2 FlaB3

MKVKEFMNNKKG MNIKEFLSNKKG MKIKEFMSNKKG MLKNFMKNKKG

ATGVGTLIVFIAMVLVAAVAASVLINTSGFLQQK ASGIGTLIVFIAMVLVAAVAASVLINTSGFLQQK ASGIGTLIVFIAMVLVAAVAASVLINTSGFLQQK AVGIGTLIIFIALVLVAAVAASVIINTAGKLQHK

M. acetivorans

Fla Fla Fla FlaB1

MKGIFSIFRKDDKA MWNRLIKNERG MWNTFSKDEKG MKGIFSIFRKDDKA

FTGLESAIVLTAFVVVAAVFSYVVLGAGFTTSDT FTGLEAAIVLIAFVVVAAVFSYVMLGAGFYTTQK FTGLEAAIVLVAFVVVAAVFSYVMLGAGFYTTQK FTGLESAIVLTAFVVVAAVFSYVVLGAGFTTSDT

M. barkeri M. mazei

Fla Fla

MWNRFIKNDKG MWKMFSKDEKG

FTGLEAAIVLIAFVVVAAVFSYVMLGAGFYTTQK FTGLEAAIVLVAFVVVAAVFSYVMLGAGFYTTQK

N. magadii

FlaB1 FlaB2 FlaB3 FlaB4

MFEQNDDRDRG MFTNDTDDGRG MFTSNTDDDRG MFVNETTDDRG

QVGIGTLIVFIAMVLVAAIAAGVLINTAGMLQTQ QVGIGTLIVFIAMVLVAAIAAGVLMNTAGMLQSQ QVGIGTLIVFIAMVLVAAIAAGVLINTAGMLQTQ QVGIGTLIVFIAMVLVAAIAAGVLINTAGMLQSQ

Archaeal Flagella, Bacterial Flagella and Type IV Pili


179

Table 2 (continued)

% N. pharaonis

FlaB1 FlaB2 FlaB3

MFETLTETKERG MFEQFTESEERG MFEQFTESEERG

QVGIGTLIVFIALVLVAAIAAGVLINTAGFLQTQ QVGIGTLIVFIALVLVAAIAAGVLINTAGFLQTQ QVGIGTLIVFIALVLVAAIAAGVLINTAGFLQTQ

P. abyssi

FlaB1 FlaB2 FlaB3

MRRG MHRKG MKNLQGGAWQMARRG

AIGIGTLIVFIAMVLVAAVAAGVLISTSGYLQQR AIGIGTLIVFIAMVLVAAVAAGVIIGTAGYLQQK AIGIGTLIVFIAMVLVAAVAAAVLINTSGFLQTR

P. furiosus

Fla Fla

MKKG

AIGIGTLIVFIAMVLVAAVAAGVLIATSGYLQQK MLVAAVAAGVIIGTAGYLQQK

P. horikoshii

FlaB1 FlaB2 FlaB3 FlaB4 FlaB5

MRRG MRKG MKKG MRRG MTVVPRKG

AIGIGTLIVFIAMVLVAAVAAAVLINTSGYLQQK AIGIGTLIVFIAMVLVAAVAAAVLINTSGYLQQK AVGIGTLIVFIAMVLVAAVAAAVLINTSGYLQQK AVGIGTLIVFIAMVLVAAVAAGVIIGTAGYLQQK AVGIGTLIVFIAMVLVAAVAAGVLITTSGYLQQK

S. solfataricus

Fla

MNSKKMLKEYNKKVKRKG

LAGLDTAIILIAFIITASVLAYVAINMGLFVTQK

S. tokodaii

Fla

MGAKNAIKKYNKIVKRKG

LAGLDTAIILIAFIITASVLAYVAINMGLFVTQK

T. kodakarensis

FlaB1 FlaB2 FlaB3 FlaB4 FlaB5

MKTRTRKG MFRGLKKRG MRFLKKRG MRRRG MRRG

AVGIGTLIVFIAMVLVAAVAAAVLINTSGYLQQK AVGIGTLIVFIAMVLVAAVAAAVLINTSGYLQQK AVGIGTLIVFIAMVLVAAVAAAVLINTSGYLQQK AIGIGTLIVFIAMVLVAAVAAGVIIGTAGYLEQK AIGIGTLIVFIAMVLVAAVAAGVLISTSGYLQQK

T. acidophilum

FlaB2 Fla

MYTVKQMPILKSLKKLRKIFQTDDSKS MRKVFSLKADNKA

ESGIGVLIVFIAMILVAAVAASVLIHTAGILQQK ETGIGTLIVFIAMVLVAAVAATVLIHTAGTLQQK

T. volcanium

Fla Fla

MYIVKKMPILKLLNSIKRIFKTDDSKA MNKLLRKVRKAFSLKADNKA

ESGIGVLIVFIAMILVAAVAASVLIHTAGILQQK ETGIGTLIVFIAMVLVAAVAATVLVHTAGTLQQK

KKG RR

A*GIGTLIVFIAMVLVAAVAASVLINTSGYLQQK G A F A

CONSENSUS SEQUENCE

The vertical arrow indicates the demonstrated or predicted sites of signal peptide cleavage from the preflagellins. In some cases, analyses of the surrounding amino acid sequences of the signal peptides with unusual lengths revealed in-frame methionines or alternative start sites that, if they represent the true translation start site, would result in signal peptides of more typical lengths. For S. solfataricus, Albers et al. [2003] used the inter-

nal start site to give a signal peptide of 13 amino acids and demonstrated signal peptide processing. Note that a few flagellins have annotated start sites that begin at a conserved methionine located 13 amino acids into the mature flagellin. Whether more typical start sites including a signal peptide are present upstream is not known at present but seems likely.

S. solfataricus sugar-binding proteins that contain type IV prepilin-like sequences were absent in the genome of another species of Sulfolobus, S. tokodaii, it is speculated that S. solfataricus PibD has undergone a specialization allowing for a broader substrate specificity [Albers et al., 2003]. Thus, the broad substrate specificity observed here is most likely an exception rather than the rule.

ger signal peptides (25 residues). In all the flagellated archaea for which sequence data is available, the typical flagellin signal peptide is 11–12 amino acids in length although longer and shorter sequences have been reported in complete annotated genomes (table 2). It is speculated that while a certain extent of flexibility might exist; some optimum length probably exists that is crucial for the juxtaposition of the signal peptide and signal peptidase with respect to each other and the membrane. However, some other archaeal preflagellins contain signal peptides of peculiar lengths. Some are annotated to be unusually long (e.g. MJ0893 of M. jannaschii and Ta1407 of T. acidophilum). These sequences, however,

Functional Limitations on Signal Peptide Length Members of the type IV pilin superfamily can be divided into two groups. The majority belongs to type 4a and contains signal peptides that are short (5–8 residues). Type 4b pilins, on the other hand, are proteins with lon180


Ng/Chaban/Jarrell

contain in-frame alternative translational start sites that, if they correspond to true translation start sites, would result in signal peptides more typical in length. On the other hand, organisms with preflagellins predicted to possess unusually short signal peptides include Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii and Aeropyrum pernix, with predicted signal peptides of only 4–6 amino acids. In addition, in the unusual case of S. solfataricus, where the preflagellin peptidase PibD was suggested to have broader substrate specificity, some predicted substrates (sugar-binding proteins) have predicted signal peptides as short as 2 amino acids. These unusual signal peptides are deduced exclusively from gene sequences with no biochemical or genetic verification. To systematically address the relationship between signal peptide length and proper processing by preflagellin peptidase, M. voltae mutant FlaB2 flagellins with truncated signal peptides were generated and used as substrates in an in vitro preflagellin peptidase assay utilizing M. voltae membranes as an enzyme source. In all the mutant proteins, the conserved amino acids surrounding the cut site were unaltered. Significant processing was observed in mutated FlaB2 proteins containing signal peptide shortened to 5 amino acids, whereas mutants with signal peptides at or below 4 amino acids in length were not processed. To study the unusual preflagellin peptidase of S. solfataricus, a his-tagged version of PibD was expressed in E. coli. Crude membranes were used as enzyme source to test against the collection of M. voltae flagellins with varying signal peptide lengths in vitro. Interestingly, the shorter signal peptides not processed by M. voltae FlaK were recognized and processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted towards cleaving shorter signal peptides. Whether the Sulfolobus PibD can complement in vivo a non-flagellated flaK knockout mutant in M. maripaludis, leading to properly processed flagellins and flagellated cells, is being investigated [S.Y.M. Ng, B. Chaban, D. VanDyke and K.F. Jarrell, unpubl. data].

Flagellin/Pilin Subunit Glycosylation

Glycosylation has been identified to various degrees among bacterial flagellins, archaeal flagellins and bacterial type IV pilins. Once thought to be a strictly eukaryotic modification, glycosylation has become recognized within all three domains of life. Several reviews have summarized current advancements in prokaryotic glycobiology [Messner, 2004; Moens and Vanderleyden, 1997; Archaeal Flagella, Bacterial Flagella and Type IV Pili

Power and Jennings, 2003; Schmidt et al., 2003; Sumper and Wieland, 1995; Upreti et al., 2003]. It is now apparent that surface-exposed proteins make up the majority of known prokaryotic glycoproteins. Despite the growing list of genera that are known or strongly suspected to have glycans modifying their flagellins or pilins, the number of well-characterized systems remains limited. This is due, in part, to the large amount of work required to identify and characterize glycan structures (through mass spectrometry and NMR analysis) and identify underlying genes and proteins responsible for glycan synthesis and attachment. Fortunately, there are well-studied examples of glycosylation in all three systems. These include bacterial flagella from Campylobacter jejuni, Helicobacter pylori and P. aeruginosa and bacterial pili from P. aeruginosa, Neisseria meningitidis and Neisseria gonorrhoeae. From archaeal flagella, glycosylation structures have been determined in H. salinarum [Sumper, 1987] and M. voltae [Voisin et al., 2005]. In addition, preliminary evidence for modification of flagellins has been presented for M. jannaschii [Giometti et al., 2001]. Indeed, an aberrantly high apparent molecular mass determined by SDS-PAGE compared to the predicted mass from the gene sequence is a regular finding in many, if not all, archaeal flagellins, indicating that posttranslational modification of some sort may be the rule for these organisms [Thomas and Jarrell, 2001]. The major information for selected glycosylation examples is summarized in table 3. One must be cautious in attempting to make generalizations about the mechanisms and requirements pertaining to each system, as available data are still limited in some cases. N- versus O-linkage Glycans can be attached to proteins through one of two linkages: N-link or O-link. N-linkages involve glycan attachment to an Asn residue within the consensus sequence Asn-Xaa-Ser/Thr, where Xaa can be any amino acid except Pro. This sequon is regarded as necessary but not sufficient for N-glycosylation in eukaryotes [Gavel and von Heijne, 1990] as well as in bacteria [Nita-Lazar et al., 2005]. In the archaeon H. salinarum, changing the serine in the Asn-Ala-Ser sequon does not prevent N-glycosylation at that site in the cell surface glycoprotein [Zeitler et al., 1998]. It is speculated that this organism has two different saccharyltransferases, one that attaches the glycan via a GalNAc (1-N) Asn linkage and a second one that attaches the glycan via a Glc (1-N) Asn linkage. The latter is a conventional N-glycosyltransferase which requires a hydroxyamino acid in the N-glycosylation seJ Mol Microbiol Biotechnol 2006;11:167–191

181

Table 3. Summary of glycosylated bacterial flagellins, bacterial type IV pilins and archaeal flagellins Species

Linkage type; site(s)

Glycan structure

Genes involved

Campylobacter jejuni

O-linkage; 19

Pseudaminic acid with heterogeneity added by substitution of acetamido groups with acetamidino and hydroxyproprionyl groups

Flagellar glycosylation locus; up to 50 genes (variable between strains) including Cj1316, Cj1293, neu and ptm genes

Helicobacter pylori

O-linkage; 7 on FlaA, 10 on FlaB

Pseudaminic acid

4 genes confirmed; neuA (HP0326a), flmD (HP0326b), neuB (HP0178), HP0114

Pseudomonas aeruginosa

O-linkage; 2 (type a flagellin only)

Heterogeneous structure with linking rhamnose; 2–7 sugars comprised of pentose, hexose, deoxyhexose or hexuronic acid; deoxyhexosamine; deoxyhexose and terminal 174 Da unknown modification

Glycosylation island containing orfA-orfN (long island) or orfA-orfH and orfN (short island)

Pseudomonas aeruginosa

O-linkage; 1 (Ser148)

Trisaccharide with terminal -5-N-hydroxybutyryl-7-N-formyl-pseudaminic acid-(2-4)--xylose-(1-3)--Nacetylfucosamine-(1-3)--Ser

pilO and O-antigen pathway

Neisseria meningitidis


Galactose-(1,4)-galactose-(1,3)-2,4diacetamido-2,4,6-triedoxyhexose-Ser

Polymorphic locus containing 4–8 genes; pglA-H

Neisseria gonorrhoeae


Galactose-(1,3)-N-acetylglucosamine-Ser

Polymorphic locus containing 4–8 genes; pglA-H

Halobacterium salinarum

N-linkage; 3 on each of FlaA1, FlaA2, FlaB1, FlaB2 and FlaB3

Four sugars: Asn-glucose-(1-4)-HexUA-(1-4)HexUA-(1-4)-HexUA or glucose where HexUA is sulfated either glucuronic acid or iduronic acid (3:1 ratio)

Unknown

Methanococcus voltae

N-linkage; 2 on FlaA, 5 on FlaB1, 6 on FlaB2 and 2 on FlaB3

Trisaccharide with 2-acetamido-2-deoxymannuronic acid, with the carbonyl group at position C-6 forming an amide bond with the amino group of threonine, linked (1-4) to 2,3-diacetamido-2,3-dideoxy-glucuronic acid linked (1-3) to 2-acetamido-2-deoxy-glucose

Two known; STT3 homolog and glycosyl transferase

Bacterial flagella

Type IV pili

Archaeal flagella

quon while the former enzyme is not dependent on the hydroxyamino acid. Archaeal flagellins possess N-linkages and of all the available archaeal flagellin sequences, 61 have at least one N-glycosylation sequon. The most are found in M. thermolithotrophicus with 16 in FlaB2, 9 in FlaB1, 5 in FlaB3 and 3 in FlaB4. None are found in the flagellins of H. marismortui, although one flagellin has an apparent incorrect start site (GenBank accession No. YP_137938). No N-glycosylation sequons are present in the flagellins of M. mazei or M. barkeri although each of the 3 flagellins of M. acetivorans has 2 sites. A. pernix has 1 flagellin with 1 site and the other flagellin has none. In Natriaba, 1 of the 4 flagellin lacks an N-glycosylation site. Alternatively, the attachment sites for O-linkages are less clearly defined, with the glycan covalently linked to a Ser 182


or Thr residue. To date, the well-characterized glycan structures on bacterial flagellins and pilins are Olinked. Very little is known regarding where in the cell the different glycan linkages are assembled in prokaryotes. In eukaryotic cells, N-linked glycans are preassembled on lipid carriers in the endoplasmic reticulum membrane using nucleotide-activated sugars. Following a ‘flipping’ of the carrier to the lumen side of the endoplasmic reticulum, the glycan is transferred to the target protein via a membrane-bound oligosaccharyltransferase [Alberts et al., 2002; Moens and Vanderleyden, 1997]. A similar system has been proposed for N-linked glycans on proteins (not flagellins) of Campylobacter [Szymanski et al., 2003], where the final transfer of the assembled glycan to the Ng/Chaban/Jarrell

target protein occurs on the periplasmic side of the cytoplasmic membrane, the equivalent of the lumen of the endoplasmic reticulum in eukaryotes. Direct evidence that N-linked glycosylation occurs in the periplasm was recently obtained with AcrA in E. coli [Nita-Lazar et al., 2005]. In archaea, several lines of evidence also suggest an extracellular transfer of the glycan to the target proteins in the N-linked glycosylation of flagellins and the cell surface glycoprotein. Bacitracin, which has been demonstrated to inhibit flagellin glycosylation in M. maripaludis [Bayley et al., 1993] and Halobacterium spp. has also been documented as being unable to enter cells [Lechner and Wieland, 1989]. It did not interfere with the transfer of sulfated oligosaccharides that are O-linked to the cell surface glycoprotein [Sumper, 1987]. Furthermore, in vivo studies with a synthetic hexapeptide containing the N-linked acceptor sequon demonstrated that the peptide could be glycosylated but could not enter the cell, again indicating an extracellular site of transfer of the glycan via the oligosaccharyltransferase [Sumper, 1987]. It has been suggested that N-linked glycosylation in eukaryotes is derived from a similar system in archaea, based on the observation of clusters of orfs in Archeoglobus potentially involved in this process [Burda and Aebi, 1999]. The cellular location of O-linked glycosylation, especially in prokaryotes, is poorly studied. O-linked -Nacetylglucosamine found on Ser and Thr residues in cytoplasmic proteins of eukaryotes occurs via the activity of enzymes found in the cytosol [Vosseller et al., 2001]. Biosynthesis of the O-glycans of the N-acetylgalactosaminyl-Ser/Thr linkage type (so-called mucin-type) involves glycosyltransferases possessing a signal anchor which is embedded in the Golgi membrane with an orientation such that the transferase reactions occur on the inside of the lumen of the Golgi [Brockhausen, 1995]. The generation of a lipid-linked oligosaccharide precursor for transfer to a protein is not required [Alberts et al., 2002; Varki et al., 1999]. In bacteria, the location of the glycosylation system is not known. However, in the case of the AAH heptosyltransferase responsible for the carbohydrate attachment of the AIDA-I adhesion of E. coli, its location in the cytoplasm [Moorman et al., 2002] might indicate that the O-glycosylation in this system occurs in the cytoplasm [Benz and Schmidt, 2002]. Gerwig et al. [1992] reported different stages of an O-linked glycan on the glycoproteins from cellulosomes in Bacteroides cellulosolvens and Clostridium thermocellum, suggesting that the mechanism involved addition of monosaccharides directly to the target protein, analogous to eukaryotic O-glyco-

sylation [Moens and Vanderleyden, 1997]. Where in the cell the attachment of O-linked glycans occurs on bacterial flagellins is completely unknown, although it has been hypothesized that this is a cytoplasmic event [Doig et al., 1996; Upreti et al., 2003]. Another possibility is that the glycosylation occurs near the site of export into the central channel at the base of the MS ring. The fact that no bacterial flagellins have yet been reported with N-linked glycosylation makes the finding of the N-linked glycans in the two archaeal cases (H. salinarum and M. voltae) intriguing from an assembly viewpoint. If O-linked glycosylation occurs in the cytoplasm or on the cytoplasmic side of the cytoplasmic membrane, then bacterial flagellins could be glycosylated prior to their entry into the central channel of the flagellum. However, N-linked glycosylation, which occurs on the periplasmic side of the cytoplasmic membrane, requires that the flagellin target be inserted into the cytoplasmic membrane with its glycosylation sequons accessible to the oligosaccharide transferase on the external side of the cytoplasmic membrane. Unless this machinery is present in the patch of membrane located within the MS ring, the same location as the type III secretion apparatus, then this would not be a viable option for N-linked glycosylation in bacterial flagellins. The fact that archaeal flagellins are N-linked indicates that they are inserted into the cytoplasmic membrane for the glycosylation to occur (as well as for signal peptide removal). This would allow for incorporation of the subunits at the base from their membrane location as is hypothesized for the type IV pilin subunits. An alternative, that de-insertion of the glycosylated flagellins then occurs for subsequent passage through the interior of the filament, for growth at the distal tip, seems much less likely. The lack of a detectable central channel in archaeal flagellins would further preclude this. The fact that the M. voltae S-layer proteins carry the same N-linked glycan as the flagellins indicates that the glycosylation process occurs as a general cytoplasmic membrane function, not one localized to the base of flagella. The possibility that the glycosylation machinery also occurs at the base of the flagella as part of the export process for the flagellins exists. Again, however, the lack of a hollow flagellum structure in archaea suggests that glycosylation and signal peptide removal simply occur in the cytoplasmic membrane.



Glycan Synthesis Pathways Although a unique glycan is constructed by each organism (table 3), generalizations can be made as to how the glycans are assembled. It appears that both archaeal 183

flagella and bacterial pili tap into other cellular pathways for the synthesis of their glycans. Structural analyses have revealed that the structures found on the flagellin of H. salinarum [Sumper, 1987] and M. voltae [Voisin et al., 2005] are identical to the S-layer protein glycans of the cell. Consistent results were seen in mutation analysis, where mutations causing alterations of the M. voltae glycan found on flagellins also caused corresponding alterations in the S-layer protein [Chaban et al., 2006]. Although in both cases it is known or suspected that the S-layer proteins contain further unique glycan modifications, the one glycan assembly pathway is shared by both proteins. The glycans found on type IV pili are identical to the O-antigens in P. aeruginosa. Mutants lacking functional genes involved in O-antigen formation (wbpM and wbpL) synthesized no O-antigen and produced only non-glycosylated pilin. Furthermore, the introduction of an alternate O-antigen biosynthetic gene cluster (LPS serotype O11) into P. aeruginosa 1244 (LPS serotype O7) resulted in 1244 pili that contained both O7 and O11 antigens, clearly indicating the sharing of the precursor glycan pool [DiGiandomenico et al., 2002]. The pathway used by N. meningitidis is less clear. The gene locus pglA-D has been identified and characterized as part of the N. meningitidis pilin glycosylation pathway [Power et al., 2000]. There is homology and similar gene order of this N. meningitidis pgl locus to the pgl locus found in C. jejuni. The pgl locus in Campylobacter has been characterized as a general N-linkage glycosylation pathway used for several different proteins (not including flagellin) [Szymanski et al., 2003]. This homology exists even though this cluster is used for O-linked glycosylation in N. meningitidis, as opposed to N-linked glycosylation in C. jejuni. Whether the pgl pathway is dedicated for pilin glycosylation in N. meningitidis, or whether it also represents a general glycosylation pathway for proteins as yet unrecognized, remains to be determined. On the other hand, dedicated glycosylation genes and loci for bacterial flagellin modification have been discovered. Campylobacter species contain several variations of a flagellar glycosylation locus, independent of the pgl locus. With up to 50 genes implicated in the glycosylation process, the complete locus is found in C. jejuni 11168; truncated versions with various alterations are found in C. jejuni 81–176 and Campylobacter coli VC167 [Szymanski et al., 2003]. At the present time, several genes within this locus are still hypothetical orfs. Similarly, a glycosylation island in P. aeruginosa has been found. Fourteen genes, orfA-N, have been identified and were shown to be 184


essential for glycosylation [Arora et al., 2001]. Further characterization of this locus determined that two versions existed: a long island containing all 14 genes, leading to a A1-subtype flagellin and a short island, in which orfD, -E and -H are polymorphic and orfI, -J, -K, -L and -M were absent, leading to a mixture of A1- and A2-subtype flagellins [Arora et al., 2004]. Finally, although the genes involved in H. pylori glycosylation do not appear to be part of a larger locus, at the present time, their known activity is limited to flagellar glycosylation [Schirm et al., 2003]. Glycan Assembly and Attachment In eukaryotic glycosylation, O-linked glycan structures are assembled sugar by sugar directly onto the target protein. Conversely, N-linked glycans are assembled separate from the target protein and are attached en bloc after completion [Alberts et al., 2002]. For N. meningitidis pilins, mutants defective in either pglA (attach galactose to glycan) or galE (synthesize galactose) have truncated glycans containing only the linking sugar [Power et al., 2000]. These mutations had no effect on LPS. Alternatively, knockouts of intermediate enzymes in P. aeruginosa glycan assembly (wbpM and wbpL) completely prevented pilin glycosylation as well as proper LPS synthesis [DiGiandomenico et al., 2002]. Within the bacterial flagellin glycosylation systems of C. jejuni, H. pylori and P. aeruginosa, mutational studies that have inactivated various glycosylation genes in the pathways have abolished flagellin glycosylation of either assembled (for P. aeruginosa) or unassembled (for C. jejuni and H. pylori) flagellin [Arora et al., 2001; Goon et al., 2003; Schirm et al., 2003]. Why knockouts of genes within bacterial flagellin glycosylation pathways have not yielded truncated glycans remains to be explained. Finally, within M. voltae, the inactivation of the homologue of the STT3 protein (known to transfer complete glycans to their target proteins in eukaryotes) completely inhibits archaeal flagellin glycosylation, as expected [Chaban et al., 2006]. Attachment Specificity Attachment specificity of the glycan to the flagellin/ pilin is variable. In the case of P. aeruginosa pilin, a complete glycan of a different serotype (O11 vs. O7) could readily be attached. In this case, it was hypothesized that the recognition of the glycosylation substrate may be based on a combination of molecule size and the presence of some common structural elements [DiGiandomenico et al., 2002]. As well, M. voltae flagellins have been detected that contain an intermediate glycan structure. Ng/Chaban/Jarrell

When a glycosyltransferase gene is disrupted, flagellins migrate through SDS-PAGE faster than wild-type flagellin (fully modified) but slower than exogenously produced M. voltae flagellins from E. coli (fully unmodified). Mass spectrometry analysis indicates that these flagellins are lacking the terminal sugar of the trisaccharide structure [Chaban et al., 2006]. This indicates that the transfer of the glycan (most likely by the M. voltae STT3 homolog) is not specific for the entire structure, since this partial structure can also be used as a substrate. Glycan Location on Target Protein Bacterial flagellins, archaeal flagellins and type IV pilins all appear to have surface-exposed glycans. The bacterial flagellin linkage sites for C. jejuni, H. pylori and P. aeruginosa have all been identified within the central, surface-exposed domain of the flagellin protein [Schirm et al., 2003, 2004; Thibault et al., 2001]. The X-ray crystallographic structure of the N. gonorrhoeae pilin illustrated that the glycan on Ser63 is also surface-exposed [Forest et al., 1999]. As well, a glycan-specific monoclonal antibody was able to inhibit twitching motility of P. aeruginosa pili, also indicating the pilin glycan is surface-exposed under physiological conditions [Castric et al., 2001]. Within the archaeal flagellin, the 15 N-linkage sites on 5 flagellins for H. salinarum glycans and the 15 N-linkage sites on the 4 flagellins on the M. voltae are likely to be surface-exposed as well, although this remains to be proven.

ternatively, P. aeruginosa is able to assemble functional flagella without glycosylation, assessed by the faster migration of flagellins through SDS-PAGE and the lack of any impairment of motility on swarming plates [Arora et al., 2001]. It is worth noting that P. aeruginosa has only two glycan modifications per protein whereas C. jejuni and H. pylori have from 7 to 19 glycans per protein (table 3). Whether this decrease in overall glycosylation is a factor in this phenomenon remains unknown. Finally, glycosylation of type IV pili appears to be independent of pili assembly. For example, many species of Pseudomonas, including widely used laboratory strains PAO and PAK, produce only non-glycosylated pili [Castric et al., 2001; Frost and Paranchych, 1997; Paranchych et al., 1979]. For P. aeruginosa, glycosylation-defective mutants showed no adverse effects in twitching motility [Castric et al., 2001]. In addition, N. meningitidis glycosylation mutants also showed no adverse effects on motility or structure, with electron microscopy revealing no differences between wild-type and mutant pili [Power et al., 2000]. Instead of assembly, it has been proposed that glycosylation of pili plays an important role in immune avoidance. For example, glycosylation of Neisseria pili may play a significant role in the resistance of meningococci to complement-mediated lysis [Power and Jennings, 2003]. As well, the terminal sugar in P. aeruginosa has the same basic structure as sialic acid (a common host modification) and could cause biological masking [Castric et al., 2001].

Requirement of Glycan for Structure Assembly One hypothesis for flagellin/pilin glycosylation is that the glycan is necessary for the assembly of the flagella/pili filament. Whether this holds true depends on the system being studied. In archaeal flagella, the complete glycan appears necessary for proper assembly. In M. voltae, a mutant that contained completely unmodified flagellin (STT3 homologue mutant) was non-motile by light microscopy and non-flagellated by electron microscopy and a mutant in which attachment of the terminal sugar to the glycan was disrupted (glycosyltransferase mutant) was only very poorly flagellated [Chaban et al., 2006]. In bacteria, glycosylated flagellins are necessary for flagella assembly for some species, but not all. Both C. jejuni and H. pylori have impaired flagella assembly without glycosylation of their flagellins. In each case, unglycosylated flagellin was detectable intracellularly, but no motility was observed with swarm plates [Schirm et al., 2003] and no flagella filaments were seen by electron microscopy [Goon et al., 2003; Schirm et al., 2003]. Al-

Other Posttranslational Modifications Although not as commonly recognized or studied, other posttranslational modifications exist on flagella and pili. The N. gonorrhoeae pilin contains a glycerophosphate on Ser94 and a phosphoethanolamine on Ser68 in a wild-type background or a phosphocholine on Ser68 in a pilV– background [Forest et al., 1999; Hegge et al., 2004]. This is in addition to the glycan at Ser63 (table 3). Phosphorylation has also been detected on Pseudomonas flagellin a- and b-types [Kelly-Wintenberg et al., 1993]. In archaea, the possibility of phosphorylation of the flagellins in N. magadii based on specific staining has been suggested [Polosina et al., 1998].



Model of Assembly

Limited information exists pertaining to the assembly of the archaeal flagella. Thus far, only one gene cluster consisting of up to 13 genes has been identified that is in185

volved in archaeal flagellation, compared to the estimated 35 and 50 genes required for the assembly, structure and function of type IV pili and bacterial flagella, respectively [Donnenberg et al., 1997; Mattick, 2002]. Even among this fla gene cluster, the functions of many genes remain unknown. However, we have proposed a mechanism of assembly of the archaeal flagella that includes several fundamental features of the bacterial type IV pilus system. This is based on the following reasons. First, the archaeal flagellins share significant sequence similarities with the type IV pilins and both require signal peptide cleavage prior to assembly into the growing filament. Second, the presence or absence of a hollow internal channel and its size has critical implications for potential models of assembly. It has long been known that bacterial flagella grow by insertion of the new flagellin subunits at the distal tip, i.e. the point furthest from the cell surface [Iino, 1969]. This is done via a type III secretion system with a specific export system for flagellar structural proteins located in the patch of membrane within the MS ring of the basal body [Macnab, 1999] and is possible due to the hollow nature of the entire flagellum structure. Recently, a complete atomic model of the bacterial flagellar filament has been determined by electron cryomicroscopy [Yonekura et al., 2003]. Strikingly, it reveals a hydrophilic channel of only 2 nm diameter (rather than the earlier estimate of 3 nm) to allow the passage of the flagellin monomers. Since main chain and side chain atoms extend into the central channel, its effective diameter is even smaller, suggesting that considerable unfolding of the flagellin monomers must occur to allow passage through the filament. Structural work has been reported for several type IV pili. Structural and X-ray fiber diffraction analyses of P. aeruginosa PAK and the closely related strain PAO pili indicate hollow cylinders with a 5.2 nm outer diameter and 1.2 nm inner diameter [Folkhard et al., 1981; Keizer et al., 2001]. This hole is again effectively smaller due to the extension of side chains into it. Unlike the case with flagella however, the center of the pilus is essentially hydrophobic [Keizer et al., 2001]. A structure-based model of the PAK pilus filament by Craig et al. [2004] determined that it has a 5.8-nm outer diameter and a central channel of 1.8 nm, 1.6 nm at its narrowest. For the V. cholerae TCP, the outer diameter is larger at 8 nm but the internal diameter is thinner at 1 nm, only 0.5 nm at its narrowest [Craig et al., 2003, 2004]. The smaller hollow channel is believed to be too small and hydrophobic for the passage of DNA or RNA as was once proposed but may allow for the passage of small organic molecules [Craig et al., 2003; Keizer et al., 186


2001]. We speculate the small diameter and hydrophobicity of the channel to also be too small for the passage of protein subunits. In contrast to both bacterial flagella and type IV pili, the limited data available for archaeal flagellar filaments indicates that there is no evidence for a central channel [Cohen-Krausz and Trachtenberg, 2002; Trachtenberg et al., 2005]. Third, the N-linkage of glycans on archaeal flagellins means that glycosylation occurs at the external face of the cytoplasmic membrane, a location seemingly more easily reconciled to a cell proximal site of incorporation of new subunits rather than the distal site of growth in bacterial flagella (see previous section). In our working model (fig. 4), it is assumed, based on the aforementioned reasons, that assembly of the archaeal flagellum occurs by insertion of new subunits at the base. Flagellin-specific chaperones bind to the preflagellins in the cytoplasm, which prevents their aggregation through their hydrophobic N-termini. The monomers are escorted to the cytoplasmic membrane, where processing of the signal peptide by the membrane-bound preflagellin peptidase occurs. Posttranslational glycosylation also takes place, and this modification occurs on the exterior of the cytoplasmic membrane. It is notable that while the exact order of events is unknown, it has been demonstrated that glycosylation of flagellins is independent of signal peptide removal and vice versa [Bardy and Jarrell, 2003; Chaban et al., 2006]. Almost all type IV pilus and archaeal flagella systems contain multiple pilins/flagellins or pilin-like proteins, nucleotide-binding proteins, a polytopic cytoplasmic membrane protein and a specific signal peptidase. In addition, the pilus systems contain an outer membrane secretin [Mattick, 2002], not found in the archaea which generally have no outer membrane. Furthermore, there are proteins of unknown function that potentially could form minor parts of the pilus/flagellum structure or be involved in the assembly process. This is in contrast to bacterial flagella systems where the identification and function of almost all the proteins is established [Minamino and Namba, 2004]. The roles of FlaCDEFGH in archaeal flagellation remain unknown. Overexpression data indicates that these proteins fractionate to the membrane. Taken together with the observation that several archaeal species have one large gene with significant similarity to flaC, D and E, it is possible that in species where these are found as separate proteins they interact directly forming some kind of a complex basal structure or subassembly structure required for biogenesis. Evidence for numerous proNg/Chaban/Jarrell

S.L.

5,6 C.M.

1

2

FlaK

4

7 FlaJ FlaI FlaH

3 C FlaA FlaB1 FlaB2 FlaB3

Fig. 4. A speculative model for the structure and assembly of the M. voltae flagellum. Steps 1–3: the assembly of the glycan onto a lipid carrier, one sugar at a time; step 4: the assembled glycan is translocated to the periplasmic side of the cytoplasmic membrane; steps 5 and 6: upon the arrival of the preflagellin at the cytoplasmic membrane (escorted by a chaperone), glycosylation and preflagellin peptide removal takes place. The exact order of these two events is unknown; step 7: the processed flagellin is incorporated into the growing flagellar filament.

teins of the type IV pilus system interacting has been presented in different systems. Recently, for example, Crowther et al. [2004] showed that at least 10 of the 14 proteins encoded by the E. coli bfp operon can be chemically crosslinked, suggesting that they form an oligomeric complex, possibly forming a type IV pilus subassembly complex of cytoplasmic proteins and cytoplasmic ATPbinding proteins. Using a yeast two-hybrid system, multiple interactions (through the N-terminus) were demonstrated between BfpC, D, E and F. BfpC and E are cytoplasmic membrane proteins, while BfpD and F (putative nucleotide-binding proteins) are believed to be cytoplasmic. It has been previously suggested that the ATPase and conserved polytopic membrane-spanning protein of the

pilus system may interact [Alm and Mattick, 1997]. By analogy, the archaeal homologues of these proteins in the flagella system would be expected to also interact. Recent evidence in many type IV pili systems has shown that surface translocation is accomplished by extension and attachment of the pili followed by pili retraction which pulls the cell forward [Li et al., 2003; Merz et al., 2000; Skerker and Berg, 2001]. Evidence has been presented in Neisseria that the retraction of the pilus fiber involves translocation of the pilin subunits into the cytoplasmic membrane [Morand et al., 2004]. It has been speculated that these retracted pilin monomers can be recycled into new fibers at a later point.



187

While rotation and not extension/retraction is known to be the mode of action of the archaeal flagella, the flagella gene cluster does contain a bacterial pilB homologue, flaI [Bayley and Jarrell, 1998]. FlaI contains an ATP-binding domain, and could act to supply the energy for filament assembly in a way that is analogous to the PilB mediated pilus assembly at the base. This cell proximal process is completely different from the type III secretion apparatus located at the base of the bacterial flagellar MS ring, which is responsible for sending subunits through the hollow growing structure for assembly at the distal end.

Concluding Remarks

Despite recent exciting progress, the current knowledge on the archaeal flagella system is but the tip of the iceberg. While the known characteristics allow us to draw parallels to the bacterial type IV pilus system, and in a few limited cases the bacterial flagella system, it is apparent that the archaeal flagella system contains unique features, many of which remains to be unveiled. First, it is of great interest to unmask the functions of flaC, D, E, F and G, which have no bacterial homologues yet are found between flagellin genes and flaHIJ in many flagellated archaeal species. Interestingly, none of these genes have yet been found in any archaea lacking flagellin genes. To establish the roles each of these genes play in flagellation, a recently developed technique [Moore and

Leigh, 2005] is being employed to generate in-frame deletional mutants of these genes in M. maripaludis. Secondly, it seems unlikely that the one major cluster of genes containing the flagellins and the fla-associated genes is all that are required for flagella assembly and function. For type IV pili assembly and function in P. aeruginosa, about 35 genes [Mattick, 2002] are needed (although much smaller numbers needed for the bfp of E. coli [Stone et al., 1996; Tobe et al., 1996]) and bacterial flagella require the contribution of more than 50 genes for assembly and function [Chilcott and Hughes, 2000]. Since these additional genes in archaea cannot be found through homology searches, other approaches must be taken in identifying these genes. All archaea have a significant percentage of their genes that are of unknown function and specific to archaea. It is expected that some of these are probably involved in flagellation. Experiments are underway to identify some of these genes in M. voltae. Random insertional mutagenesis has been employed to generate several stable non-motile mutants in M. voltae [unpubl. data]. The location of the insertion event is currently being determined to identify the genes affected, hopefully identifying additional clusters of novel archaea specific flagellation/motility genes. Finally, the paramount question of polarity of growth of the archaeal flagella still remains, and efforts should be dedicated to generating direct evidence to prove/disprove the current assembly model.

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