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Keywords:

  • Archaea;
  • Transcriptional analysis;
  • Evolution;
  • Flagellin;
  • Hyperthermophile;
  • Pyrococcus

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

Five clustered genes (flaB1, flaB2, flaB3, flaB4 and flaB5) for multiple subunits of flagellar filaments from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 were cloned and sequenced. Deduced amino acid sequences were aligned and it was revealed that five flagellin genes are homologous especially in the N-terminal hydrophobic region which might be important for interaction among flagellin subunits. Phylogenetic analysis was performed among archaeal flagellins from methanogens, an extreme halophile and our hyperthermophile, indicating that KOD1 flagellins were grouped together with methanogenic counterparts and were distinguishable from halophilic flagellins. Northern analysis of transcripts from flagellin genes from P. kodakaraensis KOD1 revealed that four major transcripts (0.98, 3.7, 5.4 and 9.2 kb) initiating from immediately upstream of flaB1 encode different combinations of five flagellins.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

The phylogenetic tree based on 16S rRNA or protein sequences shows that all organisms are divided into three domains, bacteria, eukarya and archaea [1,2]. Many proteins produced in the archaeal domain have eukaryal features and that fact indicates a close evolutionary relationship between archaea and eukarya. Members of the hyperthermophilic group within the archaeal domain have evolved most slowly retaining many ancestral features of higher eukaryotes [3]. Pyrococcus kodakaraensis KOD1 is a newly isolated hyperthermophilic archaeon from a solfatara at a wharf of Kodakara Island, Kagoshima, Japan [4].

Flagellation is common to all the three domains of bacteria, eukarya and archaea. Although the bacterial flagellar filament is usually composed of a single component named flagellin, archaeal and eukaryal flagellar filaments are composed of multiple components [5–10]. Studies of archaeal flagella have been concentrated on methanogens and extreme halophiles [5–7]. Transcriptional units have been studied for multiple flagellin genes from Halobacterium salinarum and Methanococcus voltae. In H. salinarum, the flagellin genes are transcribed as two transcriptional units, one for two flagellin genes (flgA1 and flgA2) and the other for three flagellin genes (flgB1, flgB2 and flgB3) [5]. In M. voltae, the flagellin genes are transcribed as two transcriptional units, one for one flagellin gene (flaA) and the other for three flagellin genes (flaB1, flaB2 and flaB3) with unknown open reading frames (ORFs) [6]. The similar arrangement of flagellin genes is found in Methanococcus jannaschii. However, M. jannaschii lacks a flaA homologue [11]. Limited information is available for hyperthermophilic flagellins [8,9], although it would be interesting to know how the flagella of hyperthermophiles can be stably maintained on the cell surface under an extremely high temperature.

In the present study, we report cloning, sequencing and transcriptional analysis of the five multiple flagellin genes from the hyperthermophilic archaeon P. kodakaraensis KOD1. In addition, based on phylogenetic analysis, we investigated the relationship among flagellins from three archaeal groups (methanogens, extreme halophiles and hyperthermophiles).

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

2.1Bacterial strains, plasmids and growth conditions

P. kodakaraensis KOD1 was cultured as previously described [4]. Escherichia coli strain JM109 was used as a host for plasmids. E. coli strain XL1-Blue MRA (P2) (Stratagene, La Jolla, CA, USA) was used for infecting phage lambda. Plasmids pUC18, pUC19 and pUC119 were used as vectors for cloning and sequencing of flagellin genes.

2.2DNA manipulation

General DNA manipulations including plasmid preparations, digestion with restriction enzymes, ligation, transformation of E. coli and gel electrophoresis were performed according to standard protocols [12]. Plasmids for DNA sequencing were extracted and purified by Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI, USA). A GeneClean II kit (Bio 101, La Jolla, CA, USA) was used for extraction of DNA fragments from agarose gels. Polymerase chain reaction (PCR) [13] was performed on a GeneAmp PCR System 2400 (Perkin Elmer) using Ex Taq DNA polymerase (Takara Shuzo, Kyoto, Japan). PCR for obtaining flagellin genes was performed using two primers designed referring to both 5′- and 3′-ends of flagellin genes from various microorganisms (forward primer: 5′-CCATCCATGGCACAAGTCATTAATAC-3′, reverse primer: 5′-CCGAATTCTTAACCCTGCAGCAGAGACA-3′) as follows: 94°C for 1 min, 30 cycles of 94°C for 30 s/55°C for 30 s/72°C for 1 min [14,15].

2.3Construction of a genomic library in phage lambda and screening

P. kodakaraensis KOD1 genomic DNA was prepared as previously described [16]. A lambda phage library of P. kodakaraensis KOD1 was constructed by ligating genomic DNA partially digested with EcoRI into EcoRI-digested arms of lambda EMBL4 (Stratagene, La Jolla, CA, USA). The positive phage clone was screened by plaque hybridization using the PCR product as a probe according to the DIG DNA Labeling kit (Boehringer Mannheim, Mannheim, Germany). The phage DNA containing entire flagellin genes were digested with different restriction enzymes, separated on 1% agarose gels and fragments of appropriate sizes were excised and purified. The fragments were then cloned into pUC19 for sequencing.

2.4DNA sequence analysis

The nucleotide sequence was determined by the dideoxy chain termination method [17] using the ALF Auto-Read Sequencing kit (Amersham Pharmacia Biotech UK, Buckinghamshire, UK). The DNA sequence was analyzed using the DNASIS software (Hitachi software, Yokohama, Japan). The phylogenetic tree was constructed using the ODEN program (National Institute of Genetics, Mishima, Japan) and Clustal W [18]. The sequence of the P. kodakaraensis KOD1 flagellin genes has been deposited in the EMBL/DDBJ/GenBank data library under accession number AB018434.

2.5Isolation of RNA and Northern hybridization

Exponential phase cells from 1 l culture were harvested by centrifugation (7000×g for 15 min). Then, total cellular RNA was isolated by the method of Chomczynski and Sacchi [19]. Isolated RNA was electrophoresed through 1% agarose, 2.2 M formaldehyde gels and transferred to a nylon membrane Hybond-N+ (Amersham Pharmacia Biotech UK, Buckinghamshire, UK). Following transfer, the blots were dried at room temperature and then baked at 80°C for 2 h. Blots were pre-hybridized in Rapid-hyb buffer (Amersham Pharmacia Biotech UK, Buckinghamshire, UK) at 42°C for 1 h. The probes were labelled by T4 polynucleotide kinase (Gibco BRL, Rockville, MD, USA) or the rediprime DNA labelling system (Amersham Pharmacia Biotech UK, Buckinghamshire, UK). The labelled probes were added to hybridization solution and allowed at 42°C for 2 h. Membranes were washed at room temperature for 20 min in 2×SSC, 0.1×SDS and then at 42°C for 15 min in 1×SSC, 0.1×SDS and exposed on an X-ray film.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

3.1Nucleotide sequence of flagellin genes

In order to obtain homologues of flagellin genes from P. kodakaraensis KOD1, two primers were designed for PCR referring to 5′- and 3′-ends of nucleotide sequences of bacterial flagellin genes. The 600-bp DNA fragment was amplified by PCR and the fragment was sequenced. The nucleotide sequence revealed that part of the flagellin gene was contained. A lambda phage library was constructed from chromosomal DNA of P. kodakaraensis KOD1 and a positive clone containing the entire flagellin genes was selected from the library. The obtained fragment (14 kb) was subcloned into pUC19 and sequenced.

Five clustered flagellin genes could be identified by nucleotide sequence analysis (Fig. 1). Since the deduced amino acid sequences of these flagellin genes exhibited a higher similarity to those of M. voltae than those of H. salinarum on the whole and exhibited a higher identity to flaB genes from M. voltae rather than the flaA gene from M. voltae as shown in Table 1, these genes were designated as flaB1, flaB2, flaB3, flaB4 and flaB5, respectively, from the upstream region (Fig. 1). Among archaeal flagellin genes, three tandemly arranged flagellin genes (flaB1, flaB2 and flaB3) were found in M. voltae, M. jannaschii, Methanococcus vannielii and H. salinarum[5–7,11]. In the case of P. kodakaraensis KOD1, however, five flagellin genes, flaB1flaB5, are tandemly arranged and separated by a short distance, suggesting that the flagellin genes are expressed by polycistronic mRNA, which is distinct from the reported flagellin genes from methanogens and a halophile.

image

Figure 1. Schematic map of KOD1 flagellin genes. The numbers above the thick lines indicate the length (bp) between ORFs. The numbers in boxes indicate the length of amino acid sequences encoded by flagellin genes. The numbers under gene names denote the length of flagellin genes. The arrows show the transcriptional direction of flagellin genes.

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Table 1.  The overall amino acid sequence similarities of KOD1 and archaeal flagellins
P. kodakaraensis KOD1P. kodakaraensis KOD1M. voltaeH. salinarum
 FlaB2FlaB3FlaB4FlaB5FlaAFlaB1FlaB2FlaB3FlgA1FlgA2FlgB1FlgB2FlgB3
FlaB133534547394144423333323433
FlaB2 332731252726272322232323
FlaB3  4749394647443534353534
FlaB4   49394346423533363636
FlaB5    363942383132323331
The values indicate the percentage identities.

3.2Sequence comparison of archaeal flagellins

17 Deduced amino acid sequences of N-terminal regions of archaeal flagellins from P. kodakaraensis KOD1, M. voltae, M. jannaschii and H. salinarum were aligned as shown in Fig. 2. Amino acid sequences of the N-terminal regions of archaeal flagellins are highly conserved. The hydropathy profiles [20] of five flagellins, FlaB1, FlaB2, FlaB3, FlaB4 and FlaB5, of KOD1 were compared as shown in Fig. 3. The conserved N-terminal region of each flagellin was very hydrophobic and this hydrophobic N-terminal region could be found in halophilic and methanogenic flagellins [6]. This N-terminal region may have an important function for assembly of flagellins through hydrophobic interactions.

image

Figure 2. Conserved N-terminal amino acid sequences of five flagellins from P. kodakaraensis KOD1 aligned with four flagellin sequences from M. voltae, three flagellin sequences from M. jannaschii and five flagellin sequences from H. salinarum. Identical amino acid acids in the same position for all the flagellins are shaded dark, while similar amino acids are lightly shaded.

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image

Figure 3. Hydropathy plots from deduced amino acid sequences for KOD1 flagellins. The hydropathy plots were performed by the method of Kyte and Doolittle [20] with a window size of 11 amino acids.

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3.3Phylogenetic analysis

The alignment for 17 amino acid sequences of archaeal flagellins from P. kodakaraensis KOD1, M. voltae, M. jannaschii and H. salinarum was based on pair-wise. From total amino acid sites (639 amino acid positions), 473 sites with at least one gap in any sequence were excluded. Based on the remaining 166 amino acid sites, a phylogenetic tree of archaeal flagellins was constructed. As a consequence, an unrooted tree was drawn based on amino acid sequences by the NJ (Neighbor Joining) method [21] as shown in Fig. 4. The bootstrap percentage values from 1000 resamplings of the data set were given at each node. KOD1 flagellins were grouped with methanogenic counterparts and were distinguishable from halophilic flagellins. The result of phylogenetic analysis may suggest that the difference in amino acid sequences may reflect their different living environments. The profile of the phylogenetic tree agreed with that of the universal tree based on 16S rRNA sequences [22].

image

Figure 4. Phylogenetic analysis of archaeal flagellins. A phylogenetic tree was constructed using the ODEN program (National Institute of Genetics, Mishima, Japan) and Clustal W. The scale represents the estimated number of amino acid substitutions per site. The bootstrap percentage values from 1000 resamplings of the data set were given at each node.

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3.4Transcriptional analysis

As mentioned above, we have found that five KOD1 flagellin genes are arranged in a tandem. Then, it would be interesting to study how these five genes are expressed. Northern blot analysis against total RNA purified from exponentially growing cells was performed (Fig. 5). A specific probe A, which was designed to hybridize an ORF which is located upstream of flaB1, was obtained from a 250-bp EcoRI-EcoRV fragment as shown in Fig. 5a. Since a single signal with a size of 1.0 kb was obtained using the probe A (Fig. 5c), the probe A specified a single transcript (1.0 kb) of the ORF as shown in Fig. 5b. This result suggested that transcription of the flagellin genes starts from the downstream region of probe A. The promoter of flaB1 was considered to locate immediately upstream of flaB1.

image

Figure 5. Transcriptional analysis of flagellin genes from P. kodakaraensis KOD1. (a) Arrangement of flagellin genes and the positions of the probes. Probe A is the EcoRI-EcoRV digest specific for the upstream ORF of flaB1 and probe B is a synthetic oligonucleotide specific for flaB1. (b) Predicted sizes and locations of transcripts hybridized with probe A or B. (c) Signals by Northern hybridization. The left and right panels denote hybridization patterns with probe A and B, respectively. Size markers (Perfect RNA Markers, Novagen, Madison, WI, USA) are shown between panels. The sizes of signals detected by Northern hybridization with probe A and B are indicated with arrows.

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Then, a specific probe B, which was designed to hybridize transcript of the flaB1 region, was obtained from a synthetic oligonucleotide (Fig. 5a). Five signals of sizes 0.98, 2.1 (minor band), 3.7, 5.4 and 9.2 kb were obtained (Fig. 5c). The minor band of 2.1 kb was an artifact because the signal at the same position was detected even when non-specific probe was used (data not shown). The other signals of 0.98, 3.7, 5.4 and 9.2 kb correspond to the length of flaB1 (0.98 kb), flaB1flaB3 (3.7 kb), flaB1flaB5 (5.4 kb) and flaB1 to a further downstream region of flaB5 (9.2 kb), respectively. Hence, flagellin genes from P. kodakaraensis KOD1 were considered to be transcribed as shown in Fig. 5b. Although further experiments must be performed, these four transcripts with different sizes appeared to be initiated from the same transcription start point. In M. voltae, three kinds of transcripts including unknown ORFs besides flaB1, flaB2 and flaB3 are produced (Fig. 6) [6]. It was speculated that the unknown ORFs were involved in transporting or processing the flagellin molecule into a mature flagellar filament. The longest mRNA of P. kodakaraensis KOD1 could cover the downstream region of the flaB5. Therefore, the region may encode proteins responsible for transporting or processing of flagellins. H. salinarum also possesses five flagellin genes like P. kodakaraensis KOD1. However, the clustered structure and transcription profiles are completely different (Fig. 6) [5]. The transcription mechanism of flagellin genes from P. kodakaraensis KOD1 appears to be similar to that of M. voltae, because three different sizes of transcripts (1.5, 4.2 and 5.4 kb) from the same transcription start site encode three or more flagellar genes (Fig. 6). Phylogenetic analysis also revealed that flagellins from P. kodakaraensis KOD1 are closer to those from M. voltae than those from H. salinarum.

image

Figure 6. Comparison of transcriptional units of flagellin genes from P. kodakaraensis KOD1, M. voltae and H. salinarum. (top) The profile of P. kodakaraensis KOD1. (middle) The profile of M. voltae. (bottom) The profile of H. salinarum.

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Acknowledgments

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References

This work was supported by a grant from CREST (Core Research for Evolutional Science and Technology).

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgments
  7. References
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