Isolation and characterization of flagella and flagellin proteins from the Thermoacidophilic archaea Thermoplasma volcanium and Sulfolobus shibatae.

Isolated flagellar filaments of Sulfolobus shibatae were 15 nm in diameter, and they were composed of two major flagellins which have M(r)s of 31,000 and 33,000 and which stained positively for glycoprotein. The flagellar filaments of Thermoplasma volcanium were 12 nm in diameter and were composed of one major flagellin which has an M(r) of 41,000 and which also stained positively for glycoprotein. N-terminal amino acid sequencing indicated that 18 of the N-terminal 20 amino acid positions of the 41-kDa flagellin of T. volcanium were identical to those of the Methanococcus voltae 31-kDa flagellin. Both flagellins of S. shibatae had identical amino acid sequences for at least 23 of the N-terminal positions. This sequence was least similar to any of the available archaeal flagellin sequences, consistent with the phylogenetic distance of S. shibatae from the other archaea studied.     

Evidence for posttranslational modification and gene duplication of Campylobacter flagellin.

A gene encoding a flagellin protein of Campylobacter coli VC167 has been cloned and sequenced. The gene was identified in a pBR322 library by hybridization to a synthetic oligonucleotide probe corresponding to amino acids 4 to 9 of the N-terminal sequence obtained by direct chemical analysis (S. M. Logan, L. A. Harris, and T. J. Trust, J. Bacteriol. 169:5072-5077, 1987). The DNA was sequenced and shown to contain an open reading frame encoding a protein with a molecular weight of 58,945 and a length of 572 amino acids. The deduced amino acid sequence was identical to the published N-terminal amino acid sequence of VC167 flagellin and to four internal regions whose partial sequences were obtained by direct chemical analysis of two tryptic and two cyanogen bromide peptides of VC167 flagellin. The C. coli flagellin protein contains posttranslationally modified serine residues, most of which occur within a region containing two 9-amino-acid repeating peptides separated by 34 unique amino acids. Comparisons with the sequences of flagellins from other bacterial species revealed conserved residues at the amino- and carboxy-terminal regions. Hybridization data suggest the presence of a second flagellin copy located adjacent to the first on the VC167 chromosome.     

Isolation and characterization of Campylobacter flagellins.

Sequential acid pH dissociation, differential ultracentrifugation, and neutral pH reassociation were used to partially purify serotypically distinct flagella from three strains of Campylobacter jejuni and the two antigenic phases of flagella of Campylobacter coli VC167. Each C. jejuni flagellin and C. coli VC167 antigenic phase 1 flagellin were purified to homogeneity by reverse-phase high-performance liquid chromatography with a C8 Spheri-10 column. C. coli VC167 antigenic phase 2 was purified to homogeneity by ion-exchange chromatography with a Mono-Q column. Amino acid compositional analysis put the C. jejuni flagellin molecular weight in the range 63,200 to 63,800 and the C. coli antigenic phase 1 and 2 flagellins at 61,500 and 59,500, respectively. The amino acid compositions of the C. jejuni were similar to each other and to the C. coli VC167 antigenic phase 1 and phase 2 flagellins. One-dimensional peptide mapping of the C. jejuni flagellins by partial digestion with trypsin or chymotrypsin confirmed the structural similarities of the C. jejuni flagellins and the C. coli VC167 antigenic phase 1 flagellin and showed that C. coli VC167 antigenic phase 2 flagellin was structurally distinct from the phase 1 flagellin. The antigenic phase 2 flagellin was especially sensitive to digestion by chymotrypsin. Amino-terminal sequence analysis showed that the 20 N-terminal amino acids of the Campylobacter flagellins were highly conserved. The Campylobacter flagellins also shared limited sequence homology with the N-terminal sequences reported for Salmonella and Bacillus flagellins.     

Identification, characterization, and spatial localization of two flagellin species in Helicobacter pylori flagella.

Flagellar filaments were isolated from Helicobacter pylori by shearing, and flagellar proteins were further purified by a variety of techniques, including CsCl density gradient ultracentrifugation, pH 2.0 acid disassociation-neutral pH reassociation, and differential ultracentrifugation followed by molecular sieving with a Sephacryl S-500 column or Mono Q anion-exchange column, and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to an Immobilon membrane. Two flagellin species of pI 5.2 and with apparent subunit molecular weights (Mrs) of 57,000 and 56,000 were obtained. N-terminal amino acid analysis showed that the two H. pylori flagellin species were related to each other and shared sequence similarity with the N-terminal amino acid sequence of Campylobacter coli, Bacillus, Salmonella, and Caulobacter flagellins. Analysis of the amino acid composition of the predominant 56,000-Mr flagellin species isolated from two strains showed that it was comparable to the flagellins of other species. The minor 57,000-Mr flagellin species contained a higher content of proline. Immunoelectron microscopic studies with polyclonal monospecific H. pylori antiflagellin antiserum and monoclonal antibody (MAb) 72c showed that the two different-Mr flagellin species were located in different regions of the assembled flagellar filament. The minor 57,000-Mr species was located proximal to the hook, and the major 56,000-Mr flagellin composed the remainder of the filament. Western immunoblot analysis with polyclonal rabbit antisera raised against H. pylori or Campylobacter jejuni flagellins and MAb 72c showed that the 56,000-Mr flagellin carried sequences antigenetically cross-reactive with the 57,000-Mr H. pylori flagellin and the flagellins of Campylobacter species. This antigenic cross-reactivity did not extend to the flagellins of other gram-negative bacteria. The 56,000-Mr flagellin also carried H. pylori-specific sequences recognized by two additional MAbs. The epitopes for these MAbs were not surface exposed on the assembled inner flagellar filament of H. pylori but were readily detected by immunodot blot assay of sodium dodecyl sulfate-lysed cells of H. pylori, suggesting that this serological test could be a useful addition to those currently employed in the rapid identification of this important pathogen.     

Flagellin inhibits Myoviridae phage φCTX infection of Pseudomonas aeruginosa strain GuA18: purification and mapping of binding site

PhiCTX is a double-stranded DNA phage of the Myoviridae family that converts Pseudomonas aeruginosa into a cytotoxin producer. A 42-kDa phiCTX-inhibiting protein was purified from the outer membrane fraction of P. aeruginosa strain GuA18 by octyl-beta-glucoside extraction, DEAE-chromatography, and mono-Q HPLC. This protein had an isoelectric point of 5.4 and bound specifically [125I]-labeled phiCTX. The N-terminal amino acid sequence of six out of seven Lys-C fragments was highly similar (87%) to that of the entire of type-a flagellin of P. aeruginosa strain PAK. At a concentration of 14 nM, purified flagellin protein caused a 50% decrease in the phage titer after a 20-min incubation at 37 degrees C (PhI50). The presence of ethanol was necessary to reconstitute the inhibitory activity. In contrast, no ethanol treatment was necessary for the inhibitory activity of the sheared flagellin filaments from P. aeruginosa strain GuA18, which consists of the 42-kDa flagellin subunits and the synthesized 17-mer phage-binding-peptide NGSNSDSERTALNGEAK, representing flagellin residues 100-116 of P. aeruginosa strain PAK. The PhI50 was 10 nM and 200 nM, respectively. Antisera against the flagellin filament protein as well as against the 17-mer peptide neutralized phage infection. These results indicated that the amino acid region 100-116 of the flagellin subunit of strain GuA18 is involved in phiCTX binding. This region might play a role in phage attachment.     

Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as in-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ;C,D' complex and a presumed ;AB' fragment. 4. The sum of the amino acid analyses of fragments A and B and the ;C,D' complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 in-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [(125)I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ;C,D' complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B-A-D-C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.     

Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti.

The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395- and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar filaments. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N- and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.     

pH-dependent flagella formation by facultative alkaliphilic Bacillus sp. C-125.

A facultative alkaliphilic strain of Bacillus sp. C-125 grown at alkaline pH had many sinuous peritrichous flagella and was highly motile. However, most of the cells grown initially at pH 7 were non-motile and possessed few straight flagella. The amount of flagellin was low when the organism was grown at pH 7, suggesting that non-motility is due to poor synthesis of flagellin. The molecular mass of the flagellin was 37 kDa and the isoelectric point was pH 5.0. The amino acid composition of the flagellin was similar to that found in the flagellin from neutrophilic Bacillus subtilis 168.     

Structural studies on the subunits of glutamate synthase from Azospirillum brasilense

The amino acid composition and the N-terminal sequences of the two dissimilar subunits of glutamate synthase from Azospirillum brasilense have been determined along with the sequences of selected CNBr peptides. Comparison of our data with those available for Escherichia coli glutamate synthase revealed an overall good homology between the enzymes from the two sources. This is more evident for the heavy subunits where the highly conserved N-terminal sequence containing Cys-1, suggests that this region may be involved in catalysis. However, it appears that the light subunits are different with respect to both their amino acid composition and their N-terminal region, suggesting that the latter may not be part of the enzyme active site. Finally, an extinction coefficient at 444 nm of 62.66 +/- 4.61 mM-1.cm-1 was determined.     

Characterization of a New (R)-Hydroxynitrile Lyase from the Japanese Apricot Prunus mume and cDNA Cloning and Secretory Expression of One of the Isozymes in Pichia pastoris

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.     

Cloning of the flagellin gene from Bacillus subtilis and complementation studies of an in vitro-derived deletion mutation.

The flagellin promoter and structural gene from Bacillus subtilis I168 was cloned and sequenced. The amino-terminal protein sequence deduced from the coding sequence of the cloned gene was identical to that of the amino terminus of purified flagellin, indicating that the export of this protein is not directed by a posttranslationally processed N-terminal signal peptide. A sequence that was homologous to that of a consensus sigma 28 RNA polymerase recognition site lay upstream of the proposed translational start site. Amplification of this promoter region on a multicopy plasmid resulted in the formation of long, filamentous cells that accumulated flagellin intracellularly. The chromosomal locus containing the wild-type flagellin allele was replaced with a defective allele of the gene (delta hag-633) that contained a 633-base-pair deletion. Transport analysis of various flagellin gene mutations expressed in the hag deletion strain suggest that the extreme C-terminal portion of flagellin is functionally involved in export of the protein.     

Common and variable domains of the flagellin gene, flaA, in Campylobacter jejuni

The organization of the flagellin gene locus in Campylobacter jejuni strain IN1 (Lior 7) was determined using the polymerase chain (PCR) reaction and a series of oligonucleotide primers. Two tandemly arranged flagellin genes of approximately 1.7 kb were found to be joined by an intervening segment of c.0.2kb, similar to that reported for Campylobacter coli. The 5' flagellin gene, flaA, was generated by PCR and both strands sequenced. Comparison of the deduced amino acid sequence for C. jejuni FlaA with the published sequence for C. jejuni FlaA with the published sequence for C. coli FlaA showed 77% identical amino acids between the proteins. Two common regions, C1 and C2, comprising the N-terminal 170 amino acids and C-terminal 100 amino acids, exhibit amino acids 94% and 96% identical to those of C. coli, respectively. The variable region, V1, comprising the middle of the protein, shows 61% identical residues with C. coli. Comparison of these regions with other bacterial flagellins reveals a similar pattern but with much less identity. Several areas within the V1 region correspond to predicted surface-exposed regions and may represent areas in which surface epitopes are located.     

蜘蛛大壶状腺丝蛋白基因的克隆和原核表达

以悦目金蛛(Argiope amoena)丝腺SMARTRACEcDNA文库为模板进行RT-PCR,克隆了1条大壶状腺丝蛋白(major ampullate spidroin,MaSp)基因cDNA序列。该条cDNA序列编码的氨基酸序列可区分为两部分(1)富含丙氨酸的片段和富含甘氨酸的片段相间排列构成的重复氨基酸序列区,并且富含甘氨酸的片段中有脯氨酸分布;(2)约100个氨基酸残基组成的C末端非重复氨基酸序列区。把MaSp基因cDNA序列亚克隆到质粒pET28b( )中,构建原核表达质粒pET28b( )-MaSp,表达质粒转化大肠杆菌BL21(DE3),用IPTG诱导表达。SDS-PAGE、氨基酸组成测定和N末端氨基酸序列测定的结果表明,表达产物为重组MaSp,表达量约为40mg/L。还对C末端非重复氨基酸序列对重组MaSp在水媒介中溶解性的影响进行了探讨。     

Analysis of the flagellin (hag) gene of alkalophilic Bacillus sp. C-125.

Motility of the alkalophilic Bacillus sp. C-125, a flagellate bacterium, was demonstrated to be Na(+)- and pH-dependent. Flagellin protein from this strain was purified to homogeneity and the N-terminal sequence determined. Using the hag gene of Bacillus subtilis as a probe, the hag gene of Bacillus sp. C-125 was identified and cloned into Escherichia coli. Sequencing of this hag gene revealed that it encodes a protein of 272 amino acids (M(r) 29,995). The predicted N terminal sequence of this protein was identical to that determined by N-terminal sequencing of the flagellin protein from strain C-125. The alkalophilic Bacillus sp. C-125 flagellin shares homology with other known flagellins in both the N- and C-terminal regions. The middle portion, however, shows considerable differences, even from that of flagellin from the related species, B. subtilis.     

吸血诱导的埃及伊蚊海口株后期胰蛋白酶基因测序及与美国株同源性比较

应用逆转录-聚合酶链式反应（RT-PCR）技术从吸血后24 h埃及伊蚊海口株总RNA中扩增出了后期胰蛋白酶编码区cDNA序列。采用自动DNA分析仪进行序列分析,并与已知埃及伊蚊美国株后期胰蛋白酶基因及推导的氨基酸序列进行了同源性比较。结果表明：埃及伊蚊海口株后期胰蛋白酶基因序列与美国株同源性达98%,有11个碱基发生变异;氨基酸同源性达99%,仅有3个氨基酸发生变异,但与催化位点密切相关的氨基酸及N末端氨基酸序列完全一致。以上结果显示,埃及伊蚊胰蛋白酶不同地理株间存在微小的差异。     

Chromogranin from normal human adrenal glands: purification by monoclonal antibody affinity chromatography and partial N-terminal amino acid sequence

A method is presented for the purification of human chromogranin from adrenal glands obtained at autopsy. The procedure involved homogenization of whole glands in aqueous buffer, salt precipitation, affinity chromatography using a highly specific monoclonal antibody (LK2H10) and reverse-phase high-pressure liquid chromatography. Chromogranin purified from autopsy adrenal glands revealed a high degree of polypeptide heterogeneity when analyzed by silver-stained SDS polyacrylamide gels. Greater than 90% of the protein was represented by a cluster of polypeptides with an Mr = 70 000 (i.e. chromogranin A), while the remaining protein was highly disperse in molecular weight. That these various polypeptides were in fact chromogranin was shown by Western blotting using monoclonal antibody LK2H10. About 6 nmol of chromogranin were obtained from 97 g of starting adrenals which was estimated to be a 25% yield and a 250-fold enrichment from adrenal homogenates. Critical to achieving reasonable yields of this protein was the need for particular low pH buffers for resuspension of chromogranin after solvent removal steps. Chromogranin purified from human adrenal glands was similar in amino acid composition, and identical in the N-terminal amino acid sequence (24 residues) to bovine chromogranin A. A secondary sequence representing 25% of the total protein and missing the first three residues of the N-terminus suggested the possibility of N-terminal processing of chromogranin in situ. The conservation of the N-terminal amino acid sequence of human and bovine chromogranin contrasts with the strong sequence variability predicted by antisera cross-reactivity and suggests that the N-terminus of chromogranin may be critical for its biological activity.     

Isolation of Tyrosinase From Bovine Eyes

Pigmented tissues from bovine eye were used as a source for isolation of tyrosinase from normal melanocytes. Tyrosinase is highly hydrophobic and the isolation procedure is mainly based on the use of hydrophobic interaction chromatography. The bovine enzyme is, in contrast to the human melanoma tyrosinase, mainly soluble. The predominant part of the ocular enzyme from cow has a molecular weight and isoelectric behavior similar to that of the soluble tyrosinase in the human melanoma cells. The N-terminal amino acid sequence of isolated bovine tyrosinase was determined by automated Edman degradation. The N-terminal amino acid sequence from normal bovine tyrosinase was identical to the sequence of an N-terminal region of mouse melanoma tyrosinase predicted from a c-DNA clone by Kwon et al. (1988). The amino acid sequence of bovine tyrosinase shows homology to that of human tyrosinase (Wittbjer et al., 1989), but three amino acids of the 16 residues determined by us differed. Histidine was the N-terminal amino acid.