Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO.

Pseudomonas aeruginosa strains PAO and PAK bear polar pili which are flexible filaments having a diameter of 6 nm and an average length of 2500 nm. Both types of pili are retractile and promote infection by a number of bacteriophages. The present communication describes the partial biochemical characterization of PAO pili isolated from a multipiliated nonretractile mutant of PAO. The observed properties are compared to those of PAK pili which were characterized previously. PAO pili were found to contain a single polypeptide subunit of 18 700 daltons. This is similar to PAK pili which contain a single polypeptide of 18 100 daltons. The amino acid composition of PAO pilin was also similar to that of PAK pilin. Neither protein contained phosphate or carbohydrate residues and both were found to contain N-methylphenylalanine at the amino terminus. Sequencing of 20 amino acid residues at the amino terminal end of PAO pilin revealed the sequence to be identical with that of PAK pilin, while tryptic peptide analyses of PAO and PAK pilin indicated that the two proteins probably contain a number of homologous regions within the polypeptide. It was concluded that PAO and PAK pili were closely related structures.     

Sequence of pilin from Bacteroides nodosus 351 (Serogroup H) and implications for serogroup classification

The nucleotide sequence of the pilin gene from Bacteroides nodosus strain 351, currently classified as serogroup H, subgroup 2 (H2) has been determined. The gene encodes a single polypeptide (prepilin) of 160 amino acids and Mr 17,150. However, pilin isolated from B. nodosus 351 migrates as two distinct bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, due to an internal peptide bond cleavage. Amino acid sequence studies of pilin from B. nodosus 351 have established that the cleavage occurs between 72Ala and 73Ser of the mature protein sequence. Comparisons of gene and amino acid sequences of pilin from B. nodosus 351 with the corresponding sequences from strains of serogroups D and H1 indicate that these sequences share a close relationship. However, the level of sequence identity between B. nodosus 351 pilin and pilin from strain 265 of serogroup H1 is lower than anticipated for strains within a serogroup and suggests that B. nodosus 265 and B. nodosus 351 should not be classified within the same serogroup.     

The complete primary structure of pilin fromHaemophilus influenzae type b strain Eagan

Adherence ofHaemophilus influenzae type b (Hib) to human oropharyngeal cells is mediated by pili which are proteinaceous filaments that extend outward from the bacterial cell surface. Pili from Hib strain Eagan were purified, and the primary structure of the major subunit, pilin, was determined. Sequencing of overlapping peptides showed the mature protein to be comprised of 196 amino acids and to have an M_r of 21,152. The amino terminal sequence was found to be homologous with the sequence previously reported for Hib strain M43 and also to have significant homology to pilins of other gram-negative pathogenic bacteria. Furthermore, Hib pilin had two cysteinyl residues in the amino terminal portion of the protein which were separated by 40 residues (positions 21 and 61); a motif found in other bacterial pilins. The data show that Hib pilin has structural features common to other bacterial pilins.     

The amino acid sequence of the cowpea strain of tobacco mosaic virus protein.

The amino acid sequence of the coat protein of the cowpea strain of tobacco mosaic virus (cowpea virus) has been determined. The tryptic peptide overlaps were obtained by digesting the protein with chymotrypsin and separating and analysing the lysine-and arginine-containing chymotryptic peptides. The primary structure of cowpea virus protein has been found to differ markedly from that of any other known strain of tobacco mosaic virus, and contains 3 amino acid residues more and 96 amino acid changes from the type strain. The significance of the distribution of those areas of the protein in which the amino acid residues are the same for all naturally occurring strains and chemically induced mutants of tobacco mosaic virus so far studied and the residues that form the important carboxyl-carboxylate pairs are discussed.     

Nucleotide sequence of the 30K protein cistron of cowpea strain of tobacco mosaic virus

The nucleotide sequence of cloned cDNA copies of cowpea strain of tobacco mosaic virus RNA including the 30K protein cistron was determined. The 30K protein cistron was located at residue 676-1,527 from the 3' end of the genomic RNA. The 30K protein was composed of 282 amino acid residues and was basic, similar to the 30K protein of common strain OM. However, homology of the amino acid sequences between the two strains was only 27%.     

Nucleotide sequence of the gene encoding the two-subunit pilin of Bacteroides nodosus 265.

The nucleotide sequence of the gene encoding pilin from Bacteroides nodosus 265 has been determined. The pilin is encoded by a single-copy gene, from which can be predicted a prepilin comprising a single protein chain of Mr 16,637. The prepilin sequence differs in several respects from the mature protein sequence. Seven additional N-terminal amino acid residues are present in prepilin, whereas residue 8, phenylalanine, undergoes posttranslational modification to become the N-methylated amino-terminal residue of mature pilin. In addition, further processing occurs through internal cleavage to produce two noncovalently linked subunits characteristic of pilins from serogroup H of B. nodosus, of which strain 265 is a member. The position of cleavage has been identified between alanine residues at positions 72 and 73 of the mature 149-residue pilin protein. The predicted pilin sequence of B. nodosus 265 shows extensive N-terminal amino acid sequence homology with other pilins of the N-methylphenylalanine type. In addition this sequence also shows homology with these N-methylphenylalanine-type pilins in the C-terminal region of the molecule, especially with pilin from Pseudomonas aeruginosa PAK.     

Cable (cbl) type II pili of cystic fibrosis-associated Burkholderia (Pseudomonas) cepacia: nucleotide sequence of the cblA major subunit pilin gene and novel morphology of the assembled appendage fibers.

Previous studies have shown that appendage pili of Burkholderia cepacia strains isolated from patients with cystic fibrosis (CF) at The Hospital for Sick Children, Toronto, Canada, mediate adherence to mucus glycoproteins and also enhance adherence to epithelial cells. The specific pilin-associated adhesin molecule is a 22-kDa protein. In the present study we purified the major subunit pilin (17 kDa) and immunolocalized it to peritrichously arranged pili. On the basis of their novel morphological appearance as giant intertwined fibers, we refer to them as cable (Cbl) pili. Using an oligonucleotide probe corresponding to regions of the N-terminal amino acid sequence of the pilin subunit, we detected the encoding cblA gene in a chromosomal DNA library. Sequencing revealed this structural gene to be 555 bp in length, encoding a leader sequence of 19 amino acids, a cleavage site between the alanine at position 19 and the valine at position 20, and a mature pilin sequence of 165 amino acids. The calculated molecular mass is 17.3 kDa. Hydrophobic plus apolar amino acids account for 60% of the total residues. The pilin exhibits some similarities in its amino acid sequence to colonization factor antigen I and CS1 fimbriae of Escherichia coli. With the cblA gene used as a probe, hybridization assays of 59 independent isolates, including those from several geographically separated CF centers, plus environmental and clinical (non-CF) strains, gave positive results with all of the 15 CF-associated B. cepacia isolates from Toronto, plus a single strain from one other CF center (Jackson, Mississippi). The cblA gene is the first pilin subunit gene of B. cepacia to be identified.     

Primary structure of the F-gene from Rinderpest virus strain K

Synthesis, cDNA cloning, and nucleotide sequencing of F gene of rinderpest virus strain K was carried out. Analysis of nucleotide sequence showed the only open reading frame coding for protein from 546 a.o. with mol. weight 58.6 kDa. The mean percentage of identical nucleotide residues between F genes of strains K, Kabete O, and L is 76.4% for 5'-untranslated region and 90.5% for translated region, the share of similar amino acid residues in the respective proteins is 92.9%. The structure of restriction site of F0 precursor protein in rinderpest strains with different virulence is similar. Protein F of rinderpest virus strain K has 3 potential glycosylation sites and 13 cystein residues in positions identical to those of F protein of rinderpest strains Kabete O and L.     

Isolation of the gene encoding pilin of Bacteroides nodosus (strain 198), the causal organism of ovine footrot

The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin-producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence.     

Nucleotide sequence of the pilin gene of Bacteroides nodosus 340 (serogroup D) and implications for the relatedness of serogroups

The gene encoding pilin of Bacteroides nodosus 340 has been isolated and the nucleotide sequence determined. The gene is present as a single copy within the B. nodosus genome and a protein of Mr 16683 can be predicted from the proposed coding region. A comparison of the predicted amino acid sequence with pilin from other strains of B. nodosus indicated that the protein of strain 340 (serogroup D) has a high degree of similarity with pilin of strain 265 (serogroup H). The degree of similarity between pilins from these strains and from other B. nodosus serogroups is no greater than that between B. nodosus pilins and the homologous proteins of several different bacterial species. These findings suggest that serogroups D and H may form a subset of B. nodosus serogroups.     

The primary structure of the coat protein of alfalfa mosaic virus strain VRU. A hypothesis on the occurrence of two conformations in the assembly of the protein shell

The complete primary structure of the coat protein of strain VRU of alfalfa mosaic virus (AMV) is reported. The strain is morphologically different from all other AMV strains as it contains large amounts of unusually long virus particles. This is caused by structural differences in the coat protein chain. The amino acid sequence has mainly been established by the characterization of peptides obtained after cleavage with cyanogen bromide and digestion with trypsin, chymotrypsin, thermolysin or Staphylococcus aureus protease. The major sequencing technique used was the dansyl-Edman procedure. The VRU coat protein consists of 219 amino acid residues corresponding to a molecular weight of 24056. Compared to the coat protein of strain 425 [Van Beynum et al. (1977) Eur. J. Biochem. 72, 63-78], 15 amino acid substitutions were localized. Most of them have a conservative character and may be explained by single-point mutations. A correction is given for the AMV 425 coat protein: Asn-216 was shown to be Asp-216. The prediction of the secondary structure for the two viral coat proteins was not significantly influenced by the various amino acid substitutions except for the region containing residues 65-100. This led us to the hypothesis that the AMV coat protein may occur in two different conformations favouring its incorporation into either a pentagonal or hexagonal quasi-equivalent position in the lattice of the protein shell. The substitutions in the above-mentioned region of the VRU coat protein may have caused a strong preference for the hexagonal lattice conformation. The model is supported by preliminary sequence data of the same coat protein region in AMV 15/64, a strain morphologically intermediate between 425 and VRU.     

Neuraminidase gene from the early Asian strain of human influenza virus, A/RI/5-/57 (H2N2)

The complete structure of the neuraminidase gene from the A/RI/5-/57 strain of influenza virus has been determined. It is 1467 nucleotides long and codes for a protein of 469 amino acid residues. Comparison with the gene sequence for the N1 strains A/WSN/33 and A/PR/8/34, the N2 strain A/Udorn/72 and the protein sequence for the N2 strain A/Tokyo/3/67 shows the amino acid sequence changes that have occurred during antigenic shift (60%) and drift (7-9%).     

Comparative studies of genes encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp. strain YL, other dehalogenases, and two related hypothetical proteins from Escherichia coli.

We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Moraxella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%).     

N-Terminal amino acid sequence of pilin isolated from Pseudomonas aeruginosa.

The amino-terminal amino acid sequence of the pili protein from Pseudomonas aeruginosa K pili is presented. The sequence is compared with those reported by others for pilin obtained from Neisseria gonorrhoeae and Moraxella nonlique-faciens. All three sequences are highly homologous, contain only two hydrophilic residues in the first 22 positions, and contain an unusual amino acid, N-monomethylphenylalanine, at the amino terminus.     

Comparative studies of the amino acid and nucleotide sequences of pilin derived from Pseudomonas aeruginosa PAK and PAO.

The entire amino acid sequence for Pseudomonas aeruginosa PAO pilin was determined through peptide sequencing and from the complete nucleotide sequence encoding the pilin gene. The precursor PAO pilin is 149 amino acids in length which includes a 6-amino-acid positively charged leader sequence. Comparison of the amino acid sequences of pilin produced by P. aeruginosa PAO and PAK reveals a region of high homology corresponding to the leader peptide and residues 1 to 54 of the mature pilin. The amino acid sequence of the peptide encompassing the major antigenic determinant of PAK differs greatly from that of the equivalent region in PAO. The C-terminal regions of these proteins are semiconserved. Few major differences were found when the predicted secondary structures for PAO and PAK pilins were compared. Major nucleotide sequence variation between the equivalent restriction fragments from PAO and PAK occurred within the areas coding for the peptides containing the immunodominant site for PAK pilin and the C termini.     

Complete amino acid sequence of the B875 light-harvesting protein of Rhodopseudomonas sphaeroides strain 2.4.1. Comparison with R26.1 carotenoidless-mutant strain

The complete amino acid sequence was determined for the alpha- and beta-chains of the B875 light-harvesting protein purified from photosynthetic membranes of Rhodopseudomonas sphaeroides 2.4.1. The sequence of the B875-alpha-polypeptide was identical to that reported for the R26.1 carotenoidless mutant [(1985) Biochim. Biophys. Acta 806, 185-186] and contained 58 amino acid residues with a blocked methionine and a glutamic acid at the N- and C-termini, respectively. The B875-beta-polypeptide contained 48 amino acid residues with alanine and phenylalanine as respective N- and C-termini; although otherwise identical, the leucine at position 29 in the wild-type strain was replaced by proline in the mutant. This radical amino acid substitution occurred within the central hydrophobic domain of the beta-polypeptide chain and is thought to result in a weakening of the structure of the alpha/beta heterodimer since it was not possible to isolate the intact pigment-protein complex from the R26.1 mutant strain.     

Antigen-antibody interactions: elucidation of the epitope and strain-specificity of a monoclonal antibody directed against the pilin protein adherence binding domain of Pseudomonas aeruginosa strain K.

The C-terminal region of Pseudomonas aeruginosa strain K (PAK) pilin comprises both an epitope for the strain-specific monoclonal antibody PK99H, which blocks pilus-mediated adherence, and the adherence binding domain for buccal and tracheal epithelial cells. The PK99H epitope was located in sequence 134-140 (Asp-Glu-Gln-Phe-Ile-Pro-Lys) by using a single alanine replacement analysis on the 17-residue synthetic peptide corresponding to the PAK C-terminal sequence 128-144. Indeed, a 7-residue peptide corresponding to this sequence was shown to have a similar binding affinity to that of the native conformationally constrained (disulfide bridged) 17-residue peptide. This epitope was found to contain two critical residues (Phe137 and Lys140) and one nonessential residue (Gln136). Interestingly, the peptide, Phe-Ile-Pro-Lys, which constitutes the four most important side chains for antibody binding did not bind to PK99H. It was of interest to investigate the structural basis of the strain-specificity of PK99H utilizing naturally occurring pilin sequences. Therefore, all different residues found in the sequence corresponding to the PK99H epitope of the four other strains (PAO, CD4, K122-4, and KB7) were substituted one at a time in the PAK sequence and the changes in binding affinity of these analogs to the antibody PK99H were determined by competitive ELISA. The strain-specificity of PK99H for strains PAO, K122-4, and KB7 can be explained by the accumulated sequence changes in these strains, and at least two amino acid changes were required to explain the strain-specificity of PK99H. Similarly, cross-reactivity of PK99H with CD4 can be explained by the fact that there was only one side chain responsible for decreasing binding affinity compared to the PAK sequence.     

The preparation and properties of gonococcal pili.

Pili have been isolated from Neisseria gonorrhoeae by controlled homogenization followed by selective disaggregation in sucrose and purification by CsCl density gradient centrifugation. Pili from six gonococcal strains had buoyant densities of 1-30 to 1-31 g ml-1 on CsCl. The pili were immunologically distinct when tested with rabbit antisera to purified pili. The amino acid composition of pilin from strains P9 and 201 was very similar, consisting of 208 and 212 amino acid residues respectively giving molecular weights of 22 600 and 22352. The pili contained a high proportion (46%) of non-polar amino acids. Further analysis of strain P9 pili revealed the presence of 1 to 2 phosphate groups and 1 to 2 hexose groups per pilin subunit; no amino sugars were detected. Pili from strain P9 were resolved into two bands by equilibrium density gradient centrifugation or column isoelectric focusing, suggesting the presence of more than one kind of pilus.     

Cloning and characterization of a novel gene cry9Ec1 encoding lepidopteran-specific parasporal inclusion protein from a Bacillus thuringiensis serovar galleriae strain

A novel delta-endotoxin gene from a lepidopteran-specific Bacillus thuringiensis serovar galleriae strain was cloned, and the full sequence of the cry gene was determined. The cloned 6.5-kb DNA fragment included the full sequence of the cry gene and three open reading frames located upstream of the cry gene. The gene, designated cry9Ec1, encodes a polypeptide of 1154 amino acid residues with a predicted molecular weight of 130 237. The deduced amino acid sequence of the Cry9Ec1 protein had the highest homology (77.7%) with the Cry9Ea1 protein when compared with existing Cry proteins. The expression, in an acrystalliferous B. thuringiensis strain, of the cry9Ec1 gene was high when controlled by the cyt1A2 promoter, leading to the formation of large spherical inclusions. The purified crystals from the recombinant strain were toxic when tested against two lepidopteran species, Bombyx mori and Plutella xylostella. However, the Cry9Ec1 protein gave no toxicity against Spodoptera litura, Spodoptera exigua, Plodia interpunctella, Helicoverpa zea, and Culex pipiens molestus.     

Primary structure of a zinc protease from Bacillus mesentericus strain 76

The amino acid sequence of the neutral zinc protease from Bacillus mesentericus strain 76 (MCP 76) has been determined by using peptides derived from digests with trypsin, chymotrypsin, and cyanogen bromide and from cleavage with o-iodosobenzoic acid. The peptides were purified by means of gel filtration and reversed-phase high-performance liquid chromatography and analyzed by automatic sequencing. The protein contains 300 amino acid residues. It proved to be identical with the neutral protease deduced from the DNA precursor sequence of Bacillus subtilis. The residues for zinc and substrate binding are conserved, whereas the number of calcium binding sites is reduced compared to thermolysin. A classification of the neutral zinc protease is discussed.