Isolation, cloning, and expression of a 70-kilodalton plasminogen binding protein of Borrelia burgdorferi

Surface receptors for plasminogen are expressed by many gram-positive and gram-negative bacteria and may play a role in the dissemination of organisms by binding plasminogen, which upon conversion to plasmin can digest extracellular matrix proteins. Two plasminogen binding proteins have been identified for Borrelia burgdorferi, outer surface protein A and a 70-kDa protein (BPBP). We purified BPBP by plasminogen affinity chromatography and obtained its amino acid sequence by Edman degradation of a tryptic digest. The gene coding for BPBP was isolated from a lambda-ZAP II genomic library with probes developed from sequenced portions of the protein. This gene was expressed in Escherichia coli; the recombinant product was seen by antibody raised against native BPBP and also bound 125I-labeled plasminogen. The experimentally derived amino acid sequences corresponded to the predicted sequence encoded by the BPBP gene. The deduced amino acid sequence for BPBP revealed significant similarity to p30, a 30-kDa protein of B. burgdorferi (54% identity and 65% similarity), to a 60-kDa protein in Borrelia coriaceae (66% identity and 80% similarity), to oligopeptide binding protein A of E. coli (34% identity and 57% similarity), and, more generally, to the periplasmic oligopeptide binding family of proteins.     

Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii

A Coxiella burnetii Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [35S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70, 800. The -10 promoter sequence was shown to be identical with the consensus heat shock sigma32 promoter sequence. The base composition at the presumed -35 region revealed an EcoRI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in Escherichia coli as an intact protein. The C. burnetii 71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g., Legionella pneumophila and Francisella tularensis, as well as E. coli DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in C. burnetii cell fractions, using purified antibodies directed to the 71-kDa protein.     

Identification and characterization of the guinea-pig cytomegalovirus glycoprotein H gene

Subunit vaccines which target viral envelope glycoproteins offer promise for the prevention of congenital cytomegalovirus (CMV) infection. The guinea pig model of CMV infection is uniquely well suited to testing vaccines for prevention of congenital infection, since, in contrast to other animal cytomegaloviruses, the guinea pig CMV (GPCMV) crosses the placenta, producing intrauterine infection. Antibody to the CMV glycoproteins B (gB) and H (gH) appears to be important in conferring protective immunity. Unfortunately, little is known about specific GPCMV envelope glycoproteins. Sequencing of GPCMV genome fragments was therefore undertaken to test whether GPCMV encodes a gH homologue. Partial sequencing of the Hind III A fragment of the GPCMV genome revealed an open reading frame of 2,169 nucleotides capable of encoding a protein of 723 amino acids. Computer matrix analyses demonstrated identity between this ORF and the gH coding sequences of other herpesviruses. The GPCMV gH ORF encodes 12 highly conserved cysteine residues, contains 9 potential N-linked glycosylation sites, and has a predicted M(r) of 81.6 kDa. Northern blot hybridizations with gH-specific probes identified an abundant 5.1 kb mRNA with expression kinetics of an "early" gene. A polyclonal antiserum raised against a synthetic peptide derived from the deduced amino acid sequence of the gH ORF identified a virion-associated protein with an approximate M(r) of 85-kDa, the putative GPCMV gH, in immunoblot assays.     

Sequence analysis and characterization of a 40-kilodalton Borrelia hermsii glycerophosphodiester phosphodiesterase homolog

We report the purification, molecular cloning, and characterization of a 40-kDa glycerophosphodiester phosphodiesterase homolog from Borrelia hermsii. The 40-kDa protein was solubilized from whole organisms with 0.1% Triton X-100, phase partitioned into the Triton X-114 detergent phase, and purified by fast-performance liquid chromatography (FPLC). The gene encoding the 40-kDa protein was cloned from a B. hermsii chromosomal DNA lambda EXlox expression library and identified by using affinity antibodies generated against the purified native protein. The deduced amino acid sequence included a 20-amino-acid signal peptide encoding a putative leader peptidase II cleavage site, indicating that the 40-kDa protein was a lipoprotein. Based on significant homology (31 to 52% identity) of the 40-kDa protein to glycerophosphodiester phosphodiesterases of Escherichia coli (GlpQ), Bacillus subtilis (GlpQ), and Haemophilus influenzae (Hpd; protein D), we have designated this B. hermsii 40-kDa lipoprotein a glycerophosphodiester phosphodiesterase (Gpd) homolog, the first B. hermsii lipoprotein to have a putative functional assignment. A nonlipidated form of the Gpd homolog was overproduced as a fusion protein in E. coli BL21(DE3)(pLysE) and was used to immunize rabbits to generate specific antiserum. Immunoblot analysis with anti-Gpd serum recognized recombinant H. influenzae protein D, and conversely, antiserum to H. influenzae protein D recognized recombinant B. hermsii Gpd (rGpd), indicating antigenic conservation between these proteins. Antiserum to rGpd also identified native Gpd as a constituent of purified outer membrane vesicles prepared from B. hermsii. Screening of other pathogenic spirochetes with anti-rGpd serum revealed the presence of antigenically related proteins in Borrelia burgdorferi, Treponema pallidum, and Leptospira kirschneri. Further sequence analysis both upstream and downstream of the Gpd homolog showed additional homologs of glycerol metabolism, including a glycerol-3-phosphate transporter (GlpT), a glycerol-3-phosphate dehydrogenase (GlpD), and a thioredoxin reductase (TrxB).     

Cloning and characterization of mycovirus double-stranded RNA from the plant pathogenic fungus, Fusarium solani f. sp. robiniae

A mycovirus (named FusoV) from the phytopathogenic fungus, Fusarium solani f. sp. robiniae SUF704, has two kinds of double-stranded (ds) RNA genomes, designated M1 and M2. The cDNAs were constructed from FusoV genomic dsRNAs. The sequences of M1 and M2 cDNAs comprised 1645 and 1445bp, respectively. Sequence analysis showed that each dsRNA had a single long open reading frame (ORF) on only one of the strands. M1 ORF encodes a 519-amino acid residue polypeptide with a predicted molecular mass of 60 kDa. RNA-dependent RNA polymerase-conserved motifs were identified in the predicted amino acid sequence, and the polymerase synthesized dsRNA in vitro. The M2 ORF encodes a polypeptide of 413 amino acid residues with a predicted molecular mass of 44 kDa. The predicted amino acid sequence contained the sequence corresponding to those found in the purified 44-kDa capsid protein of FusoV.     

Cloning and sequencing of the gene for the Thiobacillus ferrooxidans ATCC33020 glutamate synthase (GOGAT) small subunit and complementation of an Escherichia coli gltD mutant

A recombinant plasmid which contains the gltD gene coding for the glutamate synthase (GOGAT) small subunit was isolated from a Thiobacillus ferrooxidans ATCC33020 gene bank by complementation of an Escherichia coli gltD mutant. The sequence of gltD was determined. The deduced amino acid sequence shows strong similarity to the two other prokaryote gltD sequences available, namely those of E. coli and A. brasilense (53% and 45% identity, respectively). A cosmid containing the gltBD region was isolated from a T. ferrooxidans cosmid gene bank, but was unable to complement an E. coli gltB mutant.     

Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.     

Cloning and sequencing of the gene encoding a 31-kilodalton antigen of Haemophilus somnus

Immunoblots using bovine antibody against Haemophilus somnus as the primary antibody consistently identified 31-, 40- and 78-kDa proteins in Sarkosyl-insoluble extracts of H. somnus. A genomic library of H. somnus 8025 DNA was constructed in plasmid pUC19, and 45 recombinants expressed proteins which were recognized by bovine antiserum in Western blots (immunoblots). Ten of the recombinants expressing a 31-kDa protein caused the lysis of bovine erythrocytes. Restriction endonuclease mapping indicated that the hemolytic recombinants shared an approximately 1.7-kb BglII fragment. Southern blot analysis using the BglII fragment as a probe revealed homology among the recombinants and the presence of an identically sized BglII fragment in the chromosome of all H. somnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion of this open reading frame resulted in the loss of hemolytic activity and protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium, and Shigella dysenteriae, with P6 of Haemophilus influenzae, and with PIII of Neisseria gonorrhoeae. An amino acid analysis of the recombinant 31-kDa protein agreed with the amino acid composition deduced from the DNA sequence.     

The mutK gene of Vibrio cholerae: a new gene involved in DNA mismatch repair

A new gene, mutK, of Vibrio cholerae, encoding a 19-kDa protein which is involved in repairing mismatches in DNA via a presumably methyl-independent pathway, has been identified. The product of the mutK gene cloned in either high- or low-copy-number vectors can reduce the spontaneous mutation frequency of Escherichia coli mutS, mutL, mutU, and dam mutants. The spontaneous mutation frequency of a chromosomal mutK knockout mutant was almost identical to that of wild-type V. cholerae cells, indicating that when the methyl-directed mismatch repair is blocked, the repair potential of MutK becomes apparent. The complete nucleotide sequence of the mutK gene has been determined, and the deduced amino acid sequence showed three open reading frames (ORFs), of which the ORF3 represents the mutK gene product. The mutK gene product has no significant homology with any of the proteins deposited in the EMBL data bank. ORF2, located upstream of mutK, encodes a 14-kDa protein which has more than 70% homology with a hypothetical protein found only downstream of the E. coli vsr gene. ORF1, located farther upstream of mutK, has more than 80% homology with a major cold shock protein found in several bacteria. Downstream of mutK, a partial ORF having 60% homology with an RNA methyltransferase has been identified. The mutK gene has recently been positioned in the ordered cloned DNA map of the genome of the V. cholerae strain from which the gene was isolated (10).     

Proteolytic processing of the coronavirus infectious bronchitis virus 1a polyprotein: identification of a 10-kilodalton polypeptide and determination of its cleavage sites

Proteolytic processing of the polyprotein encoded by mRNA 1 is an essential step in coronavirus RNA replication and gene expression. We have previously reported that an open reading frame (ORF) 1a-specific proteinase of the picornavirus 3C proteinase group is involved in processing of the coronavirus infectious bronchitis virus (IBV) 1a/1b polyprotein, leading to the formation of a mature viral protein of 100 kDa. We report here the identification of a novel 10-kDa polypeptide and the involvement of the 3C-like proteinase in processing of the ORF 1a polyprotein to produce the 10-kDa protein species. By using a region-specific antiserum, V47, raised against a bacterial-viral fusion protein containing IBV sequence encoded between nucleotides 11488 and 12600, the 10-kDa polypeptide was detected in lysates from both IBV-infected and plasmid DNA-transfected Vero cells. Coexpression, deletion, and mutagenesis studies showed that this novel polypeptide was encoded by ORF 1a from nucleotide 11545 to 11878 and was cleaved from the 1a polyprotein by the 3C-like proteinase domain. Evidence presented suggested that a previously predicted Q-S (Q3783 S3784) dipeptide bond encoded by ORF 1a between nucleotides 11875 and 11880 was responsible for the release of the C terminus of the 10-kDa polypeptide and that a novel Q-N (Q3672 N3673) dipeptide bond encoded between nucleotides 11542 and 11547 was responsible for the release of the N terminus of the 10-kDa polypeptide.     

Isolation, characterization and sequence analysis of the scrK gene encoding fructokinase of Streptococcus mutans

A gene encoding an ATP-dependent fructokinase from Streptococcus mutans GS-5 was identified within a 2 kb DNA fragment immediately downstream from the scrA gene. The gene cloned in Escherichia coli also expressed mannokinase activity. Insertional inactivation of this gene in S. mutans markedly decreased both fructokinase and mannokinase activities. Nucleotide sequence analysis of the 2 kb fragment revealed an ORF starting 199 bp downstream from the scrA gene, preceded by potential ribosome-binding (Shine-Dalgarno) and promoter-like sequences. This ORF specified a putative protein of 293 amino acids with a calculated M(r) of 31,681. The deduced amino acid sequence of the fructokinase gene, scrK, from S. mutans exhibited no significant similarity to fructokinase genes from Klebsiella pneumoniae, E. coli plasmid pUR400 or Vibrio alginolyticus, but was similar to a comparable gene from Zymomonas mobilis.     

Identification of DNA gyrase inhibitor (GyrI) in Escherichia coli

DNA gyrase is an essential enzyme in DNA replication in Escherichia coli. It mediates the introduction of negative supercoils near oriC, removal of positive supercoils ahead of the growing DNA fork, and separation of the two daughter duplexes. In the course of purifying DNA gyrase from E. coli KL16, we found an 18-kDa protein that inhibited the supercoiling activity of DNA gyrase, and we coined it DNA gyrase inhibitory protein (GyrI). Its NH2-terminal amino acid sequence of 16 residues was determined to be identical to that of a putative gene product (a polypeptide of 157 amino acids) encoded by yeeB (EMBL accession no. U00009) and sbmC (Baquero, M. R., Bouzon, M., Varea, J., and Moreno, F. (1995) Mol. Microbiol. 18, 301-311) of E. coli. Assuming the identity of the gene (gyrI) encoding GyrI with the previously reported genes yeeB and sbmC, we cloned the gene after amplification by polymerase chain reaction and purified the 18-kDa protein from an E. coli strain overexpressing it. The purified 18-kDa protein was confirmed to inhibit the supercoiling activity of DNA gyrase in vitro. In vivo, both overexpression and antisense expression of the gyrI gene induced filamentous growth of cells and suppressed cell proliferation. GyrI protein is the first identified chromosomally nucleoid-encoded regulatory factor of DNA gyrase in E. coli.     

cDNA sequence and overexpression of chloroplast chaperonin 21 from Arabidopsis thaliana

Higher plant chloroplasts contain a 21-kDa protein, chaperonin 21 (Cpn21), that is a functional homolog of the chaperonin 10 (Cpn10). The chloroplast Cpn21 polypeptide consists of two Cpn10-like domains fused together in tandem. We describe here the cDNA sequence of the Cpn21 (AtCpn21) precursor protein from Arabidopsis thaliana. The deduced amino acid sequence of the AtCpn21 precursor protein, 253 amino acids long, shows 61% identity with the spinach Cpn21 protein. The AtCpn21 precursor protein contains the typical chloroplast transit peptide of 51 amino acids at its aminoterminus and the two Cpn10-like domains which exhibits 46% sequence identity to each other. The predicted mature-sized polypeptide of AtCpn21 was expressed in Escherichia coli as a soluble 21-kDa protein. Gel-filtration and chemical cross-linking analyses showed that the recombinant mature AtCpn21 protein forms a stable homo-oligomer composed of three or four polypeptides.     

Pyrrolidone carboxyl peptidase from the hyperthermophilic Archaeon Pyrococcus furiosus: cloning and overexpression in Escherichia coli of the gene, and its application to protein sequence analysis

A gene for a pyrrolidone carboxyl peptidase (Pcp: EC 3.4.19.3, pyroglutamyl peptidase), which removes amino-terminal pyroglutamyl residues from peptides and proteins, has been cloned from the hyperthermophilic Archaeon Pyrococcus furiosus using its cosmid protein library, sequenced, and expressed in Escherichia coli. The DNA sequence encodes a protein containing 208 amino acid residues with methionine at the N-terminus. Analysis of the recombinant protein expressed in E. coli, including amino acid sequence analysis from the N-terminus by automated Edman degradation and ionspray mass spectrometric analysis of the peptides generated by enzymatic digestions with lysylendopeptidase and Staphylococcus aureus V8 protease, showed its primary structure to be completely identical with that deduced from its cDNA sequence. Comparison of the amino acid sequence of P. furiosus Pcp (P.f.Pcp) with those of bacterial Pcps revealed that a high degree of sequence identity (more than 40%) and conservation of the amino acid residues comprising the catalytic triad, Cys142, His166, and Glu79. On the other hand, a unique short stretch sequence (positions around 175-185) that is absent in bacterial Pcps was found in P.f.Pcp. A similar stretch has also been reported recently in the amino acid sequence of Pcp from the hyperthermophilic Archaeon Thermococcus litoralis [Littlechild et al., in abstracts of the "International Congress on Exthermophiles '98" p. 58 (1998)]. To elucidate their contribution to the hyperthermostability of these enzymes, further structural studies are required.     

Identification of an Escherichia coli protein impurity in preparations of a recombinant pharmaceutical

A host-cell protein impurity found in preparations of recombinant human acidic fibroblast growth factor (aFGF) was identified. Samples of aFGF examined by western blot analysis employing antiserum raised against an Escherichia coli cell lysate contained an immunoreactive protein with a molecular weight of approximately 26,000. The impurity was chromatographically isolated and the N-terminal sequence was determined. Comparing the sequence to a protein database provisionally identified the isolated impurity as the S3 ribosomal protein of E. coli. Monoclonal antibodies recognizing three separate epitopes of S3 confirmed the identity of the impurity in western blots of aFGF samples. The monoclonal antibodies were also used to estimate S3 levels in various preparations of aFGF.     

Osteopontin is the 55-kilodalton fertility-associated protein in Holstein bull seminal plasma

Previously we identified four proteins in seminal plasma that were associated with bull fertility. The purpose of this study was to identify the 55-kDa protein prevalent in seminal plasma of higher-fertility males. The 55-kDa protein was quantified by video densitometry in two-dimensional electrophoresis gels of seminal plasma from 26 bulls of known fertility. Relative density of the 55-kDa protein was positively correlated (r = 0.48) with bull fertility. The 55-kDa (pI 4.5) fertility-associated protein spot was isolated by electroelution after two-dimensional PAGE separation of seminal plasma of 36 bulls. N-terminal sequence analysis of the pure protein yielded a 15-amino acid sequence (VKPXSSGXSEEKQLN) that was 86% homologous to bovine osteopontin-k precursor. Polyclonal antiserum generated against the 55-kDa protein reacted with a single spot in two-dimensional PAGE Western blots of seminal plasma. Western blot analyses using polyclonal antisera generated against the amino terminus (LF123) and carboxyl terminus (LF124) of human recombinant osteopontin confirmed that the 55-kDa polypeptide was osteopontin. Partially purified 55-kDa protein was obtained by HPLC-MonoQ column chromatography. Protein characterization revealed that the 55-kDa protein was glycosylated, but not phosphorylated, consistent with the identity of the 55-kDa protein as osteopontin.     

Isolation and characterization of two genes, waaC (rfaC) and waaF (rfaF), involved in Pseudomonas aeruginosa serotype O5 inner-core biosynthesis

Recent studies have provided evidence to implicate involvement of the core oligosaccharide region of Pseudomonas aeruginosa lipopolysaccharide (LPS) in adherence to host tissues. To better understand the role played by LPS in the virulence of this organism, the aim of the present study was to clone and characterize genes involved in core biosynthesis. The inner-core regions of P. aeruginosa and Salmonella enterica serovar Typhimurium are structurally very similar; both contain two main chain residues of heptose linked to lipid A-Kdo2 (Kdo is 3-deoxy-D-manno-octulosonic acid). By electrotransforming a P. aeruginosa PAO1 library into Salmonella waaC and waaF (formerly known as rfaC and rfaF, respectively) mutants, we were able to isolate the homologous heptosyltransferase I and II genes of P. aeruginosa. Two plasmids, pCOREc1 and pCOREc2, which restored smooth LPS production in the waaC mutant, were isolated. Similarly, plasmid pCOREf1 was able to complement the Salmonella waaF mutant. Sequence analysis of the DNA insert of pCOREc2 revealed one open reading frame (ORF) which could code for a protein of 39.8 kDa. The amino acid sequence of the deduced protein exhibited 53% identity with the sequence of the WaaC protein of S. enterica serovar Typhimurium. pCOREf1 contained one ORF capable of encoding a 38.4-kDa protein. The sequence of the predicted protein was 49% identical to the sequence of the Salmonella WaaF protein. Protein expression by the Maxicell system confirmed that a 40-kDa protein was encoded by pCOREc2 and a 38-kDa protein was encoded by pCOREf1. Pulsed-field gel electrophoresis was used to determine the map locations of the cloned waaC and waaF genes, which were found to lie between 0.9 and 6.6 min on the PAO1 chromosome. Using a gene-replacement strategy, we attempted to generate P. aeruginosa waaC and waaF null mutants. Despite multiple attempts to isolate true knockout mutants, all transconjugants were identified as merodiploids.