Nucleotide sequence of the 5S ribosomal RNA gene of Bartonella bacilliformis.
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Bartonella↗
Biomedical subjects
M F Minnick
Publications and source records attributed to M F Minnick.
Characterization of Bartonella bacilliformis flagella and effect of antiflagellin antibodies on invasion of human erythrocytes.
Bartonella bacilliformis is the etiologic agent of Oroya fever in humans. Flagellum-mediated motility has been postulated as a major virulence factor for invasion of host cells. To address this hypothesis, we purified and characterized flagella from strain KC584 and then assessed their role in human erythrocyte association and invasion. Electron microscopy of the flagellar preparation showed a high concentration of filaments with a mean wavelength of 800 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot analysis, and KBr density gradient centrifugation indicated that the flagellar filament is composed of a polypeptide of 42 kDa. The flagellin is partially (ca. 50%) resistant to treatment with trypsin. The first 17 amino acid residues of the N terminus of the mature flagellin protein are GAAILTNDNAMDALQDL and show approximately 46% sequence identity to the residues of the N termini of two Caulobacter crescentus flagellin proteins. A monospecific polyclonal antibodies to the flagellin protein was generated, and its specificity was verified by both immunoblot and immunogold analyses. Human erythrocyte invasion assays performed with bartonellae exposed to the antiflagellin antiserum showed a significant decrease in bacterial association with and invasion of human erythrocytes in comparison with that in bartonellae exposed to preimmune rabbit serum or phosphate-buffered saline (PBS) controls. These results suggest that flagella are an important component in the invasiveness of B. bacilliformis.
Amino Acid Sequence↗
A highly repetitive DNA sequence possibly unique to canids.
A short interspersed nucleotide (nt) element (SINE) was cloned from the genomic DNA of the domestic dog, Canis familiaris. Southern-blot analysis of canine DNA digested with four restriction endonucleases indicated that the SINE is widely dispersed throughout the genome. Hybridizations also indicated that the element may be unique to canids and is absent in a variety of other mammals, including members of four closely-related carnivore families. Three examples of the SINE have been located and sequenced. The 130-bp SINE contains putative RNA polymerase III transcriptional control sequences. The SINE is flanked at the 3' end by a (TC)8-repeat region followed by a poly(A) tract of 35-65 nt. Computer database searches located two homologous sequences with approx. 80% identity to the SINE. These sequences were located in untranslated regions of the canine genes encoding interferon-omega and clotting factor IX.
Animals↗
Analysis of the cbhE' plasmid gene from acute disease-causing isolates of Coxiella burnetii.
A gene termed cbhE' was cloned from the QpH1 plasmid of Coxiella burnetii. Expression of recombinants containing cbhE' in vitro and in Escherichia coli maxicells, produced an insert-encoded polypeptide of approx. 42 kDa. The CbhE protein was not cleaved when intact maxicells were treated with trypsin. Hybridizations of total DNA isolated from the six strains of C. burnetii indicate that this gene is unique to C. burnetii strains associated with acute disease, i.e., Hamilton[I], Vacca[II], and Rasche[III]. The cbhE' gene was not detected in strains associated with chronic disease (Biotzere[IV] and Corazon[V]) or the Dod[VI] strain. The cbhE' open reading frame (ORF) is 1022 bp in length and is preceded by a predicted promoter/Shine-Dalgarno (SD) region of TCAACT(-35)-N16-TAAAAT(-10)-N14-AGAAGGA (SD) located 10 nucleotides (nt) before the presumed AUG start codon. The ORF ends with a single UAA stop codon and has no apparent Rho-factor-independent terminator following it. The cbhE' gene codes for the CbhE protein of 341 amino acid (aa) residues with a deduced Mr of 39,442. CbhE is predominantly hydrophilic with a predicted pI of 4.43. The function of CbhE is unknown. No nt or aa sequences with homology to cbhE' or CbhE, respectively, were found in searches of a number of data bases.
Acute Disease↗
A plasmid-encoded surface protein found in chronic-disease isolates of Coxiella burnetti.
The cbbE' gene codes for the E' protein of Coxiella burnetii and was detected in genomic DNA from all known human isolates of the biotzere strain but not in DNA from the other five strains of C. burnetti. The biotzere strain is strictly associated with chronic disease in humans. Extrinsic iodination of biotzere strain cells radiolabeled a 55-kDa protein which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the E' protein synthesized in vitro from recombinants containing the cbbE' gene. The 125I-labeled 55-kDa protein was immunoprecipitated with polyclonal anti-E' antiserum, confirming its identity as E'. Predicted secondary structure of the E' polypeptide shows six regions of beta-sheet structure and an alpha-helix near the C terminus with adequate lengths to span a membrane. The predicted hydropathy profile of E' is similar to profiles of known outer membrane proteins and corroborates the biochemical data, indicating that the protein is located in the outer membrane of C. burnetii.
Amino Acid Sequence↗
Analysis of strain-specific plasmid sequences from Coxiella burnetii.
Acute isolates of Coxiella burnetii possess a 36-kbp plasmid termed QpH1. DNA hybridizations show that QpH1 contains approximately 6-kbp region of DNA which is not present in the QpRS plasmid from chronic isolates. This QpH1-specific region of DNA contains the contiguous EcoRI fragments G, E, and D. The GED region was found to possess seven open reading frames (ORF's) coding for proteins ranging from 5.5 to 42.3 kDa in molecular mass when subcloned and expressed in vitro. Summing the predicted ORF's accounts for 95% of the GED coding potential. E. coli expression produced a stable 42.3-kDa protein from the pHIN19 subclone of GED. The ORF of the 42.3-kDa protein, termed cbhE', has been localized on GED by both in vitro transcription/translation and DNA sequencing. The cbhE' gene is estimated as 1142 bp in length with a putative promoter region of TCAACT (-35)-N16-TAAAAT (-10)-N14-AGAAGGA (Shine-Dalgarno)-N10-ATG.
Cloning, Molecular↗
Characterization and expression of the cbbE' gene of Coxiella burnetii.
A gene which is unique to the QpRS plasmid from chronic isolates of Coxiella burnetii was cloned, sequenced, and expressed in Escherichia coli. This gene, termed cbbE', codes for a putative surface protein of approximately 55 kDa, termed the E' protein. The cbbE' gene is 1485 bp in length, and is preceded by predicted promoter regulatory sequences of TTTAAT (-35), TATAAT (-10), and a Shine-Dalgarno sequence of GGAGAGA, all of which closely resemble those of E. coli and other rickettsiae. The open reading frame (ORF) of cbbE' ends with a UAA codon followed by a second in-frame UAG stop codon and a region of dyad symmetry which may act as a rho-factor-independent terminator. The ORF of cbbE' is capable of coding for a polypeptide of 495 amino acids with a predicted molecular mass of 55893 Da. The E' protein has a predicted pI of approximately 8.7, and contains a distinct hydrophobic region of 12 amino acid residues. In vitro transcription/translation and E. coli expression of recombinant plasmids containing cbbE' produce a protein of approximately 55 kDa. The in vivo expression of cbbE' yields a novel protein that can be detected on immunoblots developed with rabbit antiserum generated against purified outer membrane from C. burnetii. DNA hybridization analysis shows that cbbE' is unique to the QpRS plasmid found in chronic isolates of C. burnetii, and is absent in chromosomal DNA and plasmids (QpH1, QpDG) from other isolates of C. burnetii. A search of various DNA and amino acid sequence data bases revealed no homologies to cbbE'.
Amino Acid Sequence↗
Analysis of QpRS-specific sequences from Coxiella burnetii.
Coxiella burnetii from acute cases of Q fever possess a plasmid termed QpH1. Chronic isolates contain a plasmid termed QpRS or have QpRS sequences integrated into the chromosome. The correlation between an isolate's plasmid type and the chronic or acute nature of the disease has prompted analysis of unique plasmid sequences to determine if they contain virulence genes. DNA hybridization has determined that a portion of a 3.6-kb EcoR I fragment (epsilon') is unique to QpRS. In vitro transcription/translation (IVTT) of the epsilon' fragment yielded a 55-kDa protein regardless of the cloning orientation, suggesting that transcription resulted from a rickettsial promoter. A translational start site was mapped to the 1.2-kb Pst I-EcoR I subfragment of epsilon' by IVTT. DNA sequencing showed an open reading frame (ORF) of 1485 bp, capable of coding for a protein of ca. 55.9 kDa. This ORF was termed cbbE'. Putative promoter regions of cbbE' included TTTAAT (-35), TATAAT (-10), and a ribosome-binding site GGAGAGA. The ORF ended with a stop codon UAA and was followed by UAG and a potential factor-independent transcription-termination region. In-frame cloning of the 695-bp Pst I subfragment into pUC9 resulted in a fusion protein of ca. 37 kDa, confirming the frame and length of the ORF as predicted by DNA sequencing. The specificity of this gene to QpRS was confirmed by probing DNA from three plasmid groups of C. burnetii, using the internal 695-bp Pst I fragment of cbbE'.
Base Sequence↗
Strategy for detection and differentiation of Coxiella burnetii strains using the polymerase chain reaction.
A method for the rapid detection of Coxiella burnetii and differentiation between strains that cause endocarditis and those that cause acute Q fever is based on the observation that the different strains contain unique plasmid sequences. This method employs the polymerase chain reaction (PCR) and requires knowledge of specific DNA sequences in the region (target) of DNA to be amplified. To detect and differentiate between C. burnetii isolates, two sets of primers are required. The first set was derived from a fragment of plasmid QpH1 which has been detected in all C. burnetii isolates. A second PCR reaction was conducted using primers specific for DNA sequences that are shared only by QpRS plasmid-containing strains of C. burnetii. The first reaction detects the presence of C. burnetii. The second PCR is necessary to determine whether the isolate contains DNA sequences associated with strains causing chronic disease. These procedures detect as few as one to ten organisms.
Coxiella↗
A bacterial-induced lectin which triggers hemocyte coagulation in Manduca sexta.
An inducible hemagglutinin termed M13, was purified from M. sexta hemolymph. M13 is a glucose-specific lectin which in addition to erythrocyte agglutination, can activate dedifferentiation of various hemocytes into a filamentous coagulation network. When lectin activity was inhibited with glucose or antiserum, neither erythrocyte agglutination or hemocyte coagulation occurred. When M13 was boiled or trypsin treated, hemocyte activation was lost, but erythrocyte agglutination remained. Hence M13 activity appears to be bimodal, possessing both a lectin activity and a hemocyte-coagulating activity.
Agglutinins↗