Sequence and Expression Analysis of the ompA Gene of Rickettsia peacockii, an Endosymbiont of the Rocky Mountain Wood Tick, Dermacentor andersoni

ABSTRACT The transmission dynamics of Rocky Mountain spotted fever in Montana appears to be regulated by Rickettsia peacockii, a tick symbiotic rickettsia that interferes with transmission of virulent Rickettsia rickettsii. To elucidate the molecular relationships between the two rickettsiae and glean information on how to possibly exploit this interference phenomenon, we studied a major rickettsial outer membrane protein gene, ompA, presumed to be involved in infection and pathogenesis of spotted fever group rickettsiae (SFGR) but which is not expressed in the symbiont. Based on PCR amplification and DNA sequence analysis of the SFGR ompA gene, we demonstrate that R. peacockii is the most closely related of all known SFGR to R. rickettsii. We show that R. peacockii, originally described as East Side agent in Dermacentor andersoni ticks from the east side of the Bitterroot Valley in Montana, is still present in that tick population as well as in D. andersoni ticks collected at two widely separated locations in Colorado. The ompA genes of R. peacockii from these locations share three identical premature stop codons and a weakened ribosome binding site consensus sequence relative to ompA of R. rickettsii. The R. peacockii ompA promoter closely resembles that of R. rickettsii and is functional based on reverse transcription-PCR results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that OmpA translation products were not detected in cultured tick cells infected with R. peacockii. Double immunolabeling studies revealed actin tail structures in tick cells infected with R. rickettsii strain Hlp#2 but not in cells infected with R. peacockii.

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Isolation of a Spotted Fever Group Rickettsia, Rickettsia peacockii, in a Rocky Mountain Wood Tick, Dermacentor andersoni, Cell Line

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.546-552.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 546-552 ◽

Cited By ~ 50

Author(s):

Jason A. Simser ◽

Ann T. Palmer ◽

Ulrike G. Munderloh ◽

Timothy J. Kurtti

Keyword(s):

Cell Line ◽

Citrate Synthase ◽

Spotted Fever ◽

Fluorescent Antibody ◽

Rocky Mountain ◽

Embryonic Cell ◽

Host Cells ◽

Spotted Fever Group ◽

Dermacentor Andersoni ◽

Rocky Mountain Wood Tick

ABSTRACT An embryonic cell line (DAE100) of the Rocky Mountain wood tick,Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals.R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.

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Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.164-171.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 164-171

Author(s):

M J Matunis ◽

W M Michael ◽

G Dreyfuss

Keyword(s):

Hela Cells ◽

Mammalian Cells ◽

Binding Proteins ◽

Rna Binding ◽

Consensus Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Predicted Amino Acid Sequence ◽

Cdna Clones ◽

Hnrnp Proteins

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.

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Purification and Partial Amino Acid Sequence of Pentocin 31-1, an Anti-Listeria Bacteriocin Produced by Lactobacillus pentosus 31-1

Journal of Food Protection ◽

10.4315/0362-028x-72.12.2524 ◽

2009 ◽

Vol 72 (12) ◽

pp. 2524-2529 ◽

Cited By ~ 20

Author(s):

JINLAN ZHANG ◽

GUORONG LIU ◽

NAN SHANG ◽

WANPENG CHENG ◽

SHANGWU CHEN ◽

...

Keyword(s):

Molecular Mass ◽

Specific Activity ◽

Consensus Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Mass Spectrometry Analysis ◽

Gel Chromatography ◽

Original Activity ◽

Lactobacillus Pentosus ◽

Spectrometry Analysis

Pentocin 31-1, an anti-Listeria bacteriocin produced by Lactobacillus pentosus 31-1 from the traditional Chinese fermented Xuan-Wei ham, was successfully purified by the pH-mediated cell adsorption-desorption method and then purified by gel chromatography with Sephadex G-10. The purification resulted in a 1,381.9-fold increase in specific activity with a yield of 76.8% of the original activity. Using Tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), the molecular mass of the purified peptide was found to be between 3,500 and 6,400 Da, and bacteriocin activity was confirmed by overlayer techniques. When subjected to mass spectrometry analysis, the protein was highly pure and its molecular mass was 5,592.225 Da. The partial N-terminal sequence of pentocin 31-1 was the following: NH2-VIADYGNGVRXATLL. Compared with the sequence of other bacteriocins, pentocin 31-1 has the consensus sequence YGNGV in its N-terminal region, and therefore it belongs to the class IIa of bacteriocins.

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Rickettsia rickettsii and Other Spotted Fever Group Rickettsiae (Rocky Mountain Spotted Fever and Other Spotted Fevers)

Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases ◽

10.1016/b978-0-443-06839-3.00187-9 ◽

2010 ◽

pp. 2499-2507 ◽

Cited By ~ 3

Author(s):

DAVID H. WALKER

Keyword(s):

Spotted Fever ◽

Rocky Mountain ◽

Spotted Fever Group ◽

Rocky Mountain Spotted Fever ◽

Spotted Fever Group Rickettsiae ◽

Rickettsia Rickettsii

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The Typhus-Rocky Mountain Spotted Fever Group: An Epidemiological and Clinical Study in the Eastern and Southeastern States

Public Health Reports (1896-1970) ◽

10.2307/4579959 ◽

1931 ◽

Vol 46 (9) ◽

pp. 470 ◽

Cited By ~ 11

Author(s):

A. Rumreich ◽

R. E. Dyer ◽

L. F. Badger

Keyword(s):

Clinical Study ◽

Spotted Fever ◽

Rocky Mountain ◽

Spotted Fever Group ◽

Rocky Mountain Spotted Fever

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Transcriptional regulation of alpha(1b) adrenergic receptors (alpha(1b)AR) by nuclear factor 1 (NF1): a decline in the concentration of NF1 correlates with the downregulation of alpha(1b)AR gene expression in regenerating liver.

Molecular and Cellular Biology ◽

10.1128/mcb.16.11.5997 ◽

1996 ◽

Vol 16 (11) ◽

pp. 5997-6008 ◽

Cited By ~ 34

Author(s):

B Gao ◽

L Jiang ◽

G Kunos

Keyword(s):

Consensus Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Upstream Region ◽

Remnant Liver ◽

Nuclear Factor ◽

Nucleotide Substitutions ◽

P2 Promoter ◽

Hep3b Cells ◽

Nuclear Factor 1

The 5' upstream region from --490 to --540 (footprint II) within the dominant P2 promoter of the rat alpha(1b) adrenergic receptor (alpha(1b)AR) gene is recognized by a sequence-specific DNA-binding protein (B. Gao, M. S. Spector, and G. Kunos, J. Biol. Chem. 270:5614-5619, 1995). This protein, detectable in Southwestern (DNA-protein) blots of crude nuclear extracts as 32- and 34-kDa bands, has been purified 6,000-fold from rat livers by DEAE-Sepharose, heparin-Sepharose, and DNA affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV cross-linking of the purified protein indicated the same molecular mass as that in crude extracts. Methylation interference analysis revealed strong contact with a TTGGCT hexamer and weak contact with a TGGCGT hexamer in the 3' and 5' portions of footprint II, respectively. Nucleotide substitutions within these hexamers significantly reduced protein binding to footprint II and the promoter activity of P2 in Hep3B cells. The purified protein also bound to the nuclear factor 1 (NF1)/CTF consensus sequence, albeit with lower affinity. Gel mobility supershift and Western blotting (immunoblotting) analyses using an antibody against the NF1/CTF protein identified the purified 32- and 34-kDa polypeptides as NF1 or a related protein. Cotransfection into Hep3B cells or primary rat hepatocytes of cDNAs of the NF1-like proteins NF1/L, NF1/X, and NF1/Redl resulted in a three- to fivefold increase in transcription directed by wild-type P2 but not by the mutated P2. Partial hepatectomy markedly decreased the levels of NF1 in the remnant liver and its binding to P2, which paralleled declines in the rate of transcription of the alpha(1b)AR gene and in the steady-state levels of its mRNA. These observations indicate that NF1 activates transcription of the rat alpha(1b)AR gene via interacting with its P2 promoter and that a decline in the expression of NF1 is one of the mechanisms responsible for the reduced expression of the alpha(1b)AR gene during liver regeneration.

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