Characterization of the Rickettsia prowazekii pepA gene encoding leucine aminopeptidase

Wood, Douglas; Solomon, Mark J.; Speed, R R

doi:10.1128/jb.175.1.159-165.1993

Cited by 16 publications

(8 citation statements)

References 30 publications

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“…4B and not shown). PepA from Salmonella typhimurium can also function efficiently in Xer recombination at cer (Stirling et al ., 1989), whereas PepA from Rickettsia prowazekkii cannot (Wood et al ., 1993). Thus only PepA homologues from S. typhimurium and V. cholerae (i.e.…”

Section: Resultsmentioning

confidence: 99%

“…In both of these situations, the peptidase activity of PepA is not required (McCulloch et al ., 1994;Charlier et al ., 1995), and PepA appears to act as an architectural protein, bending and wrapping DNA so as to allow interactions between other proteins bound at distant sites on the DNA. PepA homologues are present in a wide variety of other bacterial species, and have been implicated in transcriptional regulation in some of these species (Wood et al ., 1993;Woolwine and Wozniak, 1999;Behari et al ., 2001;Woolwine et al ., 2001).…”

Section: Introductionmentioning

confidence: 99%

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Mutagenesis of PepA suggests a new model for the Xer/cer synaptic complex

Reijns

Leach

et al. 2005

Molecular Microbiology

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SummaryPepA is an aminopeptidase and also functions as a DNA-binding protein in two unrelated systems in Escherichia coli : Xer site-specific recombination and transcriptional regulation of carAB . In these systems, PepA binds to and brings together distant segments of DNA to form interwrapped, nucleosome-like structures. Here we report the selection of PepA mutants that were unable to support efficient Xer recombination. These mutants were defective in DNA-binding and in transcriptional regulation of carAB , but had normal peptidase activity. The mutations define extended patches of basic residues on the surface of the N-terminal domain of PepA that flank a previously proposed DNA-binding groove in the C-terminal domain of PepA. Our results suggest that DNA passes through this C-terminal groove in the PepA hexamer, and is bound by N-terminal DNA-binding determinants at each end of the groove. Based on our data, we propose a new model for the Xer synaptic complex, in which two recombination sites are wrapped around a single hexamer of PepA, bringing the crossover sites together for strand exchange by the Xer recombinases. In this model, PepA stabilizes negative plectonemic interwrapping between two segments of DNA by passing one segment through the C-terminal groove while the other is held in place in a loop over the groove.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Mutagenesis of PepA suggests a new model for the Xer/cer synaptic complex

Reijns

Leach

et al. 2005

Molecular Microbiology

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“…The tomato LAP‐A1 (LeLAP‐A1; U50151) peptide sequence was previously reported [34] and was aligned with plant, prokaryotic and animal LAPs using the pileup program from the Wisconsin Genetics Group. The Solanum tuberosum (potato) LAP (StLAP; X67845) [12], Arabidopsis thaliana LAP (AtLAP; P30184) [35], Petroselinum crispum (parsley) LAP (PcLAP; X99825), E. coli PepA (EcPepA; P11648) [36], Rickettsia prowazekii PepA (RpPepA; P27888) [37], human LAP (HsLAP; AAD17527), and bovine mature, kidney LAP (BtLAP; 1LCPA) [22,38] were included in this study.…”

Section: Methodsmentioning

confidence: 99%

Identification of residues critical for activity of the wound‐induced leucine aminopeptidase (LAP‐A) of tomato

Walling

2002

European Journal of Biochemistry

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The importance of two putative Zn2+‐binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP‐A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6–LAP‐A fusion proteins expressed in Escherichia coli. Sixty‐five percent of the mutant His6–LAP‐A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6–LAP‐As on Xaa‐Leu and Leu‐Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6–LAP‐As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+‐binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6–LAP‐A enzyme had Km and kcat values similar to the wild‐type His6–LAP‐A. One catalytic (Arg431) and one Zn‐binding (Asp347) residue were essential for His6–LAP‐A activity, as most R431 and D347 mutant His6–LAP‐As did not hydrolyze dipeptides. The R431K His6–LAP‐A that retained the positive charge had partial activity as reflected in the 4.8‐fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.

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“…Genetic analysis of R. prowazekii has been restricted to the isolation and characterization of rickettsial genes in Escherichia coli (1,2,9,11,16,17,33,(39)(40)(41), phylogenetic analyses based on selected gene sequences (22,31), analysis of genome size by pulsed-field gel electrophoresis (12), and transcriptional analysis of selected R. prowazekii genes (7,8,20,24). In addition, the R. prowazekii genome, with a size of 1,100 kb, is currently being sequenced.…”

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confidence: 99%

Transformation of Rickettsia prowazekii to Rifampin Resistance

et al. 1998

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Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism’s unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R. prowazekii. Comparison of the rpoB sequences from the rifampin-sensitive (Rifs) Madrid E strain and a rifampin-resistant (Rifr) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of theR. prowazekii RNA polymerase β subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rifs Madrid E strain of R. prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rifrregion of rickettsial rpoB. This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.

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Characterization of the Rickettsia prowazekii pepA gene encoding leucine aminopeptidase

Cited by 16 publications

References 30 publications

Mutagenesis of PepA suggests a new model for the Xer/cer synaptic complex

Mutagenesis of PepA suggests a new model for the Xer/cer synaptic complex

Identification of residues critical for activity of the wound‐induced leucine aminopeptidase (LAP‐A) of tomato

Transformation of Rickettsia prowazekii to Rifampin Resistance

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