Genetic and Biochemical Investigation of Bxb1 gp47, An Unusual Recombination Directionality Factor by Andrew Savinov BS in Chemistry and Molecular Biology, University of Pittsburgh, 2011 Submitted to the Graduate Faculty of University Honors College in partial fulfillment of the requirements for the degree of Bachelor of Philosophy University of Pittsburgh 2011
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Genetic and Biochemical Investigation of Bxb1 gp47, An Unusual Recombination Directionality Factor
by
Andrew Savinov
BS in Chemistry and Molecular Biology, University of Pittsburgh, 2011
Submitted to the Graduate Faculty of
University Honors College in partial fulfillment
of the requirements for the degree of
Bachelor of Philosophy
University of Pittsburgh
2011
UNIVERSITY OF PITTSBURGH
University Honors College
This undergraduate thesis was presented
by
Andrew Savinov
It was defended on
April 4, 2011
and approved by
Karen M. Arndt, Professor, University of Pittsburgh Department of Biological Sciences
Nigel D. F. Grindley, Professor, Yale University Department of Molecular Biophysics &
Biochemistry
Roger W. Hendrix, Distinguished Professor, University of Pittsburgh Department of
Biological Sciences
Thesis Director: Graham Hatfull, Eberly Family Professor, University of Pittsburgh
Genetic and Biochemical Investigation of Bxb1 gp47, An Unusual Recombination Directionality Factor
Andrew Savinov
University of Pittsburgh, 2011
The temperate mycobacteriophage Bxb1 infects and forms lysogens of Mycobacterium smegmatis, a fast-growing relative of and model for M. tuberculosis. In Bxb1, as in other bacteriophages, switching between the lysogenic cycle and lytic cycle depends on a site-specific DNA recombinase called an integrase. In Bxb1 the directionality of the DNA recombination process depends on the phage-encoded gp47 protein, which acts as the Recombination Directionality Factor (RDF). A number of lines of evidence suggest that Bxb1 gp47 has some additional biological role as well, however. First, very close homologues are found in 15 other mycobacteriophages, including phage L5, whose system for phage DNA integration / excision does not include the Bxb1 gp47 homologue. Second, Bxb1 gp47 and homologues are found clustered with genes for phage DNA replication.
Here we present our investigation into the putative multi-functionality of Bxb1 gp47. To
begin, we performed a bioinformatic analysis which predicts Bxb1 gp47 and its homologues to contain a calcineurin-like phosphoesterase domain; this domain is predicted to confer a metal-dependent phosphatase activity on these proteins. Further, we analyzed the phenotypic repercussions of altering the Bxb1 gp47 gene by site-specific phage mutagenesis, performed using Bacteriophage Recombineering by Electroporation of DNA (BRED). Results from this work were consistent with the hypothesis that Bxb1 gp47 has an essential function in the lytic-cycle replication of Bxb1, and also showed that gp47 RDF activity is separable from the lytic-cycle function inferred for gp47. Finally, a number of variants of Bxb1 gp47 protein were overexpressed and purified for studies of RDF and phosphatase activity. RDF activity assays suggested that Motif I of the calcineurin-like phosphoesterase domain has no role in RDF activity, but that Motif V may be involved in both RDF and phosphatase activities. The phosphatase activity assays we have performed provide support for the hypothesis that Bxb1 gp47 is a manganese-dependent phosphatase enzyme. If this result is confirmed, Bxb1 gp47 will be revealed as a highly novel RDF with a secondary phosphatase activity.
were performed in a 96-well-plate format. See Materials and Methods for details; briefly, a CIP
control or gp47 variant sample was incubated for the indicated length of time at 25 oC with FDP
in a reaction buffer from the supplier, supplemented as indicated with divalent manganese,
magnesium, calcium, or zinc as MCl2. Reactions were stopped with provided pH-shifting Stop
Solution. Detection was performed with a 473 nm laser for excitation and an LBP emission filter.
Signal is reported in arbitrary units (a.u.).
Table 1. Phosphatase Activity Assays of H6-gp47 (reaction time = 45 mins).
22
Table 2. Phosphatase Activity Assays of gp47-GST (reaction time = 45 mins).
Table 3. Phosphatase Activity Assays of H6-MBP-gp47 (reaction time = 30 mins).
Table 4. Table of gp47 protein variants used in this work.
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Figure 1.
(A)Temperate phage life-cycles and the lytic/lysogenic cycle transition mechanism in Bxb1. (B) The mechanism of serine recombinase-catalyzed DNA recombination. (C) Sequences of the attP, attB, attL, and attR DNA sites of Bxb1 and M. smegmatis, and their component half-sites. Red lines indicate the central dinucleotide of each att site.
A
B
C
attP attL
attR attB
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Figure 2.
Sequence alignments providing bioinformatic evidence of a secondary function for Bxb1 gp47. (A) Bxb1 gp47 vs. selected homologs. (B) L5 gp54 vs. the calcineurin-like phosphoesterase domain consensus. (C) L5 gp54 vs. Bxb1 gp47; the blue box indicates the Bxb1 gp47 Phe residue replacing the conserved Val of the domain consensus sequence, and the magenta box locates Bxb1 gp47 residue Ser153. The black arrows indicate residues mutated to alanines in the Motif I and Motif V K-O variants of gp47 (see also Table 4).
A
B
C
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Figure 3.
BRED mutagenesis results for Bxb1 gp47 deletion mutant. Phage samples were screened by DADA-PCR; PCR products were separated by agarose gel electrophoresis and EtBr-stained. (A) Primary plaque picks. The red box indicates the primary mutant plaque picked after recombineering which was positive for the mutation, as assayed by DADA-PCR; the red arrows indicate the position of the expected PCR product size for a sample containing deletion mutant phage DNA. (B) A representative sample of results from second-generation plaque analysis is shown; the “mutant + 1o” control is a mutant-positive primary plaque sample, as is the “Δgp47, earlier recombineering” control in (A).
A
mutant + 1o
B
mutant
mutant
(pfu)
26
Figure 4.
BRED mutagenesis results for Bxb1 gp47-S153A mutant. Samples were screened by PCR assays, and the products run on agarose gels and EtBr-stained. Red boxes indicate phage samples diluted and replated to produce next-generation samples. (A-C) Primary picks 1, 2, 3, and 4 were made off of wt M. smegmatis, RecA-:pMS47 cells, RecA-:pMS54 cells, and RecA-:pMS53-55 cells respectively and replated onto these same cell lawns to produce secondary lysates of 150-200 pfu each. The RecA-:pMS54 primary plaque ultimately yielded pure mutant phage after 5 generations of dilution & replating; the first three generations of the purification are shown here. Mutant purity was tested by whole-genome sequencing and local sequencing analysis for possible SNPs. Red arrows indicate expected position of mutant-specific MAMA-PCR product band. (D) Pure mutant phage vs. wt phage compared in a lytic propagation assay; 2 representative plates are shown. (E) 4th-, 5th-, and 6th-generation plaque sample analysis by mutant- and wt-specific MAMA-PCR. Expected positions of mutant/ wt MAMA-PCR products are indicated by red arrows. (F) 4th- and 5th-generation plaque sample analysis via restriction digestion by Bpu10I.
wt Bxb1 Bxb1 gp47-S153A
2o lysate
E
F
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Figure 5.
Purifications of Int-H6 and H6-gp47 protein variants. Yellow arrows indicate the expected position of the recombinant protein band in the SDS-PAGE analysis. F-T is used as an abbreviation for “flow-through” fraction of the column purification.
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Figure 6.
Integrative DNA recombination reactions (attP X attB) and excisive DNA recombination reactions (attL X attR) were performed as described in Materials and Methods; briefly, a plasmid DNA substrate containing attL or attP and a 50-bp attR or attB dsDNA oligonucleotide were incubated with the indicated concentrations of integrase-H6 and H6-gp47 for 1 hr (integrative reactions) or 3 hrs (excisive reactions) at 25 oC, then stopped by heat-killing integrase (75 oC, 15 mins) and run out on 0.8% agarose gels. Gels were run without EtBr, and then EtBr-stained for analysis. A linear product band of known size was the indicator of recombination reaction progress.
B
H6-gp47
A
wt
(μM H6-gp47)
(μM H6-gp47)
(μM H6-gp47)
C
H6-gp47
(μM H6-gp47)
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Figure 7.
Purification of H6-gp47 protein and the pET28c control purification used to determine presence of contaminant phosphatase activity in the H6-gp47 preparation (see Table 1).
A
gp46-H6
H6-gp47
H6-gp47
B
pET28c
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Figure 8.
Purifications of H6-MBP-gp47, pLC3 control, and BL21(DE3):pLysS expressor strain cells control used to determine phosphatase activity of H6-MBP-gp47 (see Table 3).
A H6-MBP-47 (small-scale test)
B H6-MBP-47 (1-L purification)
C pLC3 Vector Control (1-L purification)
D Expressor Strain Control (1-L purification)
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