UNM TVDC UTSA - UNM Tech Call Minutes: 10/17/06
Prepared 10/20/06: Barbara Griffith
Sent to UTSA for review: 10/20/06
Reviewed: Karl Klose and Bernard Arulanadam 10/25/06
Distributed to NIAID on: 10/26/06
Present: Karl Klose, Bernard Arulanandam, Barbara Griffith, Rick Lyons, Mindy Tyson, Vicki
Pierson, Marlene Hammer, Joe Breen
Absent: Kristin DeBord, Freyja Lynn
Action Items:
Monday 10/23 10am CT, (9am MT) Barbara will call Karl to discuss the Milestone Completion
Reports for MS 16 and 39.
The meeting was recorded for the purposes of the minutes.
A. Milestone Progress
 Active Milestones are 43, 48, 49A 50A
B. Milestone #43: Creation of uvrA and uvrB mutant F. tularensis subsp. holarctica
(LVS) strains
 Slightly different genetic techniques needed to knock out uvrA and uvrB genes in
LVS than for F. novicida
 We have cloned large flanking fragments (1.5 kbp) both upstream and downstream of
uvrA from LVS individually, done in low copy number plasmid. Francisella DNA is
fairly stable in this low copy number plasmid.
 When we try to clone the second fragment (i.e. the upstream fragment with the
downstream, or vice versa) to make the deletion constructs, we get deletions.
 Can’t get uvrB deletion construct into a high copy number plasmid.
 We have cloned uvrB (LVS) with 1.5 kbp flanking homology, but have been so far
unsuccessful to insert FpKan into the deletion to create uvrB::Kan. Introducing new
promoter upstream of Kan but something is deleterious to the cells.
 We are creating new plasmid for mutant construction in LVS/Schuh4, utilizing
pDS132, this is low-copy number vector, conjugative, conditionally replicative. So it
requires a specific host factor that is not available in normal bacterial cell. UTSA uses
this vector a lot making mutants in cholera and salmonella; it works well. This
conjugation works well in Fn. Introducing a new promoter into this plasmid upstream
of sac B, because sac B has it’s own natural promoter, which won’t be expressed in
Francisella. Under the new promoter sacB will be used as a counter-selectable
marker.
 We need to insert groELp to drive sacB (counterselective marker) and CmR (will
replace with KanR for Schuh4) (documented in UTSA TVD notebook #2)
C. Milestone #48: Characterization of uvrA and uvrB mutant F. tularensis subsp.
novicida strains for intramacrophage growth
 2 month long milestone
 In general, Francisella is very sensitive to UV irradiation and the difference between
single gene uvr mutants and wild types was not as great as Cerus had hoped. Cerus
wanted the double uvrA uvrB mutant , hoping there would be a greater difference
with the wild type.
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DuvrA::Kan plasmid (pKEK1007)created by ligating FpKan into uvrA plasmid
pKEK952
pKEK1007 transformed into uvrB::ermC strain by cryotransformation, KanR
colonies obtained
KanR colonies screened by PCR, a colony that had lost the plasmid but maintained
uvrA::Kan in chromosome selected
uvrA and uvrB double Mutation confirmed by sequencing
uvrA::Kan; uvrB::ermC strain (KKF100) sent to Cerus
Three strains (uvrA, uvrB, uvrAB) being tested for intramacrophage growth.
(documented in UTSA TVD notebook #2). uvrA and uvrB completed. Double mutant
is in progress for determining intramacrophage growth.
D. Milestone #49A: Construction of iglC F. tularensis subsp. tularensis strain
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CDC inspection is on 10/18 Wed so BSL 3 lab has been closed down for a couple of
weeks. No level 3 work done on this milestone in this period but have concentrated
on the cloning strategies.
We have constructed iglC (Schuh4) with large flanking homology for construction of
Type A strain but have experienced difficulties trying to clone this into vectors for
recombination in Schuh4 (high copy). It hasn’t gone in well and have seen deletions.
A number of high copy number plasmids with large inserts of Francisella DNA haven’t
worked in high copy number plasmids in E. coli. So using lower copy number
plasmid here too (pDS132), and replacing chloramphenical marker with a Kan
marker.
We are altering a mating plasmid (pDS132) for use in Schuh4 (as detailed in
milestone 43)
We are currently ligating iglC into pDS132 to see if this technique will work (in
novicida/LVS first)
We have successfully and faithfully achieved single recombinants in Schuh4, but
could not force second recombination to achieve allelic replacement (no
counterselectable marker). It goes in and just won’t recombine out, and leave the
deletion behind. Generally, in F.novicida 1-2 generations and it recombines back
out. So this is another difference between F. novicida and SCHU S4.
We are introducing sacB into our SCHU S4 mutagenesis vectors downstream of Ftp
to provide a counterselectable marker
After CDC inspection on 10/18/06, we should be back up and running (documented
in UTSATVD Notebook 3).
E. Milestone #50A: Immunologic characterization of F. tularensis subsp. novicida,
subsp. tularensis, and LVS strains
 Determine the LD50 of Ft subsp. novicida uvrA mutant
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LD50 of uvrA in the intranasal infection model (BALB/c mice) is calculated to be
less than 10 CFU (Fig. 1).
The virulence of uvrA is not significantly reduced compared to wild-type strain.
UvrA is not attenuated at all.
Survival of mice infected with Ft subsp. Novicida uvrA mutant- Groups of BALB/c
mice (female, 6 wk old) were challenged intranasally with 4 doses (10,50,250,
1250 CRU) of uvrA to determine LD50 of this strain
Monitor Ft subsp. novicida uvrA mutant dissemination in mice
o Bacterial burden were determined in the lungs, liver and spleen from the
uvrA-infected BALB/c mice.
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The uvrA mutant replicated rapidly in the infection site (a 4 log increase by 24
h in the lungs) and disseminated to the liver and spleen by 48 h and 72 h
(Fig. 2).
Replication and dissemination of uvrA in BALB/c mice is similar to wild-type
strain. No attenuation at all.
Replication and dissemination of uvrA mutant in mice. BALB/c mice were
challenged with 200 CFU of uvrA intranasally. Bacterial burdens in lungs,
liver, and spleen were measured at 24h-, 48h- and 72h-post infection.
F. Goals for the next month.
Milestone #16: completed.
Milestone #39: completed.
Milestone #43:
1. Construct low copy number (in E coli) pDS139 plasmid with groELp for with
the goal of mutagenizing LVS/SCHU S4. It is low copy number in E. coli and it is
conjugative. UTSA anticipates that this will overcome prior problems.
2. Clone uvrB into pDS139 derivative
Milestone #48:
1. Characterize Ft novicida uvrA, uvrB, and uvrAB mutants for intramacrophage
survival and growth.
Milestone #49A:
1. Clone iglC in pDS139 derivative
2. Clone sacB into SCHU S4 mutagenesis plasmids
3. Get BSL-3 lab up and running after CDC inspection, so can work on select
agents again
Milestone #50
1. Determine the LD50 of Ft subsp. novicida uvrAuvrB double mutant
2. Look at organ burden of uvrAB mutant
G. Next UTSA Tech Call
 Next Tech call is scheduled for Tuesday, November 28th. 12pm-1pm MT, 1pm-2pm
CT and 2-3pm EST. (ASU will be on 11/21)
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Barbara- scheduled meeting with Karl to discuss Milestone completion reports for MS
16 and 39. Meeting: Monday 10/23 10am CT, 9am MT. Barbara call Karl.
H. Additional Discussion
 CDC Inspection: In the Lyons lab at UNM, the CDC inspection team- really randomly
checked your mutant inventory to verify stock locations in freezers; compared list to
what is in the freezer. Show what is in box 20 slot 5. UNM had a good CDC
inspection team.
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LVS vaccination status: Rick- got information from Bev Fogtman at USAMRIID for
costs of vaccine. Chuck Hobbs is assembling documentation to sent to NIH and to
the True Foundation. Rick expects the documents to be sent in a week or so.
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UTSA Safety hired registered nurse who had been in the army. The nurse probably
needs to work under a physician. UTSA: will give followup to UNM and UNM is
willing to help UTSA
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Biodefense Conference in ASM in Feb 2007: Terry Kohler is organizing one of the
sessions on vaccine development for biodefense and Karl is being asked to
participate to speak on Francisella vaccine development for biodefense. Should Karl
represent his lab alone or the UNM TVDC contract? Rick is fine with Karl
representing the overall contract at the ASM biodefense meeting. Vicki and Marlene
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are fine with Karl being a spokesperson. Rick wants to review Karl’s slides. Karl will
indicate the TVDC teams’ broad approaches and what we want to do. Karl- broad
description to publicize this project to a very receptive crowd.
Karl and Vicki went the first year and did’t find it useful; Terry Kohler says it is more
useful now and more organizers are knowledgeable about biodefense.
Joe: one of his contractors is helping to organize the ASM biodefense meeting too.
Trying to make it more scientifically biodefense oriented.
Woods Hole Meeting: Karl is sending Jeff Barker, who is a graduate student, to the
joint DVC/UNM/NIAID meeting. Rick will help Jeff during this conference