Download A previously uncharacterized tetratricopeptide-repeat

Microbiology (2015), 161, 148–157
DOI 10.1099/mic.0.082420-0
A previously uncharacterized
tetratricopeptide-repeat-containing protein is
involved in cell envelope function in Rhizobium
leguminosarum
Kara D. Neudorf,1 Elizabeth M. Vanderlinde,2 Dinah D. Tambalo1
and Christopher K. Yost1
Correspondence
1
Christopher K. Yost
2
[email protected]
Received 15 July 2014
Accepted 30 October 2014
Department of Biology, University of Regina, Regina, Saskatchewan S4S 0A2, Canada
Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon,
Saskatchewan S7N 5A2, Canada
Rhizobium leguminosarum is a soil bacterium that is an intracellular symbiont of leguminous
plants through the formation of nitrogen-fixing root nodules. Due to the changing environments
that rhizobia encounter, the cell is often faced with a variety of cell altering stressors that can
compromise the cell envelope integrity. A previously uncharacterized operon (RL3499–RL3502)
has been linked to proper cell envelope function, and mutants display pleiotropic phenotypes
including an inability to grow on peptide-rich media. In order to identify functional partners to the
operon, suppressor mutants capable of growth on complex, peptide-rich media were isolated. A
suppressor mutant of a non-polar mutation to RL3500 was chosen for further characterization.
Transposon mutagenesis, screening for loss of the suppressor phenotype, led to the identification
of a Tn5 insertion in an uncharacterized tetratricopeptide-repeat-containing protein RL0936.
Furthermore, RL0936 had a 3.5-fold increase in gene expression in the suppressor strain when
compared with the WT and a 1.5-fold increase in the original RL3500 mutant. Mutation of
RL0936 decreased desiccation tolerance and lowered the ability to form biofilms when compared
with the WT strain. This work has identified a potential interaction between RL0936 and the
RL3499–RL3502 operon that is involved in cell envelope development in R. leguminosarum, and
has described phenotypic activities to a previously uncharacterized conserved hypothetical gene.
INTRODUCTION
Rhizobium leguminosarum is a Gram-negative bacterium
that can form a symbiotic relationship with legumes such
as peas and lentils. R. leguminosarum infects the plant root
through an infection thread, enters root cells through
endocytosis and differentiates into bacteroids culminating
in the formation of a root nodule structure. Within the
nodule the bacteroids supply fixed atmospheric nitrogen to
the plant, while the plant supplies carbon to the bacteroids
(Gibson et al., 2008). Free-living rhizobia can be found in
the rhizosphere, where they experience a dynamic growth
environment including fluctuations in water availability,
temperature and nutrient limitation due to competition
with other microbes. Since bacteria often must respond to
changing environments, they have evolved a complex cell
Abbreviations: NPN, 1-N-phenylnaphthylamine; TPR, tetratricopeptide
repeat; vWFA, von Willebrand factor type A.
One supplementary figure is available with the online Supplementary
Material.
148
envelope that adapts under stressful conditions to maintain
cell envelope integrity in order to sustain its primary
function of permitting the select passage of nutrients
and waste products (Silhavy et al., 2010; Storz & HenggeAronis, 2000).
The genetic network involved in cell envelope development
and stress adaptation is only partially described in rhizobia,
despite its function for cell survival in the soil and during
symbiosis. Using genetic approaches, we have initiated a
programme to identify novel genes involved in cell envelope
development. Our earlier work and that of others has
identified a link between Rhizobium genes involved in cell
envelope development and their requirement for growth
on complex peptide rich media such as tryptone-yeast
extract medium (TY). Specific examples include genes
involved in the synthesis of the very long chain fatty acid
attached to the lipid A of the outer membrane lipopolysaccharide (Vanderlinde et al., 2009), a gene with homology to the cell-envelope-associated carboxyl-terminal
protease (ctpA) (Gilbert et al., 2007), a two-component
Downloaded from www.microbiologyresearch.org by
082420 G 2015 The Authors
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:21:11
Printed in Great Britain
Protein involved in cell envelope function
sensor histidine kinase (ctpA) (Gibson et al., 2008), a
two-component signal transduction system (chvG/chvI)
(Bélanger et al., 2009; Foreman et al., 2010; Vanderlinde &
Yost, 2012) and a broadly conserved four gene operon with a
MoxR AAA+ and von Wilderbrand A domain containing
gene (RL3499–RL3502) (Vanderlinde et al., 2011). The
shared mutant phenotype of an inability to grow on complex
peptide rich media suggests a possible network between these
cell envelope related genes, and further experimentation is
needed to identify potential interaction networks.
tetratricopeptide repeat (TPR)-containing protein, originally annotated as RL0936 (Young et al., 2006), and henceforth termed ttpA (tetratricopeptide repeat protein). The
results herein characterize this previously uncharacterized
ttpA gene, coding for a conserved hypothetical protein, and
provide evidence suggesting that ttpA may interact with the
cmdA–cmdD operon.
Vanderlinde et al. (2011) identified a link between the
uncharacterized, broadly conserved MoxR AAA+ operon,
and proper cell envelope function in R. leguminosarum.
The genes in this operon are subsequently referred to as
complex media growth deficient (cmdA–cmdD), based on
the mutant phenotype of an inability to grow on complex
peptide rich growth media. Mutations in the operon
resulted in a number of phenotypes related to envelope
disfunction and cell morphology (Vanderlinde et al., 2011).
The large MoxR AAA+ family contains a diverse group of
ATPases that are found throughout bacteria and are
suggested to have chaperone-like activities (Snider &
Houry, 2006; Wong & Houry, 2012). These MoxR
AAA+ proteins often function together with VWAdomain-containing proteins to form a chaperone system
involved in the folding and activation of proteins through
the insertion of metal cofactors into the substrate
molecules (Snider & Houry, 2006). The general nature of
MoxR related genes and the pleiotropic phenotypes
observed in the R. leguminosarum mutants suggests the
operon may be connected to a network of envelope related
genes. By identifying interacting partners with the cmdA–
cmdD operon, we can begin to elucidate the complex
genetic network responsible for proper cell envelope
development in rhizobia.
Plasmids, PCR primers, bacterial strains and growth conditions. All plasmids, strains and primers used in this study are listed in
Isolation of suppressor mutants is a well-established
approach to identify functional partners and elucidate
novel gene functions (Clementz et al., 1997; Driscoll &
Finan, 1993; Li et al., 2004; Ogura et al., 1991; Oresnik et al.,
1994; Wells & Long, 2003), and has been used to define
functional networks involved in cell envelope development
and function (Alonso-Monge et al., 2001; Fields et al.,
2012; Hayden & Ades, 2008). Vanderlinde et al. (2011)
noted that culturing cmdA–cmdD mutants on TY agar at
high cell density (108 c.f.u. ml21) resulted in the appearance of suppressor mutants capable of growth on the TY
agar.
In this study, suppressor mutants capable of growth on TY
medium were successfully isolated for strains with mutations in the cmdA–cmdD operon. A suppressor mutant in
the non-polar cmdB (RL3500) deletion mutant background
was chosen for further characterization. Transposon
mutagenesis of the suppressor mutant and screening for
loss of growth on TY was used to attempt identification of
the suppressor locus. This approach lead to the isolation of
a mutant carrying a Tn5 insertion in an uncharacterized
http://mic.sgmjournals.org
METHODS
Table 1. R. leguminosarum strains were grown using either Vincent’s
minimal media (VMM) and 10 mM mannitol as the carbon source
(Vincent, 1970), or tryptone-yeast extract (TY) media (Beringer,
1974). These strains were cultured with the following antibiotic
concentrations when appropriate (mg ml21): streptomycin, 500;
gentamicin, 30; tetracycline, 5; and neomycin, 100. All Escherichia
coli strains were grown using LB medium and the following antibiotic
concentrations when appropriate (mg ml21): spectinomycin, 100;
gentamicin, 15; tetracycline, 10; ampicillin, 100; and kanamycin, 50.
Isolation of suppressor mutants in cmdA–cmdD operon
mutants. Deletion of genes in the cmdA–cmdD operon results in a
mutant’s inability to grow on TY media or VMM supplemented with
3 mM glycine. Suppressor mutants that restored the ability of a cmdB
mutant (38cmdB) to grow on VMM supplemented with 3 mM glycine
were isolated by performing serial dilutions of 38cmdB (100–108) and
plating on VMM with 3 mM glycine, as well as VMM as a control.
Colonies that were able to grow on the glycine plates were purified and
PCR was performed to verify the original cmdB mutation remained
present in the suppressor mutants. Suppressor strains were then
streaked onto TY to confirm restored growth on TY. A suppressor
mutant matching these criteria was used for further study (38cmdB-S).
Transposon mutagenesis of suppressor mutants. The mini-Tn5
derivative found on plasmid pTGN (Tang et al., 1999) was used for
insertional mutagenesis of the suppressor mutant 38cmdB-S. The
transposon was introduced into 38cmdB-S via conjugation using E.
coli S17-1 containing pTGN as the donor strain and incubation at
30 uC for 48 h on VMM plates containing 1 mM proline. Mutants
were screened for sensitivity to 3 mM glycine by tooth-picking
mutants onto VMM containing 3 mM glycine, and using VMM with
streptomycin and neomycin as a positive control. Mutants that were
unable to grow on the glycine plates were also screened for an
inability to grow on the peptide rich complex media TY, which is the
phenotype in the original 38cmdB genetic background. To identify
the transposon insertion site, genomic DNA was isolated and used to
perform the thermal asymmetrical interlaced (TAIL)-PCR protocol as
described by Liu & Huang (1998). The TAIL-PCR product was sent
for sequencing and location of the Tn insertion was identified using
the RhizoDB database (http://www.xbase.ac.uk/rhizodb/) and a
BLASTN search (Altschul et al., 1997).
Molecular techniques. An alkaline lysis method as described by
Sambrook & Russell (2001) was used to perform all plasmid
preparations. A QIAEX II gel extraction kit (Qiagen) was used to
extract PCR products from agarose gels whenever needed. All
restriction enzymes were purchased from Invitrogen and were used
according to the supplier’s instructions.
Insertional mutagenesis of ttpA. To further characterize ttpA in a
WT genetic background, a single crossover mutant was constructed in
strain 3841. Insertional mutagenesis via allelic exchange using the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:21:11
149
K. D. Neudorf and others
Table 1. Summary of all plasmids, strains and primers used in this study
Strain or plasmid
E. coli
JM109
DH5a
S17-1
R. leguminosarum
3841
38cmdA
38cmdA-S
38cmdB
38cmdB-S
38cmdB-S/ttpA : : Tn5
38ttpA
Plasmids
pTGN
pGem-T easy vector
pJQ200SK
pHC41
pRL0936-comp
pfus1par
pfus : : ttpA
Primers
AD-1
Gm-TAIL-1
Gm-TAIL-2
RL0936A
RL0936B
RL0936C
RL0936H
RL0936F
RL0936R
pfusRL0936F
pfusRL0936R
Relevant characteristics
F- episome recA- endA-, AmpR TcR KmR
endA1 hsdR17 supE44 thi-1 recA1 gyrA96 relA1 D(argF-lacZYA) DlacU169
f80lacZDM15
RP4 tra region, mobilizer strain recA derivative of MM294A with RP4-2
(Tc : : Mu : : Km : : Tn7)
Promega
Invitrogen
Spontaneous streptomycin-resistant derivative of R. leguminosarum bv. viciae
strain 300, SmR, TY+, Gly+
3841 RL3499 (cmdA) deletion incapable of growth on TY media or VMM
with 3 mM glycine, SmR, TY2, Gly2
cmdA suppressor mutant, restored growth on TY and in presence of 3 mM
glycine, SmR, TY+, Gly+
3841 RL3500 (cmdB) deletion incapable of growth on TY media or VMM
with 3 mM glycine, SmR, TY2, Gly2
38cmdB suppressor mutant, restored growth on TY and in presence of 3 mM
glycine, SmR, TY2, Gly2
38cmdB-S background, RL0936 (ttpA) mutant due to Tn5 insertion 290 bp into
ORF, SmR NmR GmR, TY2, Gly2
3841 RL0936 (ttpA) single crossover insertional mutant containing pJQ200SK
suicide vector, SmR GmR, TY+, Gly+
Glenn et al. (1980), Johnston
& Beringer (1975)
Vanderlinde et al. (2011)
Tn5 derivative, GmR AmpR, promoter-less nptII, gfp
pGem TA cloning vector, 3015 bp, AmpR
Suicide vector, P15a ori mob sacB, GmR
IncP broad-host-range vector with par stabilization, TcR
pHC41 with a 2195 bp PCR fragment containing the entire ttpA region
including the promoter, TcR
pFus1 with par locus of RK2
Tang et al. (1999)
Promega
Quandt & Hynes (1993)
Cheng & Walker (1998)
This study
Simon et al. (1983)
This study
Vanderlinde et al. (2011)
This study
This study
This study
ttpA promoter fragment cloned into pfus1par with KpnI and EcoRI
Reeve et al. (1999); Yost et al.
(2004)
This study
59-AGWGNAGWANCAWAGG-39
59-CAACGCGCTTGGTGCTAATGTGAT-39
59-AGTTGGGCATACGGGAAGAAGTGA-39
59-AGTCTAGATGATCGACTTCTGCATCGAG-39
59-GTTCGGCAACAGGTTGTCAC-39
59-ATCTGAACGATGCCGTGCT-39
59-AGTCTAGACCAGCATATCGAGCAGATGA-39
59AGACTAGTGGACAGCGTGACCACTTTCT-39
59-AGCCCGGGTGCTTATTGCCTTGTGTGC-39
59-AGGGTACCGCGATCCATTACGGAACAAT-39
59-GGAGAAAGTGGTCACGCTGT-39
Liu & Huang (1998)
Vanderlinde et al. (2010)
Vanderlinde et al. (2010)
This study
This study
This study
This study
This study
This study
This study
This study
suicide vector pJQ200SK was performed using a method described by
Quandt & Hynes (1993). A 600 bp internal fragment of ttpA was
amplified using primers RL0936F with a SpeI linker and RL0936R
with a SmaI linker (Table 1). PCRs used 1 U of Taq DNA polymerase
(UBI) and the following reaction conditions: 2 mM MgSO4, 0.2 mM
dNTPs, 16 reaction buffer and 0.2 mM of each primer. Using a BioRad thermocycler the PCR was run at 94 uC for 5 min, followed by 30
cycles of 95 uC for 30 s, 68 uC for 30 s and 72 uC for 1 min 30 s, with
a final extension of 72 uC for 5 min. The PCR fragment was cloned
into a pGem-T Easy Vector (Promega) as per the supplier’s
instructions. Restriction enzymes SpeI and SmaI were used to excise
the ttpA fragment from the pGem-T Easy Vector, which was then
ligated into pJQ200SK using complementary restriction enzyme sites
150
Source
to create pJQRL0936. The new construct was first transformed into
DH5a competent cells to confirm proper insertion, and subsequently
transformed into the mobilizer strain S17-1. Conjugation was
performed over 48 h with the WT strain on VMM containing
1 mM proline. Single crossover mutants were isolated by plating the
conjugation mixture onto VMM containing streptomycin and
gentamicin to select for insertion of the pJQ200SK vector. PCR was
used to confirm the correct disruption of ttpA.
Construction of a broad-host-range plasmid containing the
RL0936 gene. Genetic complementation of ttpA mutants used a
vector constructed to contain the WT ttpA gene. The ttpA gene with
the upstream promoter region was PCR amplified from the WT strain
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:21:11
Microbiology 161
Protein involved in cell envelope function
of R. leguminosarum bv. viciae 3841 using primers RL0936A and
RL0936H (Table 1), both containing an XbaI restriction linker at the
59 end. PCR used 1 U of Taq DNA polymerase (UBI) and the
following reaction conditions: 2 mM MgSO4, 0.2 mM dNTPs, 16
reaction buffer and 0.2 mM of each primer. Using a Bio-Rad
thermocycler the reaction was run at 94 uC for 5 min, followed by
30 cycles of 95 uC for 30 s, 66 uC for 30 s and 72 uC for 3 min 30 s,
with a final extension of 72 uC for 5 min. The PCR fidelity was
confirmed though DNA sequencing of the PCR amplicon. The PCR
fragment was then cloned into a pGem-T Easy Vector (Promega) and
digested using XbaI. The digested fragment was then ligated into the
broad-host-range plasmid pHC41 using the same restriction enzyme
to cut the vector. The ligation was transformed into the mobilizer
strain S17-1 to make pRL0936comp and was used for conjugation to
complement ttpA in the mutant strain 38ttpA (Table 1). The
pRL0936comp plasmid was also conjugated into 38cmdB. Recipient
colonies were tested for growth on TY and VMM containing 3 mM
glycine.
RNA extraction and quantitative reverse transcription PCR (RTqPCR) to measure mRNA levels of ttpA. RNA was extracted using
cells that were grown on VMM with erythritol (15 mM) to an OD600
of 0.5. A volume of 0.4 was added of 5 % acid phenol in ethanol to the
cells, which were then kept on ice for 30 min. Samples were
centrifuged at 8200 g for 5 min at 4 uC. RNA extraction was
performed using an miRNeasy mini kit (Qiagen) following the
manufacturer’s protocol with a few modifications. Briefly, cell pellets
were resuspended in 100 ml TE with lysozyme (50 mg ml21) and
20 ml proteinase K (Qiagen). Samples were incubated at room
temperature for 5 min, vortexing for 10 s every 2 min. One millilitre
of pre-heated QIAzol reagent (65 uC) was added, samples were
vortexed on high for 3 min, and were incubated at room temperature
for 5 min. Chloroform was added (200 ml) with vigorous mixing for
5 s, and then incubated at room temperature for 3 min. Samples were
centrifuged at 12 000 g for 15 min at 4 uC. Aqueous phases were
transferred to new microfuge tubes and extraction was completed
using an miRNeasy mini kit, starting with the ethanol addition step
(Qiagen). DNase treatment was conducted using Turbo DNase (Life
Technologies). Reverse transcription reactions were carried out
according to a previously described protocol (Manzon et al., 2007),
with modifications described by Vanderlinde et al. (2009, 2011).
Quantitative PCR was conducted on an Applied Biosystems StepOne
Real-Time PCR System using SYBR Green PCR Master Mix (Life
Technologies) as per manufacturer’s instructions and using primers
RL0936B and RL0936C (Table 1).
Construction of an ttpA : : gusA transcriptional fusion. A 245 bp
fragment upstream of RL0936 was amplified with the primers
pfusRL0936F and pfusRL0936R under standard PCR conditions. The
fragment was subsequently cloned into a pGem-T easy vector as per
manufacturer’s instructions. The cloned promoter region was then
excised using KpnI and EcoRI and ligated into the pFus1par vector
containing the promoterless gusA reporter gene in a par-stabilized
plasmid. The PCR fidelity was confirmed through sequencing of the
PCR amplicon. The resulting plasmid pFusRL0936 was then
conjugated into the R. leguminosarum WT strain 3841 and into the
mutant strains 38cmdB, 38cmdB-S and 38cmdB-S/ttpA : : Tn5 to
measure gene expression using the b-galactosidase reporter gene assay
of Miller (1972), with modifications described by Yost et al. (2004).
All assays were carried out on cultures in the late-exponential growth
phase.
DNA sequence analysis. DNA sequencing was conducted by
Quintarabio (Albany, CA, USA). Sequence data were analysed
using EnzymeX [version 3.1; (http://nucleobytes.com/index.php/
enzymex)] and 4PEAKS software [version 1.7.2; (http://nucleobytes.
com/index.php/4peaks)]. OLIGO 4.0 software (National Biosciences)
http://mic.sgmjournals.org
was used to design all primers. Sequence alignments were done using
(Thompson et al., 1994). Phylogenies were reconstructed
using MEGA 5.05 (Tamura et al., 2011).
CLUSTAL W
Desiccation assay. Due to the importance of a functional cell
envelope in desiccation tolerance (Gilbert et al., 2007; Vanderlinde
et al., 2009, 2010), desiccation tolerance of the mutants was
investigated. A filtration method described by Ophir & Gutnick
(1994) and modified by Gilbert et al. (2007) was used to perform
desiccation sensitivity assays. Cultures were first grown in VMM
broth for 48 h and diluted to 50 ml using sterile VMM broth. The
dilution was filtered using a Millipore Vacuum Manifold, Microfil
Filtration funnels, and S-Pak 0.45 mM type HA membranes,
according to the manufacturer’s description (Millipore). Filter
membranes were cut in half and one half was placed on water agar
(1.25 %), while the other was placed in a sterile empty Petri dish. All
plates were incubated at 30 uC for 48 h. After incubation, each filter
was put in separate vials containing 2 ml of water and vortexed for
5 min to remove cells. The cell suspension was serially diluted and
spot plated on VMM to determine the number of culturable bacteria
and the percentage survival was calculated using the ration of c.f.u.
ml21 (desiccated) to c.f.u. ml21 (hydrated).
Biofilm assay. A functional cell envelope is important for biofilm
formation in R. leguminosarum (Vanderlinde et al., 2010); therefore,
the biofilm capabilities of various mutants were tested. A method
adapted from Fujishige et al. (2006) and O’Toole & Kolter (1998)
was used to culture biofilms in microtitre plates. Cells were first
scraped off from VMM plates using a sterile cotton swab and were
suspended in double-distilled water (ddH2O). To obtain an
approximate starting inoculum of about 16107 c.f.u. ml21, the
suspensions were diluted 1 in 15 using VMM broth containing 1 %
mannitol. From this dilution, 150 ml was added to each well of a flat
bottom Corning CellBIND Surface brand 96-well plate. The plates
were sealed using Parafilm and were incubated on a gyrorotary
shaker set to 100 r.p.m. for 48 h at 30 uC. Biofilm-containing wells
were rinsed with 200 ml ddH2O and stained with 200 ml 0.4 % crystal
violet in each well for 15 min at room temperature. The wells were
then rinsed three times with 200 ml ddH2O and the remaining crystal
violet was solubilized with 200 ml 95 % ethanol. Absorbance was
measured at 550 nm.
Antibiotic sensitivity assay. Sensitivity to hydrophobic antibiotics
can relay information regarding cell envelope integrity. Sensitivity
assays were conducted on VMM plates containing appropriate
antibiotics. Strain suspensions were added to 400 ml 0.75 % agar
VMM overlay tubes in and poured onto individual plates. The
overlay was allowed to solidify, and 10 ml of either 10 mg
erythromycin ml21 or 0.1 mg tetracycline ml21 was added to
sterilized 7 mm discs of blotting paper. The antibiotic-containing
discs were placed on the overlay plates and allowed to incubate for
48 h at 30 uC. Zones of inhibition were measured at the end of the
incubation period.
NPN assay. To determine outer membrane permeability changes in
the R. leguminosarum mutant strains, uptake of the fluorescent probe
1-N-phenylnaphthylamine (NPN) was measured using a method
described by Helander & Mattila-Sandholm (2000). An intact outer
membrane prevents binding of the NPN; therefore, an increase in
fluorescence indicates decreased structural integrity of the outer
membrane. Cells were added to a microtitre plate in HEPES buffer at
a concentration of 5 mmol l21, pH 7.2, with 50 ml NPN (40 mmol
l21 in HEPES buffer). The fluorescence was measured using a fluorescence microplate spectrophotometer with an excitation wavelength at 355 nm and detection of an emission wavelength at
405 nm.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:21:11
151
K. D. Neudorf and others
RESULTS AND DISCUSSION
Isolation of suppressor mutants and subsequent
transposon mutagenesis
Suppressor mutants can be valuable in identifying interactions between genes when studying a specific phenotype,
particularly when the function of the genes is not readily
apparent. Further, isolation of suppressor mutants is a
well-established strategy for identifying gene partners and
elucidating genetic networks (Clementz et al., 1997;
Driscoll & Finan, 1993; Li et al., 2004; Ogura et al., 1991;
Oresnik et al., 1994; Wells & Long, 2003). Suppressor
mutants were isolated in a cmdB non-polar mutant
background at a frequency of 1026 c.f.u. ml21 on both
TY and VMM with 3 mM glycine. Suppressor strains were
confirmed through PCR to maintain the original in-frame
deletion to cmdB, confirming that a secondary site
mutation is the result for the restored growth on TY and
VMM with 3 mM glycine phenotype. We were able to
isolate suppressor mutants for cmdA, cmdB and cmdC nonpolar mutants, as well as a polar mutant at similar
frequencies, of 1025, 1026, 1026 and 1025, respectively.
cmdB suppressors were selected for further study, as it is a
likely partner to cmdA based on canonical moxR descriptions (Snider & Houry, 2006; Wong & Houry, 2012), and
we were interested in identifying genes that could
compensate for loss of cmdB in this partnership. Future
studies into the functional partners interacting with the
other operon genes will be undertaken to provide a more
complete picture of the overall operon function. However,
a substantial effort is required to identify each specific
unmarked suppressor locus and was therefore beyond the
scope of this investigation.
Transposon mutagenesis was conducted in an attempt to
identify the suppressor locus in strain 38cmdB-S by
screening for a loss of growth on TY media phenotype
(Fig. 1). This screen led to the isolation of a mutant with a
Tn5 insertion in a broadly conserved gene, within the
alphaproteobacteria, coding for a tetratricopeptide-repeatcontaining protein (ttpA). Genes containing this motif are
predicted to code for proteins involved in a broad range of
protein–protein interactions involved in diverse cellular
functions such as cell cycle regulation, transcription and
protein folding (Cerveny et al., 2013). The conservation
of ttpA was similar to the conservation of the cmdA–
cmdD operon where both loci are conserved within the
Rhizobiales and in other Alphaproteobacteria classes such as
the Rhodobacteraceae and Rhodospirillaceae (Fig. S1,
available with the online Supplementary Material, and
Vanderlinde et al., 2011).
The transposon mutant strain, 38cmdB-S/ttpA : : Tn5, was
complemented with a plasmid carrying WT cmdB, which
restored an ability to grow on TY and VMM with 3 mM
glycine plates, suggesting that mutation of the ttpA gene
did not create a loss in TY growth that was independent of
the cmdB mutation in 38cmdB-S. Furthermore, insertional
152
mutation of ttpA in a WT background does not result in an
inability to grow on TY media or VMM supplemented with
3 mM glycine. These combined results suggested ttpA as a
putative candidate for the suppressor locus. However, ttpA
and the promoter region were PCR amplified and DNA
sequenced in all relevant strains, and no polymorphisms
were identified between the suppressor strain, the original
cmdB mutant strain or the WT strain (data not shown).
This result implies that the specific secondary site mutation
is located elsewhere in the genome. Nonetheless, the
above phenotypic data suggested a link between ttpA and
cmdB. Furthermore, bioinformatic analysis supports a
predicted interaction of ttpA with genes from the cmdA–
cmdD operon. The STRING (http://string-db.org/) algorithm
explores predicted protein interaction networks, using data
extracted from curated functional genomic and protein
structure databases. STRING weakly predicted ttpA to
interact with cmdA–cmdD (score of 0.56). It is also
interesting to note the STRING analysis predicted interaction
between RL0936 and ctpA (score of 0.65), a gene that is
known to be associated with cell envelope function in R.
leguminosarum and is required for growth on TY agar
(Gilbert et al., 2007).
ttpA expression is increased in cmdB mutant
backgrounds
To examine the expression levels of ttpA in the various
mutants, a transcriptional fusion containing the promoter
region of ttpA cloned upstream of the promoter-less gusA
reporter gene in pFus1par1 was constructed. GusA assays
were performed, and it was noted that expression of ttpA
was 3.5-fold higher in the original suppressor when
compared to the WT and 1.5-fold higher in 38cmdB
(Table 2). The increased expression of ttpA was exclusive
to the 38cmdB-S suppressor strain. Expression levels of
ttpA in the cmdA, cmdC or cmdD suppressors did not
significantly increase relative to the level of expression in
the original mutant strains. Since there was no sequence
difference for ttpA nor in its promoter region, the actual
suppressor locus may be involved in ttpA regulation.
Addition of a broad-host-range plasmid
containing the WT ttpA rescues the TY minus
phenotype in a cmdB mutant
A broad-host-range plasmid containing ttpA and its
promoter region (pRL0936comp) was conjugated into
38cmdB. This strain was able to grow on both TY and
VMM with 3 mM glycine, while the control of 38cmdB
carrying the empty vector pHC41 was unable to grow on
TY or VMM with 3 mM glycine. RT-qPCR confirmed that
the mRNA concentration of ttpA was a mean of 12-fold
higher in strains carrying the pRL0936comp plasmid
compared to strains carrying the empty pHC41 vector.
These data, along with the data in Table 2, support the
hypothesis that an increase in expression of ttpA in
38cmdB can lead to restoration of growth on TY and
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:21:11
Microbiology 161
Protein involved in cell envelope function
(a) 3841
(WT)
RL3499 RL3500 RL3501
RL3502
(b) 38cmdB
(RL3500
non-polar
mutant)
RL3499 RL3500 RL3501
RL3502
RL3499
(c) 38cmdB-S
(cmdB
suppressor)
RL3499
RL3501
TY+ Gly+
RL3502
TY– Gly–
?
RL3501 RL3502
TY+ Gly+
Suppressor
change
Tn5
(d) 38cmdBS/ttpA::Tn5
(38cmdB-S
with Tn5)
RL3499
RL3501
?
RL3502
RL0936
TY– Gly–
Suppressor
change
Fig. 1. Schematic outlining mutational steps. (a) WT strain, being able to grow on both TY (TY+) and VMM with 3 mM glycine
(Gly+). (b) In-frame (non-polar) deletion mutant 38cmdB, which is no longer able to grow on either TY (TY”) or VMM with 3 mM
glycine (Gly”); the dashed lines indicate the region of the gene deletion. (c) Suppressor mutant (38cmdB-S) of 38cmdB that
has gained the ability to grow on both TY and VMM with 3 mM glycine (TY+, Gly+). The suppressor mutation is unmarked. (d)
Transposon mutagenesis that was performed on 38cmdB-S to screen for a TY and 3 mM VMM with glycine minus phenotype
(TY”, Gly”); the Tn5 labelled arrow indicates the location of the transposon insertion site. The transposon insertion site was
mapped to ttpA, a TPR-containing protein. Schematic is not drawn to scale.
VMM with 3 mM glycine. Notably, the ttpA containing
plasmid did not rescue growth on TY or VMM with 3 mM
glycine, when conjugated into strains carrying in-frame
deletions in the other genes of the cmdA–cmdD operon
(e.g. Fig. 2c), suggesting that the interaction of ttpA with
the cmdA–cmdD operon is specific and may be complementary to the role of the cmdB gene (Fig. 2, Table 2).
cmdB codes for a conserved hypothetical protein with
a domain of unknown function (DUF) 58 and a von
Willebrand factor type A (vWFA) motif. vWFA domains
are broadly conserved and are found in both prokaryotes
and eukaryotes. Proteins that contain vWFA domains in
eukaryotes are associated with many cellular functions such
as cell adhesion and signal transduction (Colombatti et al.,
Table 2. ttpA gene expression measured by gusA fusion in various strains
Expression of ttpA is 1.5-fold upregulated in 38cmdB and 3.5-fold upregulated in 38cmdB-S and 38cmdB-S/ttpA : : Tn5.
Strain
Genotype and phenotype
Miller units
(mean±SD)
3841
38cmdB
38cmdB-S
38cmdB-S/ttpA : : Tn5
http://mic.sgmjournals.org
WT
RL3500-, TY minus, VMM 3 mM glycine minus
RL3500-suppressor, TY plus, VMM 3 mM glycine plus
Tn5 in RL0936 in cmdBS background, TY minus, VMM 3 mM glycine minus
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:21:11
1954±53
2911±71
6943±40
6911±90
153
K. D. Neudorf and others
cmdA– ttpA++
cmdB– ttpA++
cmdB– suppressed
cmdA–
(a) VMM
cmdB–
cmdB– suppressed
3841
3841
cmdB–
cmdB– ttpA++
3841
cmdA– suppressed
(b) TY
(c) TY
Fig. 2. Growth results on VMM agar (a) and TY agar (b) with WT (3841), a cmdB deletion mutant strain (38cmdB), a cmdB
suppressor strain (38cmdB-S), and 38cmdB containing plasmid pRL0936-comp, which increases expression of ttpA in the
cell. (c) Growth results on TY with WT (3841), a cmdA deletion mutant strain (38cmdA), a cmdA suppressor strain (38cmdAS), and 38cmdA containing plasmid pRL0936-comp with increased ttpA expression. All strains grew well on the VMM agar
control plate. The same growth phenotypes for TY were observed on VMM with 3 mM glycine (not shown). A control vector of
pHC41 (i.e. not containing ttpA insert) was conjugated into all strains and no difference in phenotype was observed.
1993; Whittaker & Hynes, 2002). However, the functions
for the majority of vWFA proteins within prokaryotes are
largely unknown. Whelan et al. (1997) described a vWFA
protein in E. coli that aids in protection against the toxic
effects of heavy metals. Recently, Wong et al. (2014) have
identified that the vWFA protein ViaA, which is associated
with the MoxR-like protein RavA, interacts with specific
subunits of the NADH : ubiquinone oxidoreductase I
complex. The other characterized bacterial vWFA has
magnesium-dependent enzyme interactions (Jensen et al.,
1998). The majority of prokaryotic vWFA domains have
conserved aspartic acids and serine residues, which are
characteristic of eukaryotic vWFA domains that are
important for divalent cation interactions (Ponting et al.,
1999). Notably, many of the defective phenotypes in a
cmdB mutant can be suppressed in media supplemented
with additional 3.4 mM calcium or magnesium ions
(Vanderlinde et al., 2011). The vWFA domain of CmdB
likely contains a metal ion adhesion site for incorporation
of a metal ligand, and these are often found in proteins
connected with MoxR ATPases, such as CmdA (Wong &
Houry, 2012). Based on various bioinformatic analyses and
available literature, it’s been suggested that the MoxR
AAA+ proteins function with the vWFA domain-containing proteins to form a chaperone system important for
protein folding and activation (Snider & Houry, 2006).
Bioinformatic description of ttpA, a gene coding
for a TPR-containing protein
ttpA is annotated as coding for a conserved hypothetical
uncharacterized protein. The Pfam (http://pfam.xfam.org)
and cdd (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.
shtml) databases identify a conserved TPR motif. TPRcontaining proteins are found in a diversity of organisms
154
ranging from bacteria to humans. They are predicted to
be involved in protein–protein interactions for a number of
different cellular functions, including cell-cycle regulation,
transcription and protein folding. TtpA contains a TPR
structural motif. The basic function of these TPR folds is
to mediate protein–protein interactions, which can often be
promiscuous (Cerveny et al., 2013). Since CmdB is predicted
to be involved in a protein–protein interaction with CmdA,
it is interesting to find a link to TtpA, which is also predicted
to be involved in protein–protein interactions. TPR motifs
occur in minimally conserved 34-residue-long regions,
centred around the consensus residues W4-L7-G8-Y11-A20F24-A27-P32 (Lamb et al., 1995). There is 3D structural data
available for the TPR motif, which contains two antiparallel
a-helical subdomains (helix A and helix B), which are
arranged in tandem arrays of three to sixteen TPRs that can
accommodate the complementary region of a target protein
(Cerveny et al., 2013; Das et al., 1998). There are three
predicted TPRs found in RL0936, one TPR-16 at aa 362, one
TPR-11 at aa 429 and one TPR-11 at aa 510. Since growth on
TY and VMM with 3 mM glycine can be restored through
creating a higher copy number of ttpA in 38cmdB, and since
ttpA is overexpressed in 38cmdB-S, we suggest that an
increase in expression of ttpA can compensate for the loss of
cmdB. Future collaborative work with protein structural
biologists will help to investigate potential interactions
between the TtpA protein with protein components of the
cmdA–cmdD operon, and in particular the protein coded by
cmdB.
Outer membrane integrity is not restored in the
suppressor mutant
Mutations in the cmdA–cmdD operon are known to
increase the mutant’s sensitivity to hydrophobic antibiotics
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:21:11
Microbiology 161
Protein involved in cell envelope function
The suppressor mutant and associated mutant
backgrounds with mutation of ttpA display
reduced biofilm formation and desiccation
tolerance
Biofilm formation in all mutant backgrounds was compared using the biofilm quantification assay described in
the Methods. Fig. 3(c) demonstrates that the TY suppressor
strain and strains carrying mutations in ttpA produced
significantly (Student t-test, P50.008) less biofilms than
the WT; 38cmdB-S/ttpA : : Tn5 had the greatest decrease in
biomass with a 50 % decrease, followed by 38cmdB-S with
a 38 % decrease, 38ttpA with a 33 % decrease and 38cmdB
with a 14 % decrease in biomass, when compared with
3841 (Fig. 3c). Desiccation sensitivity was also investigated,
and 38ttpA had a mean percentage cell survival of only
4.25 % when compared with the WT of 10.36 %. 38cmdB
had a cell survival mean of 8.92 %, 38cmdB-S had a mean
of 7.03 % and 38cmdB-S/ttpA : : Tn5 had the lowest cell
survival value of 1.85 %. These data, as well as data from
biofilm assays, illustrate that mutation of ttpA alters
phenotypes related to cell envelope structure.
In conclusion, the gain-of-function suppressor isolation
approach has not only provided experimental evidence in
http://mic.sgmjournals.org
Zone of inhibition (% increase)
Tc
Er
SDS
120
100
80
60
40
20
0
3841
38cmdB
38cmdB-S 38cmdBS/ttpA::Tn5
38ttpA
(b)
NPN fluorescence (% increase)
It is interesting that an inability to grow on TY often
correlates with cell envelope deficiencies (Gibson et al.,
2008; Vanderlinde et al., 2009, 2010, 2011) yet we observe
here that suppressors that restore growth on TY remain
sensitive to membrane disruptive agents. It is also interesting to note there is an increase in sensitivity when both
genes are mutated in the strain cmdB-S/ttpA : : Tn5, when
compared with the WT and other mutants. The increased
sensitivity to multiple cell envelope stressors when both
cmdB and ttpA are mutated suggests their importance in
proper cell envelope function in R. leguminosarum. The
uncoupling of the two phenotypes in the suppressor strain
also suggests a parallel genetic network for the two
phenotypes. Supporting this observation, we are able to
isolate suppressors in cmdA–cmdD operon mutants that
restore resistance to membrane disruptive agents but do
not restore growth on TY media. Future work is planned to
characterize these suppressors, and by comparing and
contrasting the two families of suppressors we hope to gain
further insight into branches within the complex genetic
networks.
(a) 140
120
100
80
60
40
20
0
3841
38cmdB 38cmdB-S 38cmdB- 38ttpA
S/ttpA::Tn5
3841
38cmdB 38cmdB-S 38cmdB- 38ttpA
S/ttpA::Tn5
(c)
Absorbance (% decrease at 550nm)
and membrane disruptors (Vanderlinde et al., 2011). To
determine if the suppressor’s restoration of TY growth is
also correlated with restored outer membrane integrity, we
examined 38cmdB-S’s sensitivity to erythromycin, tetracycline, SDS and NPN uptake into the outer membrane.
The suppressor mutation did not restore outer membrane
stability, and 38cmdB-S remained more sensitive to the
antibiotics, SDS and NPN uptake when compared to
the WT (Fig. 3a, b). Notably, 38ttpA also experienced
increased sensitivity to these stressors and NPN uptake,
further supporting a role of ttpA in a cell envelope network.
60
50
40
30
20
10
0
Fig. 3. (a) Results from mutant sensitivity tests. Ten microlitres of
the following compounds were added on separate discs: 0.1 mg
tetracycline (Tc) ml”1, 10 mg erythromycin (Er) ml”1, and 10 %
SDS. The presented data includes four independent trials, with the
mean values and SD reported. Values are normalized to the WT
strain displaying zones of inhibition as a percentage increase
relative to the WT. All mutants were more sensitive than the WT
strain. It was also observed that complementation of 38ttpA with
pRL0936-comp restored resistance levels back to WT. (b) NPN
assay results. Values are normalized to the WT strain displaying
NPN fluorescence as a percentage increase relative to the WT.
Results suggest that all mutants possess a more permeable outer
membrane when compared to the WT. (c) Biofilm assay results
displayed as a percentage decrease normalized to the WT. All
mutants had a decreased ability to attach to a solid surface when
compared to the WT strain.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:21:11
155
K. D. Neudorf and others
support of a putative interaction between two relatively
uncharacterized genes, but has also provided phenotypic
data to suggest that ttpA, previously annotated as coding
for an uncharacterized hypothetical conserved protein, is
involved in cell envelope related physiology.
Gibson, K. E., Kobayashi, H. & Walker, G. C. (2008). Molecular
determinants of a symbiotic chronic infection. Annu Rev Genet 42,
413–441.
Gilbert, K. B., Vanderlinde, E. M. & Yost, C. K. (2007). Mutagenesis of
the carboxy terminal protease CtpA decreases desiccation tolerance in
Rhizobium leguminosarum. FEMS Microbiol Lett 272, 65–74.
Glenn, A. R., Poole, P. S. & Hudman, J. F. (1980). Succinate uptake by
ACKNOWLEDGEMENTS
free-living and bacteroid forms of Rhizobium leguminosarum. J Gen
Microbiol 119, 267–271.
The research was funded through a Canada Research Chair and
Natural Sciences and Engineering Research Council Discovery Grant
to C. K. Y.
Hayden, J. D. & Ades, S. E. (2008). The extracytoplasmic stress factor,
sigmaE, is required to maintain cell envelope integrity in Escherichia
coli. PLoS ONE 3, e1573.
Helander, I. M. & Mattila-Sandholm, T. (2000). Fluorometric
assessment of gram-negative bacterial permeabilization. J Appl
Microbiol 88, 213–219.
REFERENCES
Alonso-Monge, R., Real, E., Wojda, I., Bebelman, J. P., Mager, W. H. &
Siderius, M. (2001). Hyperosmotic stress response and regulation of
cell wall integrity in Saccharomyces cerevisiae share common
functional aspects. Mol Microbiol 41, 717–730.
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z.,
Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids Res 25,
3389–3402.
Bélanger, L., Dimmick, K. A., Fleming, J. S. & Charles, T. C. (2009).
Null mutations in Sinorhizobium meliloti exoS and chvI demonstrate
the importance of this two-component regulatory system for
symbiosis. Mol Microbiol 74, 1223–1237.
Jensen, P. E., Gibson, L. C. D. & Hunter, D. N. (1998). Determinants
of catalytic activity with the use of purified I, D and H subunits of the
magnesium protoporphyrin IX chelatase from Synechocytosis
PCC6803. Biochem J 344, 335–344.
Johnston, A. W. B. & Beringer, J. E. (1975). Identification of
Rhizobium strains in pea root nodules using genetic markers. J Gen
Microbiol 87, 434–350.
Lamb, J. R., Tugendreich, S. & Hieter, P. (1995). Tetratrico peptide
repeat interactions: to TPR or not to TPR? Trends Biochem Sci 20,
257–259.
Li, Y., Wray, R. & Blount, P. (2004). Intragenic suppression of gain-of-
function mutations in the Escherichia coli mechanosensitive channel,
MscL. Mol Microbiol 53, 485–495.
Beringer, J. E. (1974). R factor transfer in Rhizobium leguminosarum.
J Gen Microbiol 84, 188–198.
Liu, Y. & Huang, N. (1998). Efficient amplification of insert end
Cerveny, L., Straskova, A., Dankova, V., Hartlova, A., Ceckova, M.,
Staud, F. & Stulik, J. (2013). Tetratricopeptide repeat motifs in the
sequences from bacterial artificial chromosome clones by thermal
asymmetric interlaced PCR. Plant Mol Biol Rep 16, 175–181.
world of bacterial pathogens: role in virulence mechanisms. Infect
Immun 81, 629–635.
Cheng, H. P. & Walker, G. C. (1998). Succinoglycan is required for
Manzon, R. G., Neuls, T. M. & Manzon, L. A. (2007). Molecular
cloning, tissue distribution, and developmental expression of lamprey
transthyretins. Gen Comp Endocrinol 151, 55–65.
initiation and elongation of infection threads during nodulation of
alfalfa by Rhizobium meliloti. J Bacteriol 180, 5183–5191.
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring
Clementz, T., Zhou, Z. & Raetz, C. R. H. (1997). Function of the
O’Toole, G. A. & Kolter, R. (1998). Initiation of biofilm formation in
Escherichia coli msbB gene, a multicopy suppressor of htrB knockouts,
in the acylation of lipid A. Acylation by MsbB follows laurate
incorporation by HtrB. J Biol Chem 272, 10353–10360.
Pseudomonas fluorescens WCS365 proceeds via multiple, convergent
signalling pathways: a genetic analysis. Mol Microbiol 28, 449–461.
Harbor, NY: Cold Spring Harbor Laboratory.
Colombatti, A., Bonaldo, P. & Doliana, R. (1993). Type A modules:
Ogura, T., Tomoyasu, T., Yuki, T., Morimura, S., Begg, K. J.,
Donachie, W. D., Mori, H., Niki, H. & Hiraga, S. (1991). Structure
interacting domains found in several non-fibrillar collagens and in
other extracellular matrix proteins. Matrix 13, 297–306.
and function of the ftsH gene in Escherichia coli. Res Microbiol 142,
279–282.
Das, A. K., Cohen, P. W. & Barford, D. (1998). The structure of
Ophir, T. & Gutnick, D. L. (1994). A role for exopolysaccharides in
the tetratricopeptide repeats of protein phosphatase 5: implications
for TPR-mediated protein-protein interactions. EMBO J 17, 1192–1199.
the protection of microorganisms from desiccation. Appl Environ
Microbiol 60, 740–745.
Driscoll, B. T. & Finan, T. M. (1993). NAD(+)-dependent malic
Oresnik, I. J., Charles, T. C. & Finan, T. M. (1994). Second site
enzyme of Rhizobium meliloti is required for symbiotic nitrogen
fixation. Mol Microbiol 7, 865–873.
mutations specifically suppress the Fix- phenotype of Rhizobium
meliloti ndvF mutations on alfalfa: identification of a conditional
NdvF-dependent mucoid colony phenotype. Genetics 136, 1233–1243.
Fields, A. T., Navarrete, C. S., Zare, A. Z., Huang, Z., Mostafavi, M.,
Lewis, J. C., Rezaeihaghighi, Y., Brezler, B. J., Ray, S. & other
authors (2012). The conserved polarity factor PodJ1 impacts multiple
Ponting, C. P., Aravind, L., Schultz, J., Bork, P. & Koonin, E. V. (1999).
cell envelope-associated functions in Sinorhizobium meliloti. Mol
Microbiol 84, 892–920.
Eukaryotic signalling domain homologues in archaea and bacteria.
Ancient ancestry and horizontal gene transfer. J Mol Biol 289, 729–
745.
Foreman, D. L., Vanderlinde, E. M., Bay, D. C. & Yost, C. K. (2010).
Quandt, J. & Hynes, M. F. (1993). Versatile suicide vectors which
Characterization of a gene family of outer membrane proteins (ropB)
in Rhizobium leguminosarum bv. viciae VF39SM and the role of the
sensor kinase ChvG in their regulation. J Bacteriol 192, 975–983.
allow direct selection for gene replacement in gram-negative bacteria.
Gene 127, 15–21.
Fujishige, N. A., Kapadia, N. N., De Hoff, P. L. & Hirsch, A. M. (2006).
Reeve, W. G., Tiwari, R. P., Worsley, P. S., Dilworth, M. J., Glenn, A. R.
& Howieson, J. G. (1999). Constructs for insertional mutagenesis,
Investigations of Rhizobium biofilm formation. FEMS Microbiol Ecol
56, 195–206.
transcriptional signal localization and gene regulation studies in root
nodule and other bacteria. Microbiology 145, 1307–1316.
156
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:21:11
Microbiology 161
Protein involved in cell envelope function
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a
Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory, NY:
Cold Spring Harbor.
Silhavy, T. J., Kahne, D. & Walker, S. (2010). The bacterial cell
envelope. Cold Spring Harb Perspect Biol 2, a000414.
Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range
mobilization system for in vivo genetic engineering: transposon
mutagenesis in Gram negative bacteria. Biotechnology 1, 784–791.
transporter required for desiccation tolerance, and biofilm formation
in Rhizobium leguminosarum bv. viciae 3841. FEMS Microbiol Ecol 71,
327–340.
Vanderlinde, E. M., Magnus, S. A., Tambalo, D. D., Koval, S. F. & Yost,
C. K. (2011). Mutation of a broadly conserved operon (RL3499-
RL3502) from Rhizobium leguminosarum biovar viciae causes defects
in cell morphology and envelope integrity. J Bacteriol 193, 2684–2694.
Vincent, J. M. (1970). A Manual for the Practical Study of Root-Nodule
Snider, J. & Houry, W. A. (2006). MoxR AAA+ ATPases: a novel
Bacteria, IBP Handb. 15. Oxford: Blackwell.
family of molecular chaperones? J Struct Biol 156, 200–209.
Wells, D. H. & Long, S. R. (2003). Mutations in rpoBC suppress the
Storz, G. & Hengge-Aronis, R. (2000). Bacterial Stress Responses, pp.
defects of a Sinorhizobium meliloti relA mutant. J Bacteriol 185, 5602–
5610.
1–30. Washington, DC: American Society for Microbiology.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar,
S. (2011). MEGA5: molecular evolutionary genetics analysis using
Whelan, K. F., Sherburne, R. K. & Taylor, D. E. (1997).
Tang, X., Lu, B. F. & Pan, S. Q. (1999). A bifunctional transposon
Characterization of a region of the IncHI2 plasmid R478 which
protects Escherichia coli from toxic effects specified by components of
the tellurite, phage, and colicin resistance cluster. J Bacteriol 179, 63–
71.
mini-Tn5gfp-km which can be used to select for promoter fusions
and report gene expression levels in Agrobacterium tumefaciens. FEMS
Microbiol Lett 179, 37–42.
Whittaker, C. A. & Hynes, R. O. (2002). Distribution and evolution of
von Willebrand/integrin A domains: widely dispersed domains with
roles in cell adhesion and elsewhere. Mol Biol Cell 13, 3369–3387.
CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Wong, K. S. & Houry, W. A. (2012). Novel structural and functional
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
Vanderlinde, E. M. & Yost, C. K. (2012). Mutation of the sensor kinase
insights into the MoxR family of AAA+ ATPases. J Struct Biol 179,
211–221.
Wong, K. S., Snider, J. D., Graham, C., Greenblatt, J. F., Emili, A.,
Babu, M. & Houry, W. A. (2014). The MoxR ATPase RavA and its
chvG in Rhizobium leguminosarum negatively impacts cellular
metabolism, outer membrane stability, and symbiosis. J Bacteriol
194, 768–777.
cofactor ViaA interact with the NADH:ubiquinone oxidoreductase I
in Escherichia coli. PLoS ONE 9, e85529.
Vanderlinde, E. M., Muszynski, A., Harrison, J. J., Koval, S. F.,
Foreman, D. L., Ceri, H., Kannenberg, E. L., Carlson, R. W. & Yost,
C. K. (2009). Rhizobium leguminosarum biovar viciae 3841, deficient
leguminosarum methyl-accepting chemotaxis protein genes are downregulated in the pea nodule. Arch Microbiol 182, 505–513.
in 27-hydroxyoctacosanoate-modified lipopolysaccharide, is impaired
in desiccation tolerance, biofilm formation and motility. Microbiology
155, 3055–3069.
Vanderlinde, E. M., Harrison, J. J., Muszyński, A., Carlson, R. W.,
Turner, R. J. & Yost, C. K. (2010). Identification of a novel ABC
http://mic.sgmjournals.org
Yost, C. K., Del Bel, K. L., Quandt, J. & Hynes, M. F. (2004). Rhizobium
Young, J. P., Crossman, L. C., Johnston, A. W., Thomson, N. R.,
Ghazoui, Z. F., Hull, K. H., Wexler, M., Curson, A. R., Todd, J. D. &
other authors (2006). The genome of Rhizobium leguminosarum has
recognizable core and accessory components. Genome Biol 7, R34.
Edited by: I. Oresnik
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Thu, 03 Aug 2017 18:21:11
157