SUPPLEMENTAL MATERIALS AND METHODS
The bacterium of E. variegatus (BEV) was discovered by Alexander Purcell in laboratory
colonies of the leafhopper in Pont-de-la-Maye, France and has been maintained at UC
Berkeley since 1984 [6]. Although BEV can be grown axenically, efforts to preserve
frozen or freeze-dried cultures of BEV have been unsuccessful. Therefore, infected
leafhoppers have been continually reared on barley and/or a mix of barley, rye and wheat
grasses. Plants are changed every one to two weeks.
Quantifying vertical transmission efficiency of BEV. We prepared ten different cages,
each containing four rye grass plants and twenty adults from a BEV-infected E.
variegatus lab colony. We left the adults on the plants for a 7-day oviposition period
before removing and freezing individuals. The plants were kept without adult insects for
5 days for egg maturation before 5–7 eggs per cage were removed and washed for 5
minutes in 95% ethanol, followed by 5 minutes in 12% bleach in water solution. DNA
was extracted from the eggs as described elsewhere [3] and screened with specific 16S
rRNA PCR primers (BEVF: 5’– GCA CAA GGG AGC TTG CTC CCC –3’, BEVR: 5’–
CAG CAA GGT TAT TAA CCT TAC TG –3’). DNA was amplified with an initial 2
min denaturation at 94°C followed by thirty cycles of 94°C for 30 s, 62°C for 1 min, and
72°C for 30 s, with a final extension of 72°C for 5 min. Amplicons were run on a 1%
agarose gel and visualized with ethidium bromide (EtBr); amplicons were randomly
selected and sequenced to confirm primer specificity and BEV identity.
Estimation of BEV Growth Rate. We isolated BEV from leafhoppers on Difco purple
broth with 1.5–2.0% agar, acidified to pH 6.3 with 0.1 N HCl and incubated at 28°C in
the dark [6]. A triple-cloned isolate (re-plated using a single colony, three times) was
plated on purple broth agar, and 1/3 of the colonies from two Petri dishes were used to
inoculate two 1 mL liquid purple broth replicates. We mixed the bacterial suspensions to
uniform turbidity and added 100 µl to 2 mL of purple broth in 10 culture tubes per
replicate. We wrapped the 20 tubes in aluminum foil to prevent light exposure and the
tubes were incubated at 28ºC and shaken at 180 rpm. Culture tubes from each replicate
were removed serially starting at day zero, plated and colony-forming units per milliliter
(CFU/mL) were counted. Three dilutions of each replicate were plated, 20 µl of a 1/1,
1/100, and 1/10,000 dilution. After 7–14 days, when the colonies grew to a visible size
under the microscope, plates were counted.
Genome size determination. BEV DNA was purified for pulsed-field gel
electrophoresis (PFGE) by scraping the colonies from purple broth agar plates with the
edge of glass coverslip and rinsing the cells off with 1X phosphate buffered saline (PBS)
pH 6.5 into a microfuge tube. The cells were spun down at 6,000 x g for three minutes
and the supernatant removed. The cell pellet was resuspended in PBS, mixed with an
equal volume of 1.5% w/v pulsed-field gel agarose (BioRad) in TE (10 mM Tris pH 8.0,
1mM EDTA pH 8.0) and solidified in plastic plug molds. The plugs, containing intact
cells, were then washed twice for 20 minutes in lysis buffer (50 mM Tris pH 8.0, 50 mM
EDTA pH 8.0, 1% w/v N-laurylsarcosine) and proteinase K (final conc. 0.4 mg/mL) at
50ºC and then twice in TE at room temperature for five minutes.
Plugs were digested with the homing endonuclease I-CeuI (NEB) for three hours.
Plugs and the appropriate size standards were separated on a PFGE CHEF DR-II rig
(BioRad) using 0.5X TBE buffer, 1% w/v pulsed-field agarose gels. To resolve DNA
fragments between 50 and 1,500 kilobases (kb), we used an initial switch time of 24s
ramping linearly to 150s over 25 hrs at 200V. For larger size fragments the gel was run
for 37.6 hrs, ramping from 47–84s at 220V. Using the same run and switch times but
decreasing the voltage to 200V allowed for the resolution of the middle band. All gels
were visualized with EtBr and fragment sizes were estimated manually by measuring
fragment migration of the size standard and plotting it by size on a semi-log plot.
BEV genome library construction. A single BEV clone was cultivated from an
infected E. variegatus adult by triple-cloning. We plated the strain on purple broth agar,
and extracted DNA using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). The
extractions were repeated until a pooled DNA sample of 20 µg was obtained. This
sample was sonicated according published protocols [7] with a Fisher Sonic
Dismembrator Model 50. DNA fragments between 1.5–2.0 kb were size-selected on a 1%
agarose gel and purified with the QiaQuick Gel Extraction Kit (Qiagen, Valencia, CA).
The purified DNA was quantified and blunt-ended with T4 DNA Polymerase (Invitrogen,
Carlsbad, CA). The pUC19 vector was digested with HincII at 37°C for 2 hours and
dephosphorylated with Bacterial Alkaline Phosphatase according to the manufacturer’s
protocol (Invitrogen, Carlsbad, CA). The BEV DNA inserts were ligated into the vector
using T4 DNA ligase and incubated overnight at 16°C, then transformed into competent
E. coli DH5α cells (Invitrogen, Carlsbad, CA). Plasmids with inserts were purified using
a boiling mini prep protocol [7], and the DNA was quantified and Sanger sequenced with
M13F primer on an ABI3730xl (Life Technologies).
Annotation of BEV genome fragments. Individual BEV reads were trimmed of vector
sequence (Cross_match; phrap.org) and low quality sites were changed to ‘N’ (Phred
score <15) [1, 2]. Putative coding sequences (CDS) were identified based on BlastX
searches against the NR and E. coli K12 protein databases. Putative gene functions were
inferred from that of identified orthologs (E value <10-10, >30 amino acids) and classified
using the MultiFun schema [8]. Non-coding RNAs were identified with BlastN
(ribosomal RNAs) and tRNAscan-SE (transfer RNAs) [4]. Reads containing identical
transposase gene fragments were clustered by BlastN searches then manually assembled
into complete insertion sequence elements in MacClade [5].
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