APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2007, p. 6748–6756
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 21
Characterization of Regulatory Pathways in Xylella fastidiosa:
Genes and Phenotypes Controlled by algU?†
Xiang Yang Shi,1C. Korsi Dumenyo,2Rufina Hernandez-Martinez,1‡
Hamid Azad,1and Donald A. Cooksey1*
Department of Plant Pathology and Microbiology, University of California, Riverside, California 92521,1and Institute of
Agricultural and Environmental Research, Tennessee State University, Nashville, Tennessee 372092
Received 2 June 2007/Accepted 29 August 2007
Many virulence genes in plant bacterial pathogens are coordinately regulated by “global” regulatory genes.
Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help
to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with
roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas
aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause
of Pierce’s disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define
its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had
reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray
analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type.
Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other
physiological processes such as virulence, competition, and survival.
Xylella fastidiosa is a xylem-limited, gram-negative, plant-
pathogenic bacterium that is transmitted by insects such as the
glassy-winged sharpshooter, Homalodisca coagulata (48).
Strains of X. fastidiosa cause many diseases, including citrus
variegated chlorosis, leaf scorch of live oak, pear leaf scorch,
and Pierce’s disease (PD) of grape (10, 46), which threatens
the grapevine industry in the United States. The characteristic
symptoms of PD are scorched leaves, matchstick (petioles
attached to the cane after the scorched leaf blades have
abscised), and green islands (patches of green tissue sur-
rounded by brown tissue on infected canes). Eventually, the
fruit desiccates, vine cordons die back, and diseased grapevines
can die 2 to 3 years after infection (47). Genetic resistance to
X. fastidiosa is not available in most commercial wine grape
varieties, although several relatives of grape, such as Vitis tili-
ifolia, can harbor high populations of X. fastidiosa without
typical symptoms of PD (16, 31). Currently, the control of
insect vectors is the main management strategy for PD. Suc-
cessful biocontrol of insect vectors depends on the insects’
ecology and field environmental conditions. The identification
of genetic factors that enable X. fastidiosa to express PD symp-
toms could lead to new disease management strategies, includ-
ing new targets for disruption of the disease process.
Although the mechanisms of X. fastidiosa pathogenicity are
not completely understood, the major symptoms of most of the
diseases caused by this pathogen are similar to water stress,
probably resulting from blockage of the xylem transport system
(37). Previous studies have shown that X. fastidiosa is embed-
ded in an extracellular translucent extracellular polysaccharide
(EPS)-matrix biofilm within xylem vessels (44). These two ob-
servations suggest that X. fastidiosa cells can form bacterial
aggregates (biofilm-like colonies) containing EPSs that oc-
clude the xylem vessels, resulting in blockage of water trans-
port and causing PD symptoms.
Sigma factors control virulence and pathogenicity factors in
various bacterial pathogens in response to different environ-
mental conditions (13, 52). algU encodes an alternate sigma
factor, AlgU, that is highly conserved in gram-negative bacteria
(17, 30) and confers tolerance to osmotic, oxidative, and heat
stresses. Its role in the regulation of the biosynthesis of the
EPS alginate has been extensively studied in the human patho-
gen Pseudomonas aeruginosa and the plant pathogen P. syrin-
gae (17, 30). Alginate may play a role in biofilm-related phe-
nomena, including contribution to adhesion and antibiotic
resistance in P. aeruginosa (17). Alginate is also involved in
colonization by and dissemination of the plant pathogen P.
syringae in planta (60). Although a homolog of algU is present
in the genome of X. fastidiosa (53), its role in X. fastidiosa is
unknown. In this study, we analyzed the effect of an insertional
mutation in the algU gene of X. fastidiosa, performed whole-
genome microarray analysis of gene expression in the mutant,
and identified genes whose expression is controlled by algU.
MATERIALS AND METHODS
Bacterial strains and growth conditions. All of the bacterial strains and plas-
mids used in this work are listed in Table 1. For growth profile, aggregation,
adhesion, colony morphology determination, and biofilm formation, bacterial
strains were cultured on PD3 medium (12) supplemented with 0.8% Gelrite
instead of agar. After 7 days at 28°C, cells were harvested with a scraper (Fisher
Scientific, CA), washed and resuspended in 1 ml of PD3 broth, and adjusted to
an optical density at 600 nm (OD600) of 0.10. Bacterial cells used for pathoge-
* Corresponding author. Mailing address: Department of Plant Pa-
thology and Microbiology, University of California, 900 University
Avenue, Riverside, CA 92521. Phone: (951) 827-3516. Fax: (951) 827-
4294. E-mail: email@example.com.
‡ Present address: Department of Microbiology, Center for Scien-
tific Research and Higher Education of Ensenada (CICESE), Km 107
Ctra. Tijuana-Ensenada, 22860 Ensenada, Baja California, Me ´xico.
† Supplemental material for this article may be found at http://aem
?Published ahead of print on 7 September 2007.
nicity tests were cultured for 5 days at 28°C on PW (27) Gelrite medium,
harvested, and adjusted to the same OD600as above with sterile water. When
required, antibiotics were added as follows: ampicillin, 100 ?g/ml; kanamycin, 10
?g/ml. All strains were stored in PD3 broth with 15% glycerol at ?80°C.
Construction of an algU::nptII mutant. A 0.891-kb region of X. fastidiosa
genomic DNA including the algU (PD1284) open reading frame (ORF) was
amplified with Vent polymerase (New England BioLabs, Ipswich, MA) with
primers algUP1 and algUP2 (see Table S1 in the supplemental material) and
cloned into the SmaI site of pUC129 (Table 1) to make pUC1284. The PCR
fragment in pUC1284 was sequenced to confirm the presence of an intact algU
ORF. pUC1284 DNA was mutagenized with the Tn5 transposon with the
EZ::TN ?KAN-2? insertion kit (Epicentre Biotechnologies, Madison, WI) as
outlined by the manufacturer. A plasmid with a Tn5 insertion within the algU
ORF in pUC1284 was named pUC12841. Tn5 insertions in pUC12841 were
precisely mapped by sequencing with transposon primers Kan-2 FP-1 or Kan-2
RP-1 (see Table S1 in the supplemental material).
X. fastidiosa electrocompetent cells of strain A05 isolated from Temecula (10)
were prepared according to previously published procedures (18). One to two
micrograms of pUC12841 DNA in a volume of 5 ?l was electroporated into the
cells in a 0.1-cm-gap cuvette at 1.8 kV, 200 ?, and a capacitance of 25 ?F in a
GenePulser (Bio-Rad, Hercules, CA) with time constants of about 4 ms. The
electrocompetent cells alone and PD3 broth with no bacterial cells served as
negative controls. Electroporated cells were grown for 24 h in PD3 broth with
shaking and plated on PD3 Gelrite medium supplemented with 10 ?g/ml kana-
mycin as previously described to select for replacement of wild-type algU with
algU::nptII by homologous recombination (20).
Genomic DNA extraction and confirmation of the algU::nptII mutant. Wild-
type X. fastidiosa or the algU::nptII mutant strain was cultured in 50 ml PD3 broth
at 28°C for 7 to 10 days with or without antibiotics. The genomic DNAs were
extracted with a MasterPure DNA purification kit (Epicentre Biotechnologies).
The insertion of the construct into the genome of the algU::nptII mutant was
confirmed by PCR with primers M13For/Rev and algUORF P1/P2, respectively
(see Table S1 in the supplemental material). A. 0.891-kb fragment from the wild
type and a 2.1-kb fragment from the mutant were cut from the gel, cloned into
pGEM-T Easy (Promega, Madison, WI) (Table 1), sequenced, and compared
with X. fastidiosa genomic sequences or Tn5 transposon sequences with Vector
NTI (Invitrogen, CA), respectively. The Tn5 insertion within the algU ORF of
algU::nptII genomic DNA was determined by sequencing with transposon prim-
ers Kan-2 FP-1 or Kan-2 RP-1 (see Table S1 in the supplemental material).
Colony morphology, growth curves, surface attachment, and cell aggregation.
The colony morphologies of the X. fastidiosa wild-type and algU::nptII mutant
strains were analyzed after 10 to 14 days of growth at 28°C by plating 100 ?l of
0.10 OD600cell suspensions on PD3 Gelrite plates. In vitro growth curves were
determined in 3 ml of PD3 broth after 3 to 21 days of growth at 28°C. Because
of the aggregation of the cells in broth, immediately after inoculation and 3, 6, 9,
12, 15, 18, and 21 days later, the cells were dispersed by repeated pipetting or
vortexing. Cell concentration was determined by measuring the OD600. For cell
aggregation analysis, strains were grown in 25 ml of PD3 broth in petri dishes and
incubated at 28°C without shaking for 4 days. After growth, the content of each
petri dish was pipetted into a tube and the cells were dispersed by vortexing or
pipetting and adjusted to an OD600of 0.10. Two hundred microliters of each
culture was then subcultured into eight tubes, each with 25 ml of fresh PD3
broth. Tubes were allowed to stand in the incubator without shaking. Three days
after incubation, the tubes were vortexed and the OD540(ODt) was measured.
The concentration of bacterial cells was also measured by determining the
OD600. These tubes were kept without shaking for 1 h to allow the bacterial cells
to clump and settle. The OD540of supernatants of the tubes (ODs) was again
measured. The relative percentage of cell aggregation was measured by using the
following formula: % aggregation ? (ODt? ODs)/(ODt? 100) (5). This pro-
cedure was repeated for the remaining seven tubes of each culture at 6, 9, 12, 15,
18, 21, and 24 days after the initial incubation.
Biofilm formation. X. fastidiosa wild-type and algU::nptII mutant cells were
cultured in PD3 broth and incubated at 28°C without shaking for 4 to 6 days.
Bacterial cells were collected and adjusted to an OD600of 0.10. One-hundred-
fifty-microliter aliquots of each culture were added to wells of 96-well microtiter
plates. The negative control consisted of PD3 broth without bacteria. Plates were
incubated at 28°C without shaking. At 3, 6, 9, 12, and 15 days after incubation,
biofilm formation on the wall of the wells was determined by a crystal violet
staining method (33). Each treatment had three replications, and the data were
LPS gel analysis. Lipopolysaccharide (LPS) fractions were prepared by a
mini-phenol-water extraction technique as described by Guihabert et al. (19).
Twenty microliters of dissolved LPS in polyacrylamide gel electrophoresis
(PAGE) sample buffer (0.3% Tris base, 0.2% glycerol, 0.05% bromophenol blue)
was loaded and separated by deoxycholic acid-PAGE with 18% acrylamide in the
bilayer stacking gel. Gels were silver stained and stored in water (28).
Tolerance of the algU::nptII mutant to desiccation stress in vitro. The sensi-
tivity of wild-type X. fastidiosa and the algU::nptII mutant to desiccation on filters
was assessed by a modification of the procedure described by Ophir and Gutnick
(43). Seven- to 10-day-old cultures were collected and adjusted to an OD600of
0.10 with sterile distilled water and serially diluted to 1 ?104CFU/ml. One
milliliter of each dilution was vacuum filtered onto Millipore filters (no.
HAWP04700; pore size, 0.25 ?m; diameter, 3.5 cm). The filters were placed in
petri dishes at 25°C for slow drying. At 0, 2, 4, 6, 8, 10, 12, and 14 days, filters were
placed onto PD3 agar plates and incubated at 28°C for 3 weeks. Filters without
dilutions, incubated for the same period of time, served as controls. The number
of colonies on each filter was recorded. Each treatment consisted of five filters
and was repeated three times.
Susceptibility to oxidative stress. Sensitivity to hydrogen peroxide (H2O2) or
sodium hypochlorite (NaOCl) was examined as described by Martin et al. (38).
Millipore filter disks (diameter, 6 mm) were soaked with 10 ?l of H2O2(3 or
12%, vol/vol) or NaOCl (3 or 6%, vol/vol) and placed on PD3 Gelrite plates on
which 100 ?l of 7-day-old cultures of wild-type X. fastidiosa or the algU::nptII
mutant were spread with a glass rod. The diameters of the inhibition zones
surrounding the impregnated disks were measured after 14 to 21 days of incu-
bation at 28°C. Three disks were placed in each treatment, each treatment was
repeated three times, and the results were averaged.
Pathogenicity assays on grapes. Wild-type X. fastidiosa and the algU::nptII
mutant were grown on PW Gelrite medium for 5 days at 28°C, suspended in
sterile deionized water, and adjusted to an OD600of 0.10. Five to 10 20-?l drops
of each suspension were used to inoculate 5 to 10 canes on plants of Vitis vinifera
var. Pinot Noir by a needle inoculation procedure as previously described (24).
Water inoculation served as a negative control. The inoculated vines were kept
on the benches in a greenhouse with 75% humidity. The vines were observed for
symptom development approximately every 2 weeks for 5 months after inocula-
tion. The symptoms were rated on a visual scale of 0 to 5 as described before
(19). Briefly, 0 represented healthy grape vines without scorched leaves (water
control) and 5 represented plants with all leaves with heavy scorching or numer-
ous matchsticks. The final disease index was an average of 10 independent
replications for each X. fastidiosa strain.
TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmidCharacteristicsSource or reference
Escherichia coli DH5?
X. fastidiosa algU::nptII
DH1 F??80dlacZ?M15 ?(lacZYA-argF)U169
Wild-type X. fastidiosa A05
Tn5 insertional mutation in algU homologue of X. fastidiosa A05
N. T. Keen
Apr; cloning vector
Apr; cloning vector
Apr; 0.891-kb fragment including algU ORF cloned into pUC129
AprKmr; Tn5 insertion within algU ORF in pUC1284
VOL. 73, 2007REGULATORY PATHWAYS IN X. FASTIDIOSA6749
Recovery and determination of populations of X. fastidiosa from inoculated
grapes. To recover and confirm the bacteria in inoculated grapes, 12 weeks after
inoculation, petiole tissues (2 to 3 cm) from each vine inoculated with either X.
fastidiosa wild-type or algU::nptII mutant cells were harvested at the inoculated
points, as well as 25 cm and 50 cm above the inoculation points. Tissues were
washed once with deionized water containing Tween 20; surface sterilized for 1
min in 20% commercial bleach, 1 min in 2% sodium hypochlorite, and 1 min in
70% ethanol; and rinsed three times in sterile deionized water. The samples were
ground in 100 ?l of sterile deionized water and cultured on PD3 and PW Gelrite
media with or without kanamycin. After incubation for 21 days at 28°C, the identity
of X. fastidiosa cells on PD3 Gelrite plates was confirmed by PCR with primers
specific for wild-type X. fastidiosa, i.e., algUORFP1/P2 and tapBPD1993P1/P2 (see
Table S1 in the supplemental material) (data not shown).
To determine the bacterial populations 16 weeks after inoculation, 2- to 3-cm
petiole tissues of each vine inoculated with the X. fastidiosa wild-type and mutant
strains were harvested and treated as described above. Tissues were tested by
enzyme-linked immunosorbent assay (ELISA) with a PathoScreenXF kit accord-
ing to the manufacturer’s (Agdia Inc., IN) instructions. The antibodies used in
the Agdia ELISA system are a mixture of polyclonal antibodies raised to whole
cells of three serologically distinct isolates of X. fastidiosa (Agdia Inc.). The
PD3-cultured X. fastidiosa wild-type and algU::nptII mutant cells were resus-
pended in phosphate-buffered saline (PBS; Agdia Inc.) and used to confirm that
the ELISA worked equally well for quantifying the wild-type and mutant popu-
lations. Developed plates were measured at 650 nm with a SpectraMax micro-
plate reader via SoftMaxPro (version 3.1.2; Molecular Devices Corp., CA).
Bacterial populations were calculated via OD650determination in comparison to
the positive control (purified X. fastidiosa cell PBS suspension).
SEM. X. fastidiosa wild-type and mutant cells in grapevine xylem were exam-
ined by scanning electron microscopy (SEM) (57). Petiole samples were col-
lected above the inoculation points from symptomatic grapevines 12 weeks after
inoculation. Preparation and observation of the samples by SEM were carried
out in the University of California Riverside Central Facility for Advanced
Microscopy and Microanalysis. Petioles of five leaves from symptomatic grape-
vines were cross-sectioned with a fine razor blade and immersed for 24 h in a
modified Karnovsky solution. Samples were washed twice with ultrapure water
for 30 min, and sections were transferred to a 1% aqueous solution of osmium
tetroxide and incubated for 24 h at 4°C. Samples were subsequently dehydrated
for 20 min each in an alcohol solution series (20, 30, 40, 50, 60, 70, 80, 90, and
100%), and the solvents were removed by vacuum (critical point dried) (57). The
samples were mounted according to the manufacturer’s instructions and ob-
served with a Philips XL30 ESEM-FEG electron microscope. All images were
recorded at a working distance of 9 mm according to the standard procedure with
an accelerating voltage of 20 kV.
RNA isolation, quantification, and RT-PCR. A modified hot-phenol RNA
preparation procedure was used to extract total RNA from X. fastidiosa wild-type
and mutant strains (7, 32). Bacterial cultures were incubated in 50 ml of PD3
broth at 28°C for 5 days under constant agitation. After the hot-phenol extrac-
tion, RNA was suspended in RNase-free distilled H2O and DNase treated with
the Turbo DNA-free DNase (2 U/?l) (Ambion, TX). To ensure that the RNA
preparation was DNA free, an aliquot of 1 ?l of RNA (500 ng/?l) was then used
to amplify the ORF of algU with algUORFP1/P2 primers (see Table S1 in the
supplemental material). The quality of isolated RNAs was determined by dena-
turing RNA formaldehyde gel electrophoresis (7). The expression of algU was
analyzed by reverse transcription (RT)-PCR with the AccessQuick RT-PCR
system by following the manufacturer’s (Promega) instructions.
Microarray hybridizations and microarray data analysis. The gene expression
profiles of wild-type X. fastidiosa and the algU::nptII mutant were analyzed with
a NimbleGen prokaryotic gene expression array (NimbleGen System Inc., WI).
DNA microarray chips were designed with 24-mer oligonucleotides according to
the available X. fastidiosa genomic sequences. The expression levels of RNAs
were averaged from three technical replications in a single hybridization exper-
iment. The raw data were analyzed with the ArrayStar FirstLight. The expression
levels of 2,188 genes under treatment (algU::nptII) and control (wild type) were
analyzed (21). The hybridization signal intensity obtained from the wild-type or
mutant RNA was normalized according to the total signal strength. The normal-
ized hybridization signals were log plot analyzed for reliability (21) and were
statistically analyzed by Student’s t test (P ? 0.001) for differential expression.
The normalized signal intensity of the mutant was divided by that of the wild type
to calculate the mutant/wild-type (M/W) ratio. M/W ratios obtained from indi-
vidual hybridization experiments were averaged to give the final M/W ratio.
Genes having ?1.5 or ?0.66 final M/W ratios were selected as mutated gene
up-regulated or mutated gene down-regulated, respectively.
Validation of microarray data. To validate the differential expression data
obtained in microarray analysis, RT-PCR and PCR experiments were performed
with specific primers designed to amplify an internal region of the ORF of each
target gene (see Table S1 in the supplemental material). Several up-regulated
and potential virulence-related genes were chosen, and primers were designed
for their ORFs according to the X. fastidiosa Temecula1 genome sequences.
cDNA was amplified from stored DNase-cleaned RNAs with the AccessQuick
RT-PCR system by following the instructions of the manufacturer (Promega).
The amplification conditions used were 45 min at 45°C for RT; 35 cycles of 2 min
at 55°C for initial denaturation, 1 min at 55°C for annealing, and 2 min at 72°C
for extension; and a final extension of 10 min at 72°C. Five microliters of the
reaction mixture was run in agarose gels, and products were stained with
Confirmation of the mutational insertion and physiological
properties of an algU::nptII mutant. The algU::nptII mutant
had the Tn5 transposon inserted in the algU ORF 79 bp down-
stream from the ATG start codon. After streaking five to eight
times on PD3 Gelrite medium with 10 ?g/ml kanamycin, the
mutant still grew well, indicating that the mutant had stable
genetic characteristics. cDNAs amplified by RT-PCR with al-
gUORFP1/P2 (see Table S1 in the supplemental material)
showed that there was no expression of algU within the
algU::nptII mutant cells, while strong expression was detected
in wild-type cells (Fig. 1). The in vitro growth curves of the
wild-type and algU::nptII strains over 21 days were similar
(data not shown). In PD3 broth, the wild-type strain formed
large aggregates whereas the algU::nptII mutant grew in less
aggregated clumps (Fig. 2A). An OD assay was used to quan-
tify the effect of the algU::nptII mutation on cell-to-cell aggre-
gation and showed that the percentage of aggregated cells of
algU::nptII was significantly lower than that of the wild type
Cells of the wild type attached to the surface of the flasks
FIG. 1. RT-PCR of genes differentially expressed between wild-
type (WT) X. fastidiosa and the algU::nptII mutant. rRNAs were de-
tected in the algU::nptII mutant and the wild type in this RT-PCR
condition. The algU, algC, algS, algR, algH, mucD, ompW, and cvaC
(PD0216) RNAs were decreased in the mutant compared to the wild
type, and the PD0521 and PD1295 RNAs were slightly increased in the
6750 SHI ET AL.APPL. ENVIRON. MICROBIOL.
and formed wide rings, while the algU::nptII mutant cells at-
tached to the surface formed lighter rings (data not shown).
This indicated that the mutant had a reduced surface attach-
ment ability, resulting in reduced biofilm formation. The ability
of the algU::nptII mutant to form biofilm was investigated
further by a crystal violet staining method. The wild type
formed more biofilm in PD3 medium than did the mutant (Fig.
3). Deoxycholate-PAGE analysis showed that there was no
significant alteration in the purified LPS profile of the
algU::nptII mutant grown in vitro compared with the LPS pro-
file of the wild type (data not shown).
Tolerance to oxidative and desiccation stresses. When wild-
type X. fastidiosa and the algU::nptII mutant were exposed to
desiccation stress in vitro in petri dishes, the survival rate of the
mutant differed significantly from that of the wild type (Table
2), indicating that the mutant died off faster than the wild type.
No significant differences in tolerance to oxidative stress (sen-
sitivity to hydrogen peroxide or sodium hypochlorite) were
observed between the mutant and wild-type strains (data not
Pathogenicity tests and recovery from infected plants.
Grapevines inoculated with the algU::nptII mutant showed sig-
nificantly less severe disease symptoms 12 to 20 weeks after
inoculation than those inoculated with the wild type (Fig. 4).
Water-inoculated control grapevines did not show any PD
symptoms. All diseased grapevines were positive, and the
asymptomatic water control grapevines were negative for the
presence of X. fastidiosa when the plants were examined by
ELISA. X. fastidiosa wild-type and algU::nptII mutant cells
were also examined by SEM in grapevine xylem vessels 12
weeks after inoculation (data not shown), confirming the suc-
cessful survival of cells of wild-type X. fastidiosa and the
algU::nptII mutant in grapevine xylem vessels.
Bacteria were reisolated from macerated inoculated grape-
vine petioles on PD3 and PW Gelrite media. The bacterial
genotypes were confirmed as wild-type X. fastidiosa or
the algU::nptII mutant by PCR amplification with primers
algUORFP1/P2 and tapBPD1993P1/P2 (see Table S1 in the
supplemental material) (data not shown).
FIG. 2. Cell-to-cell aggregation of wild-type X. fastidiosa and the
algU::nptII mutant. (A) Cell-to-cell aggregations of the algU::nptII
mutant (left) and wild-type X. fastidiosa (right) in PD3 broth in petri
dishes. (B) Quantitative assessment of cell-to-cell aggregation of wild-
type (WT) X. fastidiosa or the algU::nptII mutant by an OD assay as
previously described (5). Three replicates were used in each experi-
ment. For each assay time, different letters indicate significant differ-
ences (Student’s t test, P ? 0.05) between the wild type and the mutant.
FIG. 3. Analysis of biofilm formation of wild-type (WT) X. fastid-
iosa and the algU::nptII mutant. Three replicates were used in each
experiment. For each assay time, different letters indicate significant
differences (Student’s t test, P ? 0.05) between the wild type and the
TABLE 2. Tolerance of wild-type X. fastidiosa and the algU::nptII
mutant to desiccation stress in vitro
Survival rate (%) of X. fastidiosa cells after
drying on filtersafor:
0 days2 days4 days6 days 8 days
aData are averages of three independent replications for each treatment. Five
filters per dilution were used in each treatment. For each assay date, different
symbols indicate a significant difference (Student’s t test, P ? 0.05) between the
wild-type and mutant survival rates. The data for days 10 to 14 were all 0 and are
FIG. 4. PD progression in grapevines inoculated with wild-type X.
fastidiosa and the algU::nptII mutant. Disease severity was based on a
visual disease scale of 0 to 5 and was assessed 4, 8, 12, 16, and 20 weeks
after inoculation (19). The data are an average of 10 independent
replications. For each time point, different letters indicate significant
differences (Student’s t test, P ? 0.05) between the wild type and the
VOL. 73, 2007REGULATORY PATHWAYS IN X. FASTIDIOSA 6751
To gain further understanding of the mechanisms that could
explain why the algU::nptII mutant had reduced virulence, bac-
terial populations and bacterial movement in infected grape-
vines were estimated from ELISAs. The ELISA showed no
cross-reaction with any healthy tissue tested. Preliminary ex-
periments showed that the ELISA used to quantify the X.
fastidiosa populations worked equally well for wild-type and
mutant cultures. Bacterial populations at inoculation points
and at 25 cm and 50 cm above inoculation points were esti-
mated from ELISAs by comparing the OD650with that of the
positive control X. fastidiosa with known concentrations (Table
3). An OD650of the X. fastidiosa positive control (purified X.
fastidiosa cells in a PBS suspension) of 1 represented approx-
imately 1 ?104CFU/ml. The average bacterial populations
were calculated by comparing their OD650values to that of the
positive control and dividing by the average weight of 2- to
3-cm sampled petioles. There were no X. fastidiosa cells de-
tected in the asymptomatic water-inoculated control grape-
vines. The cell population of the algU::nptII mutant was less
than that of the wild type at 25 cm and 50 cm above inoculation
points (Table 3). The actual populations could have been
larger than we reported, since those were calculated on the
basis of X. fastidiosa cultures in PBS rather than plant sap.
Plant sap could lower ELISA detection. However, it is the
relative difference between the wild type and the mutant that is
significant (Table 3). These data suggest that the mutated algU
gene may affect the growth and possibly the movement of X.
fastidiosa inside the xylem, resulting in reduced pathogenicity.
DNA microarray analysis of gene expression in vitro. The
expression levels of 2,188 genes were monitored in the wild-
type and algU::nptII mutant strains. The expression levels of
RNAs were averaged from three technical replications in a
single hybridization experiment. The normalized hybridization
signals formed a linear pattern after the log plot analysis,
indicating that the hybridization signals were stable, repeat-
able, and reliable (data not shown). rRNA of X. fastidiosa was
detected in wild-type X. fastidiosa and the algU::nptII mutant
(Fig. 1), indicating that this RT-PCR condition was reliable.
Differential expression of algU, algC, algS, algR, algH, mucD,
ompW, cvaC (PD0216), PD0521, and PD1295 was validated by
RT-PCR (Fig. 1). Forty-three genes were differentially ex-
pressed in the algU::nptII strain compared to the wild type
(Table 4). One gene (PD1926), predicated to encode a fimbrial
protein, was shown to be negatively regulated by algU in the X.
fastidiosa wild type. The other 42 differentially expressed genes
appear to be positively regulated by algU in the X. fastidiosa
wild type, including the following: genes predicted to function
in macromolecular metabolism, intermediary metabolism, cell
structure, and cellular processes; mobile gene elements; genes
that encode conserved and hypothetical proteins; ORFs with
undefined functions; and pathogenicity, virulence, and adapta-
tion genes, according to the functional groups (Table 4).
An X. fastidiosa algU::nptII mutant did not differ in growth
rate in vitro compared to the wild type, but it had reduced
aggregation and attachment to surfaces, suggesting that algU
may play roles in the synthesis of proteins and other molecules
which are related to attachment. The algU::nptII mutant had
reduced biofilm formation and reduced virulence on grape-
vine, suggesting that algU may play a role in biofilm and viru-
lence. The mechanism of resistance of grapevines to infection
includes the restriction of X. fastidiosa to fewer xylem vessels
(15, 41). Decreased vessel-to-vessel movement of cells inside
the xylem results in a delay of systemic infection and disease
development (19, 25). The populations of the algU::nptII mu-
tant at 25 cm and 50 cm above the inoculation points were
reduced compared to those of the wild type (Table 3) but were
also significantly smaller at the inoculation points. Thus, we
cannot determine from these data specifically whether the
algU::nptII mutant had decreased vessel-to-vessel movement
inside the xylem. In vitro desiccation stress tolerance in plant
pathogens has been correlated with resistance to dehydration
in vivo and an increased ability to form microbial biofilms (22,
51). Wild-type X. fastidiosa tolerated desiccation stress for
about 2 to 3 days, while the algU::nptII mutant did not tolerate
desiccation as well as the wild type. The mechanism of toler-
ation to desiccation related to algU in X. fastidiosa remains to
AlgU is a member of a family of alternative sigma factors, ?E
(rpoE), which are only distantly related to ?70(49, 50). Envi-
ronmental stresses induce the transcription of algU in P. aerugi-
nosa (52), and virulence or persistence factors are controlled
by ?E(AlgU) (13). X. fastidiosa is exposed to a range of
variable stress factors inside the xylem of plants (1), such as
changes in osmolarity, availability of nutrients, and agents gen-
erating reactive oxygen intermediates. Gene expression pro-
files of the algU::nptII mutant of X. fastidiosa compared to
those of the wild type via microarray analysis revealed that
algU regulates various factors that could contribute to survival
under the environmental conditions present in the xylem.
Although several of the P. aeruginosa alginate genes (algA,
algD, algG, algF, algI, and algJ) were not found in the X.
fastidiosa genome (53), other genes involved in alginate bio-
genesis in P. aeruginosa, such as mucD (PD1286), algR
(PD1153), algH(PD1276), algC(PD0120), and algS (PD0347),
are present and had decreased expression in the algU::nptII
mutant of X. fastidiosa. In P. aeruginosa, the algC gene encodes
a bifunctional enzyme that is involved in alginate production
(phosphomannomutase activity) and LPS production (phos-
phoglucomutase activity) (11). Thus, the function of alginate
homolog genes in X. fastidiosa may be involved not in alginate
biosynthesis but rather in the synthesis of LPS or another form
of EPS, either of which could play a role in biofilm formation
and cell attachment. However, the purified LPS profile of the
TABLE 3. Bacterial populations in grapevines 16 weeks after inoculation
Population (106CFU/g of tissue)a
Above inoculated points
25 cm 50 cm
9.087 ? 2.6§
1.267 ? 2.1¶
0.609 ? 0.18§
0.067 ? 0.013¶
0.646 ? 0.28§
0.063 ? 0.031¶
aData are averages of 10 independent replications for each treatment. Each
treatment had five samples. For each assay point, different symbols indicate a
significant difference (Student’s t test, P ? 0.05) between the wild-type and
mutant populations. The data for asymptomatic water control were all 0 and are
6752SHI ET AL.APPL. ENVIRON. MICROBIOL.
TABLE 4. Genes differentially expressed in the X. fastidiosa algU::nptII mutant in vitro, organized by functional groups
Functional group and gened
ORF DescriptionM/W ratioa,b
clpSPD0664ATP-dependent Clp protease adaptor; posttranslational
ATP-dependent Clp protease subunit; posttranslational
ATP-dependent Clp protease subunit; posttranslational
Heat shock protein GrpE
Heat shock protein Hsp70; cochaperones are DnaJ and GrpE
RNA metabolism/ribosomal proteins
50S ribosomal protein L34; unknown function
50S ribosomal protein L23; unknown function
50S ribosomal protein L16; unknown function
50S ribosomal protein L29; unknown function
50S ribosomal protein L14; unknown function
30S ribosomal protein S14; unknown function
30S ribosomal protein S8; unknown function
50S ribosomal protein L6; unknown function
30S ribosomal protein S5; unknown function
50S ribosomal protein L30; unknown function
30S ribosomal protein S13; unknown function
50S ribosomal protein L28; unknown function
50S ribosomal protein L33; unknown function
50S ribosomal protein L31; unknown function
Energy metabolism, carbon/tricarboxylic acid
gltAPD0750 Citrate synthase; energy production and conversion0.496 Lower
Regulatory functions/sigma factors and other
csrA PD0095RsmA homologue; regulates the production of virulence
Regulatory functions/two-component systems
algRPD1153 Two-component system; regulatory protein0.617Lower
algHPD1276Transcriptional regulator 0.652Lower
PD1926 Fimbrial protein, pilus assembly protein2.478 Higher
Membrane components/inner membrane
algCPD0120 Phosphomannomutase 0.3Lower
Membrane components/outer membrane
Outer membrane protein
Outer membrane protein
Transport/protein, peptide secretion
secB PD1065Type II secretion system, preprotein translocase 0.409 Lower
Transport/carbohydrates, organic acids,
algSPD0347 Sugar ABC transporter ATP-binding protein0.41 Lower
bfr PD1672 Bacterioferritin; ferritin-like proteins0.178Lower
Mobile genetic elements
Phage-related functions and prophages
hfqPD0066 Host factor-I protein; ubiquitous RNA-binding protein Hfq0.32Lower
Continued on following page
VOL. 73, 2007 REGULATORY PATHWAYS IN X. FASTIDIOSA 6753
algU::nptII mutant grown in vitro were not significantly altered
compared with that of the LPS from the wild type by the assay
used. The mechanism by which algU regulates the synthesis of
other EPSs or LPS in X. fastidiosa remains to be determined.
Previous studies showed that unique structural components
of bacterial cells, such as the cell wall, outer membrane pro-
teins, or actively secreted proteins, may be associated with
bacterial pathogenicity or suppressing host defenses (4, 42, 45).
In X. fastidiosa, expression of the outer membrane proteins
MopB and OmpW appears to be positively regulated by AlgU.
Since SecB is also positively regulated by AlgU, the secretion
of other proteins by the type II, sec-dependent secretion system
may be affected by AlgU.
A single gene that encodes a predicted fimbrial protein
(PD1926) was shown to be negatively regulated by algU.
PD1926 is located in the gene cluster PD1922 to PD1928 (58),
which includes homologs of PilD (PD1922), PilC (PD1923),
PilA (PD1924), PilB (PD1927), PilR (PD1928), and PilS
(PD1929), which are thought to function in the biogenesis and
twitching motility of type IV pili in P. aeruginosa (26, 39).
Mutations in pilA, pilB, and pilR of X. fastidiosa resulted in a
twitching-minus phenotype (34, 40). It is predicted that
PD1926 is a gene involved in the formation or function of type
IV pili of X. fastidiosa, but its specific contribution is not
known. Since algU appears to negatively regulate PD1926, the
mutation in algU might be predicted to enhance its role in the
twitching phenotype. We did not measure twitching motility
directly, but the algU mutant had reduced populations at dis-
tances away from the inoculation points of grapevines, which is
not consistent with enhanced motility (40). However, the algU
mutant achieved significantly smaller populations in grapevine
overall, even at the inoculation points, so the effects on motility
were not necessarily apparent in our assays. The algU mutant
also had reduced cell-cell aggregation, attachment, and biofilm
formation. If PD1926 is involved in the formation of type IV
pili and has enhanced expression in the algU mutant, then the
observed adherence phenotype of the mutant is consistent with
the recent finding that mutants of X. fastidiosa lacking type IV
pili had enhanced cell-cell aggregation and biofilm formation,
which appears to be primarily the role of the shorter type I pili
(34). The long type IV pili may partially mask the adhesion
functions of shorter type I pili.
Several genes that encode ribosomal protein subunits were
shown to be positively regulated by algU in this study, indicat-
ing that algU may also be involved in regulating the normal
physiological metabolism of X. fastidiosa. Genes involved in
physiological metabolism under stress, such as heat shock pro-
tein genes cplS, clpA, clpB, dnaK, grpE, and hspA; iron storage
and detoxification gene bfr; and the energy-producing citrate
synthase gene gltA, had decreased expression in the algU::nptII
mutant. Plant pathogenic bacteria probably regulate heat
shock proteins and iron acquisition mechanisms to help them
adapt to the harsh environmental conditions present within
hosts (59). Cellular homeostasis of iron is essential for pre-
venting iron toxicity in eukaryotes and most prokaryotes. Bac-
terioferritin is one of three types of ferritin-like proteins in
bacteria (55). Bacterioferritin might be involved indirectly in
the resistance to redox stress in P. aeruginosa (35). bfr
(PD0066) of X. fastidiosa was positively regulated by algU and
is predicted to encode a bacterioferritin that may play a role in
the acquisition of iron or protection against oxidative stress. In
this study, there were no significant differences in sensitivity to
oxidative stress between wild-type X. fastidiosa and the
algU::nptII mutant, so bfr may be more likely to function in iron
acquisition than in oxidative stress in this pathogen.
cvaC (PD0216), which encodes a colicin V precursor pro-
Functional group and gened
ORF DescriptionM/W ratioa,b
Pathogenicity, virulence, and adaptation
Toxin production and detoxification
Colicin V precursor; antibacterial polypeptide toxin
Heat shock protein (Hsp)
Hypothetical/conserved hypothetical proteins
Putative transcriptional regulatory protein
Putative integral membrane protein; involved in cell shape
Putative integral membrane protein; involved in cell shape
ND PD1668 0.413 Lower
ORFs with undefined category
ND PD1667HesB-like protein; unknown function 0.462Lower
aThe hybridization signal intensity (mean of three technical replicates) obtained with the mutant was divided by that obtained with the wild type to obtain the M/W
bThe normalized hybridization signals for those genes between the wild type and mutant are all statistically significantly different as analyzed by Student’s t test (P ?
cGenes having ?1.5 or ?0.66 final M/W ratios were designated as having higher or lower expression in the mutant, respectively.
dGenes were detected on the basis of X. fastidiosa Temecula1 genomic sequences at the NCBI website.
eCurrently annotated as colicin V precursor.
fND, no designation.
6754SHI ET AL.APPL. ENVIRON. MICROBIOL.
tein, was identified as positively regulated by AlgU in X. fas-
tidiosa in this study. The colicin V precursor is an antibacterial
polypeptide toxin that acts against closely related sensitive
bacteria (23, 54). It was reported that the expression of cvaC
was detected in the pathogenic condition but not in the non-
pathogenic condition of X. fastidiosa (14). Previous studies
showed that there are diverse endophytic bacterial populations
inside the xylem of plants (3, 9); thus, it is predicted that
successful colonization of xylem of grapevine by X. fastidiosa
may depend on the ability of X. fastidiosa to compete with
other indigenous microbes for essential nutrients (2). AlgU
may play a role such competition in X. fastidiosa by regulation
RsmA is a homolog of CsrA, which is an RNA-binding
protein of Escherichia coli. Mutation of csrA in E. coli resulted
in enhanced biofilm formation (29). In a previous study, an
rsmA mutant of X. fastidiosa also formed more biofilm com-
pared with the wild type (8), suggesting that RsmA represses
biofilm formation. RsmB is an untranslated RNA molecule
that antagonizes RsmA activity in E. coli (36). rsmB (PD1761)
is present in X. fastidiosa Temecula1 (53); thus, there may be
the RsmA/RsmB posttranscriptional regulation system in X.
fastidiosa. In this study, DNA microarray analysis showed that
the expression of rsmA and another gene, hfq, that encodes an
RNA-binding protein was lower in the algU::nptII mutant than
in the wild type, indicating that rsmA and hfq are positively
regulated by the alternative sigma factor AlgU in X. fastidiosa.
A decline in RsmA expression is expected to exert a positive
effect on the production of biofilm, but the algU::nptII mutant
had a reduced ability to form biofilm.
Hfq, also called host factor I, is an abundant RNA-binding
protein and can be involved in the translational regulation of
target mRNAs by regulating the stability of RNAs (36). In P.
aeruginosa PAO1, Hfq may indirectly affect quorum sensing
(QS) and biofilm formation by regulating the RsmA/RsmY
system (6, 56). Hfq binds to and stabilizes RsmY RNA. The
stabilized RsmY RNA then binds to and inactivates RsmA,
which would release the negative effect of RsmA on the ex-
pression of QS and biofilm formation (56). The absence of Hfq
affects the expression of QS and biofilm formation in the re-
verse way (56).
RsmA (PD1208), RsmB (PD1761), and Hfq (PD0066) are
present in X. fastidiosa Temecula1 (53). In this study, the
expression of rsmA and hfq was lower in the algU::nptII mutant,
while the expression of rsmB was not significantly different.
Hfq may be involved in regulating the RsmA/RsmB system of
X. fastidiosa, as in the RsmA/RsmY system of P. aeruginosa.
Mutation of algU caused lower expression of hfq, which would
result in reduced stabilization of rsmB RNA and a lack of
inactivation of RsmA by Hfq-stabilized rsmB RNA. There
would be a lower level of RsmA in the algU::nptII mutant, but
it could be more active than if Hfq were expressed at normal
levels. This could help to explain the decreased biofilm forma-
tion in the algU::nptII mutant. Thus, it is predicted that the
biofilm formation in X. fastidiosa is regulated by algU through
a complex Hfq/rsmB/rsmA-mediated system.
The X. fastidiosa algU::nptII mutant was affected in the ex-
pression of many physiological metabolism genes, acid resis-
tance genes, and membrane-permeating protein genes, which
may contribute to maintaining normal physiological metabo-
lism and adaptation to the poor nutrient conditions of the
xylem. The specific roles of these genes in xylem colonization
should be investigated through mutagenesis and functional
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ment of Food and Agriculture (CDFA) and the University of Califor-
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