A previously uncharacterized gene, yjfO (bsmA), influences Escherichia coli biofilm formation and stress response.
ABSTRACT Bacteria growing as surface-adherent biofilms are better able to withstand chemical and physical stresses than their unattached, planktonic counterparts. Using transcriptional profiling and quantitative PCR, we observed a previously uncharacterized gene, yjfO to be upregulated during Escherichia coli MG1655 biofilm growth in a chemostat on serine-limited defined medium. A yjfO mutant, developed through targeted-insertion mutagenesis, and a yjfO-complemented strain, were obtained for further characterization. While bacterial surface colonization levels (c.f.u. cm(-2)) were similar in all three strains, the mutant strain exhibited reduced microcolony formation when observed in flow cells, and greatly enhanced flagellar motility on soft (0.3 %) agar. Complementation of yjfO restored microcolony formation and flagellar motility to wild-type levels. Cell surface hydrophobicity and twitching motility were unaffected by the presence or absence of yjfO. In contrast to the parent strain, biofilms from the mutant strain were less able to resist acid and peroxide stresses. yjfO had no significant effect on E. coli biofilm susceptibility to alkali or heat stress. Planktonic cultures from all three strains showed similar responses to these stresses. Regardless of the presence of yjfO, planktonic E. coli withstood alkali stress better than biofilm populations. Complementation of yjfO restored viability following exposure to peroxide stress, but did not restore acid resistance. Based on its influence on biofilm maturation and stress response, and effects on motility, we propose renaming the uncharacterized gene, yjfO, as bsmA (biofilm stress and motility).
- SourceAvailable from: Jintae Lee[show abstract] [hide abstract]
ABSTRACT: Analysis of the temporal development of Escherichia coli K-12 biofilms in complex medium indicates the greatest differential gene expression between biofilm and suspension cells occurred in young biofilms at 4 and 7 h (versus 15 and 24 h). The main classes of genes differentially expressed (biofilm versus biofilm and biofilm versus suspension cells) include 42 related to stress response (e.g. cspABFGI), 66 related to quorum sensing (e.g. ydgG, gadABC, hdeABD), 20 related to motility (e.g. flgBCEFH, fliLMQR, motB), 13 related to fimbriae (e.g. sfmCHM, fimZ, csgC), 24 related to sulfur and tryptophan metabolism (e.g. trpLBA, tnaLA, cysDNCJH), 80 related to transport (e.g. gatABC, agaBC, ycjJ, ydfJ, phoU, phnCJKM), and six related to extracellular matrix (e.g. wcaBDEC). Of the 93 mutants identified and studied, 76 showed altered biofilm formation. Biofilm architecture changed from thin and dense to globular and dispersed to dense and smooth. The quorum-sensing signal AI-2 controls gene expression most clearly in mature biofilms (24 h) when intracellular AI-2 levels are highest. Sulfate transport and metabolism genes (cysAUWDN) and genes with unknown functions (ymgABCZ) were repressed in young (4, 7 h) biofilms, induced in developed biofilms (15 h), and repressed in mature (24 h) biofilms. Genes related to both motility and fimbriae were induced in biofilms at all sampling time points and colanic acid genes were induced in mature biofilms (24 h). Genes related to dihydroxyacetone phosphate synthesis from galactitol and galactosamine (e.g. gatZABCDR, agaBCY) were highly regulated in biofilms. Genes involved in the biosynthesis of indole and sulfide (tnaLA) are repressed in biofilms after 7 h (corroborated by decreasing intracellular indole concentrations in biofilms). Cold-shock protein transcriptional regulators (cspABFGI) appear to be positive biofilm regulators, and deletions in respiratory genes (e.g. hyaACD, hyfCG, appC, narG) increased biofilm formation sevenfold.Environmental Microbiology 03/2007; 9(2):332-46. · 5.76 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.Nature 12/2003; 426(6964):306-10. · 38.60 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: This study was based on the hypothesis that biofilms of the opportunistic pathogen Pseudomonas aeruginosa are successfully adapted to situations of protozoan grazing. We tested P. aeruginosa wild type and strains that were genetically altered, in structural and regulatory features of biofilm development, in response to the common surface-feeding flagellate Rhynchomonas nasuta. Early biofilms of the wild type showed the formation of grazing resistant microcolonies in the presence of the flagellate, whereas biofilms without the predator were undifferentiated. Grazing on biofilms of quorum sensing mutants (lasR and rhlR/lasR) also resulted in the formation of microcolonies, however, in lower numbers and size compared to the wild type. Considerably fewer microcolonies than the wild type were formed by mutant cells lacking type IV pili, whereas no microcolonies were formed by flagella-deficient cells. The alginate-overproducing strain PDO300 developed larger microcolonies in response to grazing. These observations suggest a role of quorum sensing in early biofilms and involvement of flagella, type IV pili, and alginate in microcolony formation in the presence of grazing. More mature biofilms of the wild type exhibited acute toxicity to the flagellate R. nasuta. Rapid growth of the flagellate on rhlR/lasR mutant biofilms indicated a key role of quorum sensing in the upregulation of lethal factors and in grazing protection of late biofilms. Both the formation of microcolonies and the production of toxins are effective mechanisms that may allow P. aeruginosa biofilms to resist protozoan grazing and to persist in the environment.Environmental Microbiology 04/2004; 6(3):218-26. · 5.76 Impact Factor
A previously uncharacterized gene, yjfO (bsmA),
influences Escherichia coli biofilm formation and
Mary M. Weber,13 Christa L. French,14 Mary B. Barnes,2
Deborah A. Siegele3and Robert J. C. McLean1
Robert J. C. McLean
1Department of Biology, Texas State University-San Marcos, 601 University Drive, San Marcos,
TX 78666, USA
2Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433-8915,
3Department of Biology, Texas A&M University, College Station, TX 77843-3258, USA
Received 4 June 2009
Revised 5 October 2009
Accepted 14 October 2009
Bacteria growing as surface-adherent biofilms are better able to withstand chemical and physical
stresses than their unattached, planktonic counterparts. Using transcriptional profiling and
quantitative PCR, we observed a previously uncharacterized gene, yjfO to be upregulated during
Escherichia coli MG1655 biofilm growth in a chemostat on serine-limited defined medium. A yjfO
mutant, developed through targeted-insertion mutagenesis, and a yjfO-complemented strain,
were obtained for further characterization. While bacterial surface colonization levels (c.f.u. cm”2)
were similar in all three strains, the mutant strain exhibited reduced microcolony formation when
observed in flow cells, and greatly enhanced flagellar motility on soft (0.3%) agar.
Complementation of yjfO restored microcolony formation and flagellar motility to wild-type levels.
Cell surface hydrophobicity and twitching motility were unaffected by the presence or absence of
yjfO. In contrast to the parent strain, biofilms from the mutant strain were less able to resist acid
and peroxide stresses. yjfO had no significant effect on E. coli biofilm susceptibility to alkali or heat
stress. Planktonic cultures from all three strains showed similar responses to these stresses.
Regardless of the presence of yjfO, planktonic E. coli withstood alkali stress better than biofilm
populations. Complementation of yjfO restored viability following exposure to peroxide stress, but
did not restore acid resistance. Based on its influence on biofilm maturation and stress response,
and effects on motility, we propose renaming the uncharacterized gene, yjfO, as bsmA (biofilm
stress and motility).
When growing as surface-adherent, biofilm communities,
bacteria are typically quite resistant to a variety of adverse
environmental conditions, including antimicrobial agents,
pH extremes and oxidative stresses (reviewed by Costerton
et al., 1987). Biofilms have been implicated in a number of
problems, including many infections, industrial fouling,
and corrosion (Costerton et al., 1987; McLean et al., 1996).
Several detailed studies have been conducted on biofilms to
ascertain the differences between planktonic and biofilm
bacteria. In Gram-negative bacteria, these studies include
large-scale mutant screens using microtitre (O’Toole &
Kolter, 1998b; Prigent-Combaret et al., 1999) and com-
petition culture assays (Junker et al., 2006), promoter
identification using in vivo expression technology (Finelli
et al., 2003), proteomic analyses (Sauer et al., 2002), and
transcription profiling (Domka et al., 2007; Hancock &
Klemm, 2007; Junker et al., 2007; Ren et al., 2004;
Schembri et al., 2003; Whiteley et al., 2001a). The use of
these techniques has allowed the identification of genes
important for biofilms, such as those associated with cell
(Herzberg et al., 2006; Lee et al., 2007a), osmotic stress
and reduced oxygen (Prigent-Combaret et al., 1999), and
the global regulators rpoS (Adams & McLean, 1999), relA
and spoT (Balzer & McLean, 2002).
Abbreviation: TEM, transmission electron microscopy.
Pathogenesis, Texas A&M Health Sciences Center, College Station,
TX 77840-7896, USA.
4Present address: Applied Biosystems/Ambion, 2130 Woodward St,
Austin, TX 78744-1832, USA.
The gene array data discussed in this paper have been deposited in
Gene Expression Omnibus (GEO) under accession number GSE18362.
Microbiology (2010), 156, 139–147
031468G2010 SGMPrinted in Great Britain 139
Many of the genes expressed at higher levels in biofilms
than in planktonic cells encode proteins of unknown
function. A number of these are now being associated with
various biofilm phenotypes. In Pseudomonas aeruginosa,
ndvB is responsible for the tobramycin resistance of
biofilms (Mah et al., 2003). In Escherichia coli, ariR
(ymgB) is needed for biofilm formation and for full
expression of acid resistance genes in biofilms (Lee et al.,
2007b). The E. coli bhsA (ycfR) gene is needed for
stimulation of biofilm formation by stresses such as
hydrogen peroxide, low pH and heat stress (Zhang et al.,
2007). In the present study, we observed the uncharacter-
ized gene yjfO (b4189) to be upregulated. This same gene
has been found to be upregulated in at least two previous
transcriptional profiling studies (Beloin et al., 2004; Junker
et al., 2007). Here, we show that yjfO mutants are altered in
biofilm structure and cell motility, and in their ability as
biofilms to respond to pH and oxidative stresses.
Bacterial strains and media. The strains and plasmids used in this
study are listed in Table 1. The insertion mutation in strain B4189 was
confirmed upon receipt by PCR. Cultures were maintained on Luria–
Bertani (LB) agar (MG1655), LB supplemented with 50 mg kanamy-
cin ml21(mutant strain), or LB supplemented with 50 mg kanamy-
cin ml21and 100 mg ampicillin ml21(complemented strain). For
long-term preservation, overnight cultures were frozen at 280 uC
using glycerol [final concentration 12.5% (v/v)] as a cryoprotectant.
Prior to experimentation, cultures were revived from frozen stock,
cultured overnight on LB agar, and then transferred to MOPS serine
medium (Neidhardt et al., 1974). This minimal medium, used in
other biofilm studies (Junker et al., 2007; Schembri et al., 2003),
contained serine (1 mg ml21) as the carbon source. Additional amino
acids (Ile, Arg, Gly, His, Leu, Met, Phe, Val and Thr, each at 40 mg
ml21) were also present. All cultures were grown at 37 uC, using
laboratory facilities at Texas State University.
Chemostat culture. The biofilm culture chemostat apparatus has
been previously described (Whiteley et al., 1997). Briefly, it consists of
a chemostat from which the culture can be circulated through a
biofilm culture device (in this case 4 m Tygon laboratory tubing or a
flow cell). For culturing, the chemostat was filled with MOPS serine
medium, as described above (Neidhardt et al., 1974), inoculated with
1 ml of an overnight culture of E. coli and grown at 37 uC as a batch
culture for 24 h, after which continuous culture was initiated with a
peristaltic pump at a dilution rate of 0.025 h21. The culture was
allowed to equilibrate for one full generation (40 h). At this point,
biofilm growth was initiated as a second pump continuously
circulated the chemostat culture through the 4 m length of attached
tubing (100 ml h21) for 96 h. Bacteria attached to the tubing were
considered to be the biofilm culture, whereas unattached cells
represented the planktonic culture.
Cell harvesting. For planktonic cell harvesting, 200 ml culture was
mixed with an equal volume of ice-cold (220 uC) stop solution [5%
(v/v) water-saturated phenol in ethanol] to stop endogenous nuclease
activity, and then placed into nuclease-free 50 ml centrifuge tubes
(Falcon) (Arnold et al., 2001). Cells were harvested by centrifugation
(3200 g, 4 uC for 20 min), frozen (280 uC), and then transported in
liquid nitrogen to Texas A&M University for RNA extraction and
For biofilm cell harvesting, the biofilm-colonized tubing was removed
from the chemostat, drained to remove planktonic cells, and then
filled with ice-cold stop solution. Following this, the tubing was cut
into 2 cm sections, each section was cut in half and placed into a
sterile Petri plate, and biofilm cultures were scraped from the tubing
with a sterile scalpel into 200 ml ice-cold stop solution. The scraped
tubing sections were then placed into 200 ml ice-cold stop solution
and sonicated in a bath sonicator (Sonicor Instrument Corporation)
at 60 Hz for 10 min to further dislodge biofilm cells. Biofilm cells
were then harvested from the stop solution by centrifugation, frozen,
and transported to Texas A&M University for RNA extraction and
analysis as described above.
Biofilm growth measurements. In order to measure biofilm and
planktonic cell growth, duplicate chemostat cultures were established
for each strain as described above. Following growth, the culture was
analysed by dilution plating to enumerate the planktonic populations.
For biofilm growth, the biofilm-colonized tubing was removed and
rinsed with sterile medium, to remove loosely attached cells. The
tubing was cut into five 2 cm sections, placed into sterile PBS,
sonicated at 60 Hz for 5 min, and vortexed for 2 min to dislodge
biofilm cells (McLean et al., 1999). Cell numbers were then measured
by dilution plating onto LB agar.
RNA processing and gene array analysis. RNA extraction and
purification were conducted at Texas A&M University using a
previously described hot phenol extraction protocol (Arnold et al.,
2001) and enzymic purification. [33P]CTP (New England Biochemical)
was used to label cDNA during reverse transcription (Arnold et al.,
2001). Gene array analysis was conducted using the Sigma Genosys
macroarray protocol previously described by Arnold et al. (2001). For
differential expression patterns to be considered significant, a
minimum twofold change in expression level (compared with the
background) during both the original and replicate run was needed.
Real-time (quantitative) PCR. Transcriptional profiling results for
yjfO were validated using real-time PCR (Ju et al., 2007). RNA was
purified as described above, and reverse-transcribed to generate
cDNA using a high-capacity cDNA archive kit (Applied Biosystems)
using the manufacturer’s protocol. The primers used for quantitative
PCR were: TM-B4189-204F (59-ACC GCC AGT AAC GGA CCA T-
39) and TM-B4189-313R (59-CTA ATG CGT CAT CCG GAG AAC-
39), and 59-/5(6)-FAM/CCA TCG TGC TTA CGC TAC CTA TTC
GCT GTA/36-TAMTph/-39 (Integrated DNA Technologies) was used as
Table 1. E. coli strains and plasmids used in this study
StrainCharacteristicsSource and reference
MG1655 yjfO::Tn5(KAN-I-SceI) at position
167 in minus orientation
pGEM-T containing yjfO
M. Cashel, NIH (Hernandez & Cashel, 1995)
F.R. Blattner, University of Wisconsin (Kang et al., 2004)
pMW201 This study
M. M. Weber and others
a probe [5(6)-FAM5596-carboxyfluorescein; 36TAMpH539TAMRA].
No template and no reverse transcriptase controls were used. cDNA
products were measured using an Applied Biosystems Prism 7500 real-
time PCR system. Real-time analysis was conducted in duplicate.
Epifluorescence microscopy. To compare biofilm formation of the
various strains, each strain was grown in a chemostat coupled to a
three-chamber flow cell (Stovall Life Science). The chemostat was
inoculated and equilibrated as described above. For biofilm growth,
the culture was pumped through the flow cell at 100 ml h21. After
96 h biofilm growth, the flow cell was removed from the chemostat
and each chamber was washed with 5 ml sterile water to remove
planktonic cells. Cells were stained with 20 mM Syto 9 (Invitrogen)
for 30 min, and then rinsed with 5 ml sterile water. Biofilms were
viewed using a Nikon Eclipse 80 I microscope. Images were obtained
using a Nikon DXM 1200F digital camera and images were analysed
using Image-Pro Plus (version 5.1) (Mirza et al., 2007). Adobe
Photoshop CS3 (version 10.0.1) was used to convert the images to
greyscale, invert the image (to make bacteria appear dark against a
light background), and optimize contrast. Each flow cell experiment
was carried out in triplicate.
Transmission electron microscopy (TEM). In order to observe
whether the presence or absence of yjfO affected cell surface stability
or flagella production, cells were examined by negative-stain
preparations with TEM. Overnight cultures were grown on agar
plates and then a loop-full of culture was suspended in a water droplet
on Parafilm. The cell suspension was then mixed with 1% (w/v)
uranyl acetate, placed on a Formvar-coated grid, and examined by
TEM (Merchant et al., 2007). TEM films were scanned (2700 dots per
inch) using a photographic high-resolution scanner, and the images
were inverted and optimized for contrast using Adobe Photoshop CS3
as described above.
Twitching and flagellar motility assays. In order to elicit
differences in twitching motility between the strains, the methods
outlined by Semmler et al. (1999) were employed. Each strain was
stabbed into the centre of an LB agar plate containing 1% (w/v) agar
and incubated at 37 uC in an inverted position. To examine flagella-
based motility, cultures were stab-inoculated into the centre of an LB
soft agar (0.3%, w/v, agar) plate (O’Toole & Kolter, 1998a) and the
plates incubated in an upright position at 37 uC. Colony diameter due
to twitching motility and flagella motility was measured after 16 h
Hydrophobicity assay. Strains were tested for their ability to
partition into hexane from an aqueous suspension as described by
Zhang et al. (2007).
Biofilm stress assays. We evaluated the bacterial responses to pH,
hydrogen peroxide and elevated temperature stresses using the
experimental strategy of Zhang et al. (2007). For this purpose, each
strain was again cultured in a chemostat, coupled to laboratory tubing
as described above. In order to evaluate biofilm stress responses, the
biofilm-colonized tubing was removed and cut into 2 cm pieces.
Sensitivity to acidic and alkaline conditions was assessed by placing
five pieces of tubing into pH 2.5 or pH 12 MOPS serine medium,
which was subsequently incubated at 37 uC for 20 min. To determine
viability following exposure to hydrogen peroxide, five pieces of
tubing were incubated for 5 min at 37 uC in 20 mM H2O2. To assess
viability following exposure to heat, five pieces of tubing were placed
into MOPS serine medium and incubated for 10 min at 65 uC.
Following incubation, the tubing was placed into PBS, which was
subsequently sonicated at 60 Hz for 5 min and vortexed for 2 min.
Each sample was serially diluted and plated on LB. A total of three
biological replicates were performed for each stress measurement.
Planktonic stress assays. In order to determine strain viability
following exposure to numerous environmental stressors, the
methods outlined by Zhang et al. (2007) were employed. All strains
were incubated in MOPS serine medium at 37 uC with shaking at
100 r.p.m. to OD6000.3. To assess viability at altered pH, 2 ml culture
was incubated for an additional hour at 37 uC without shaking in
pH 2.5 or pH 12 MOPS serine medium. To determine culture
viability following exposure to hydrogen peroxide, 1 ml culture was
incubated with 20 mM H2O2at 37 uC without shaking for 15 min.
For heat sensitivity, 5 ml of each strain was removed and heated for
20 min at 65 uC. Following each treatment, cultures were serially
diluted and plated on LB, and incubated at 37 uC for 24 h. A
minimum of three biological replicates were performed.
Data analysis. On the basis of dilution plating, the planktonic and
biofilm cell concentrations were calculated as c.f.u. ml21and c.f.u.
cm22, respectively. For statistical analyses, the data were log-
transformed (Whiteley et al., 2001b) and the data analysed by one-
way ANOVA, with a minimum threshold of significance of P,0.05.
Where applicable, all pair-wise comparisons were analysed by the
Holm–Sidak method using SigmaStat v3.0 (Systat Software). Several
measurements gave no detectable survival and in these cases, the c.f.u.
data for that particular sample were assigned a value of 1 c.f.u. (the
log10of an undetectable sample was therefore zero). A minimum of
three biological replicates was performed for each measurement.
SigmaPlot v8.0 (Systat) was used to plot the results.
Complementation of E. coli mutant. The yjfO gene was amplified
fromMG1655 usingthe primers
TTACGCTTTCGT-39), yjfO-R1 (59-CCACTGTCCTGTCACGATG-
39) using AmpliTaq Gold (Applied Biosystems). Purified yjfO gene
products were cloned into the pGEM-T Easy Vector (Promega) to
generate pMW201, where yjfO could be transcribed from the lac
promoter through induction with 0.1 mM IPTG. The resulting
plasmid was subsequently electroporated into the corresponding
mutant strain. Verification of the inserted gene was accomplished
through restriction digestion using EcoRI and plasmid sequencing.
IPTG was present in the MOPS serine medium during chemostat
experiments with the complemented strain.
In order to investigate differences in biofilm and
planktonic culture gene expression, we conducted tran-
scriptional profiling on 96 h biofilms of E. coli. We found
that 43 genes were differentially expressed between biofilm
and planktonic culture. In the literature, the number of
genes that are differentially expressed within biofilms in
comparison with planktonic cultures ranges from a low of
approximately 0.5% of the genome (Whiteley et al., 2001a)
to a high of almost 20% (Hancock & Klemm, 2007; Ren
et al., 2004). The values obtained in the present study (43
genes) (GenBank accession number GSE18362) represent
approximately 1% of the genome (Blattner et al., 1997)
and are certainly consistent with other reports. As noted
elsewhere (Beloin et al., 2004; Ren et al., 2004; Schembri
et al., 2003), many of the differentially regulated genes were
uncharacterized. Several initially uncharacterized genes,
identified in earlier transcriptional profiling investigations,
have since been shown to be important in biofilm
Roles of yjfO (bsmA) in E. coli biofilms
functions (Domka et al., 2007; Zhang et al., 2007). Under
our experimental conditions, expression of yjfO (b4189)
was 3.3±0.3-fold higher in biofilms than in planktonic
culture, asmeasured by
Upregulation of yjfO in biofilms was confirmed by
quantitative PCR [D threshold cycle (Ct) 5.0±0.5]. Two
previous studies have also shown yjfO to be upregulated in
biofilms (Beloin et al., 2004; Junker et al., 2007).
To investigate the role of yjfO in biofilm formation, we
circulated a steady-state E. coli culture through a three-
chambered flow cell for 96 h. The flow cell was removed
after 96 h, stained with 20 mM Syto 9 and viewed using a
Nikon Eclipse 80 I microscope at 6100 magnification.
Clumping and microcolony formation were observed in
the wild-type (MG1655) biofilms (Fig. 1a). This phenotype
was absent in the yjfO mutant (Fig. 1b), but could be
restored upon genetic complementation (Fig. 1c). In order
to measure adherent cell populations, we grew each of the
strains in a chemostat coupled to 4 m Tygon laboratory
tubing for biofilm culture (Whiteley et al., 1997). Following
96 h of biofilm growth, the tubing was removed and
assayed for colonization by dilution plating as described
above. No statistically significant difference was noted in
planktonic or adherent cell populations among any of the
strains (Fig. 2). Based on these observations we concluded
that yjfO expression is important in microcolony forma-
tion and biofilm maturation processes, but not necessary
for planktonic growth or initial surface adhesion (Beloin et
al., 2004; Sauer et al., 2002).
Motility and ultrastructure
As twitching motility has been shown to be important for
microcolony formation (O’Toole & Kolter, 1998a), we
investigated whether the phenotype of the yjfO deletion
could be explained by loss of this characteristic. Although
there were slight differences in colony expansion (Fig. 3a),
these differences were not significant when analysed by
one-way ANOVA (P50.63), indicating that yjfO had no
effect on twitching motility. In contrast, flagella-based
motility was enhanced in the yjfO mutant and the
complemented but not induced strain (P,0.001 compared
with the wild type), but could be restored to wild-type
levels when the yjfO-complemented strains were induced
by IPTG (Fig. 3b). Using TEM, we examined negatively
stained preparations of the various strains for the presence
of flagella and found an indication of enhanced flagella in
some of the yjfO mutants (Fig. 4). In future studies, we will
explore the mechanism(s) for this yjfO-enhanced flagella-
Based on its sequence, the YjfO protein is predicted to be a
lipoprotein (Rudd et al., 1998), and as such has the
potential to be associated with cell membranes. Cell
preparation during negative-stain TEM involves suspend-
ing the bacteria in a heavy metal solution (to provide
electron contrast), and then exposing the stained, unfixed
cells to high vacuum during TEM examination. Other EM
preparation approaches, such as conventional embedding
or freeze substitution (Graham & Beveridge, 1990), employ
chemical fixation and gradual dehydration, processes
which stabilize and protect membranes and other struc-
tures from the high vacuum and electron beam in TEM. As
fixation and gentle dehydration protocols are not used
during routine negative-stain TEM preparation, one would
anticipate that membranes lacking a key structural
component would be much more prone to vacuum-
Fig. 1. Representative epifluorescence microscopy images of
96 h biofilms from chemostat cultures grown in serine-limited
MOPS medium: MG1655 (a), yjfO (b4189) (b) and complemented
yjfO (c). The magnification is the same in all figures.
M. M. Weber and others
induced damage. However, we did not observe any major
differences in the cell envelope membranes, regardless of
the presence (Fig. 4a, c) or absence of yjfO (Fig. 4b).
However, we cannot rule out subtle, membrane-associated
changes due to yjfO solely on the basis of negative-stain
Cell surface hydrophobicity
In order to assess the effect of yjfO on cell surface
hydrophobicity, aqueous suspensions of the E. coli strains
were mixed with an equal volume of hexane, and the
fraction of cells remaining in the aqueous layer was
measured as described elsewhere (Zhang et al., 2007).
Following this procedure, the percentage of cells (±SE)
remaining in the aqueous phase was: wild-type 86.5 (0.5),
yjfO 81.5 (0.5), complemented yjfO without IPTG 82.5
(0.5), and complemented and IPTG-induced yjfO 92 (3).
Based on these results, yjfO appears to have had a limited
effect on cell surface hydrophobicity in these experiments.
However, recent work by Q. Ma and T. K. Wood
(unpublished results, personal communication) has shown
that yjfO overexpression in another E. coli strain results in
greatly increased hydrophobicity. As a result, we cannot
completely rule out a contribution of yjfO to cell surface
Planktonic and biofilm stress assays
In order to determine the viability of each strain following
exposure to commonly encountered environmental stres-
ses, chemostat-grown biofilm populations were exposed to
acid, base, hydrogen peroxide (oxidative stress) or heat
stress. As shown in Fig. 2, no statistically significant
differences in overall cell numbers were noted between the
strains in the absence of stress, thus allowing for
standardization and efficient determination of biofilm cell
viability following exposure to each stressor.
Exposure of 96 h biofilms to pH 2.5 MOPS serine medium
(acid stress) resulted in a decrease in overall viability for all
strains assayed. In the planktonic populations (Fig. 5a), the
acid-treated wild-type cells were reduced in number in
comparison with the untreated control; however, the
difference was only marginally significant (P50.099). The
other two populations (yjfO mutant and complemented
strain) were significantly reduced in comparison with the
untreated control, with the yjfO strain showing the biggest
reduction and the complemented strain showing a
sensitivity intermediate between those of the wild-type
and the yjfO mutant. However, the acid-treated planktonic
strains did not differ significantly with respect to each
other. In contrast to the planktonic results, the viability
patterns in the biofilm cultures following acid stress were
quite different (Fig. 6a), with all populations differing
significantly from each other. In comparison with the acid-
treated wild-type biofilms, yjfO mutant viability was
reduced approximately 200-fold. The viability of the
Fig. 2. Planktonic (scatter plot) and biofilm growth (bar graph) of
MG1655, yjfO (b4189) (yjfO”) and complemented yjfO (Comp
yjfO) after 96 h chemostat growth. Values in all figures are
expressed as log10(c.f.u. ml”1) for planktonic cultures and
log10(c.f.u. cm”2) for biofilm cultures (±SEM). No significant
differences were seen among the planktonic populations or biofilm
populations of the three strains.
Fig. 3. Twitching (a) and flagella-based (b) motility in MG1655
(wt), yjfO mutant (yjfO) and yjfO-complemented strains in the
absence (yjfO-C) and presence (yjfO-C-IPTG) of IPTG induction.
Values with the same letter, in all figures, are not significantly
Roles of yjfO (bsmA) in E. coli biofilms
complemented strain was reduced even further (approxi-
mately sixfold in comparison with the mutant). The failure
of pMW201 to complement the acid-sensitive phenotype
of the mutant suggests that the yjfO insertion mutation has
polar effects on expression of the adjacent downstream
gene yjfN. Given that the complemented strain survives less
well than the mutant, it is also possible that high levels of
YjfO increase sensitivity to low pH.
Exposure of 96 h biofilms to pH 12 MOPS serine medium
(base stress) resulted in decreased viability of E. coli
biofilms (Fig. 6b). Here, all three strains (wild-type, mutant
and complemented strain) showed a statistically similar
reduction in viability. In contrast, there was no significant
stress-induced change in viability in planktonic cultures
(Fig. 5b). The increased susceptibility of biofilm popula-
tions to an environmental stress (in this case, alkaline pH),
in comparison with planktonic populations, is highly
unusual in that the converse is generally the case
(Costerton et al., 1987; Davey & O’Toole, 2000). We will
investigate this issue in future studies.
Exposure of planktonic and biofilm populations to
oxidative stress (20 mM H2O2) resulted in a statistically
significant decrease in viability only in the yjfO mutant
biofilm, which was complemented by the reintroduction of
the yjfO gene (Fig. 6c). In planktonic cultures, there was a
modest, and statistically insignificant, reduction in the
viability of the yjfO mutant (Fig. 5c). We interpret these
results as demonstrating that yjfO is involved in the
protection of biofilms against oxidative stress.
In contrast to the above-mentioned stresses, we observed
no significant differences in planktonic (Fig. 5d) or biofilm
(Fig. 6d) culture viability following exposure to heat
E. coli is routinely found in soil, water and intestinal
mucus, all of which impose unique stresses on the
organism in both biofilm and planktonic modes of growth
(Fabich et al., 2008). In intestinal mucus, E. coli encounters
Fig. 4. TEM micrographs of negative-stain preparations of E. coli MG1655 (a), yjfO (b), and yjfO-complemented and IPTG-
induced strains (c). Flagella (F), outer (OM) and inner (IM) membranes are indicated. Bars: 0.5 mm (a, b), 1.0 mm (c).
M. M. Weber and others
144 Microbiology 156
acetate and other volatile fatty acids, produced as metabolic
by-products of the resident flora (Arnold et al., 2001).
These organic acids are capable of traversing the membrane
into the bacterial cell, thus inducing acid stress (Arnold
et al., 2001). Typically, these weak acids decrease bacterial
viability, yet strains of E. coli, including MG1655 (Arnold
et al., 2001), are capable of combating them. Acid
resistance is one factor that allows E. coli to efficiently
colonize and inhabit the intestinal tract. In this same
largely anaerobic environment, E. coli would be exposed to
transient concentrations of reactive oxygen species from
mucosal innate defences (McLean et al., 1988). In contrast,
E. coli does not experience alkaline conditions (above
pH 8) in its normal environment, and so the low resistance
to alkaline stress is anticipated. The surprising observation
in the current study was the increased resistance of
planktonic populations (Fig. 5b) to alkali stress when
compared with biofilm populations (Fig. 6b). Also, heat
susceptibility was unaffected by biofilm (Fig. 6d) or
planktonic growth (Fig. 5d). Although biofilm growth is
normally associated with stress resistance (Costerton et al.,
1987; Davey & O’Toole, 2000), our current study certainly
shows exceptions to this phenomenon.
In this study, we focused on yjfO, a member of the yhcN
family. When first described (Rudd et al., 1998), the yhcN
family consisted of nine parahomologous, uncharacterized
genes (yjfO, yahO, ybiJ, ybiM, ycfR, ydgH, yhcN, yjfN and
yjfY) of unknown function. Members of this family are
predicted to have evolved from a common ancestor, based
on the presence of a signal peptide and a shared motif in
their N and C termini (Rudd et al., 1998). One of these
genes, ycfR, has been shown to be upregulated in biofilm
cultures and to be associated with biofilm stress responses
(Zhang et al., 2007). On that basis, Zhang et al. (2007) have
proposed that this gene be renamed to bhsA for influencing
biofilms through hydrophobicity and stress response. In
the present study, yjfO does contribute to the biofilm stress
response but unlike ycfR does not appear to contribute to
cell surface hydrophobicity.
Cell aggregation and microcolony formation of surface-
adherent bacteria via twitching motility and other
Fig. 5. Survival of E. coli planktonic cells following exposure to acid (a), base (b), oxidative stress (c) and heat stress (d). Strains
in this figure are designated untreated wild-type control (wt control), treated MG1655 (wt), yjfO (b4189) (yjfO”), and
complemented and induced yjfO (Comp yjfO). The value for the untreated control corresponds to the average of the wild-type
planktonic cells without exposure to any stressor and is included for viability comparisons.
Roles of yjfO (bsmA) in E. coli biofilms
processes are hallmarks of early biofilm maturation
(O’Toole & Kolter, 1998a; Sauer et al., 2002). In the current
study (Fig. 1), we noted that wild-type E. coli formed
microcolonies within flow cells. This feature was absent in
the yjfO mutant biofilm, although it was restored upon
genetic complementation. However, adherent cell concen-
trations were similar (Fig. 2). Aside from twitching and
flagella motility (O’Toole & Kolter, 1998a), other character-
istics involved in cell aggregation include cell–cell adhesion,
cell signalling (Domka et al., 2006) and hydrophobic
interactions (McEldowney & Fletcher, 1986). The most
striking feature of yjfO deletion was the loss of microcolony
formation (Fig. 1) and greatly enhanced flagella motility
(Fig. 3). Other investigators have shown the importance of
microcolonies, water channels and other biofilm structures
in the resistance of the component organisms to various
stresses (Matz et al., 2004; Pamp & Tolker-Nielsen, 2007).
Although flagella motility has been shown to be important
in biofilm structure development (Wood et al., 2006), our
present study suggests that unchecked flagella motility
disrupts microcolonies, a feature that certainly provides a
plausible explanation for the greatly reduced biofilm stress
response. Due to its contribution to biofilm stress response
as well as its contribution to flagella motility, we propose
renaming yjfO as bsmA (biofilm stress and motility).
This work was funded by grants from the National Institutes of Health
(1R15 AI050638) to R.J.C.M. and (RO1 GM55154) to D.A.S., and
from the Texas Higher Education Coordinating Board Advanced
Research Program (003615-0037-2007) to R.J.C.M. We thank Kerry
Fuson, Qun Ma and Patricia Zenker for assistance, and Jean Marc
Ghigo, Anthony Hay, Karl Klose, Ron Walter, Marvin Whiteley and
Tom Wood for helpful discussions and advice. R.J.C.M. would like to
dedicate this paper to the memory of Blanche A. V. McLean.
Adams, J. L. & McLean, R. J. C. (1999). The impact of rpoS deletion on
Escherichia coli biofilms. Appl Environ Microbiol 65, 4285–4287.
Arnold, C. N., McElhanon, J., Lee, A., Leonhart, R. & Siegele, D. A.
(2001). Global analysis of Escherichia coli gene expression during the
acetate-induced acid tolerance response. J Bacteriol 183, 2178–2186.
Balzer, G. J. & McLean, R. J. C. (2002). The stringent response genes
relA and spoT are important for Escherichia coli biofilms under slow-
growth conditions. Can J Microbiol 48, 675–680.
Beloin, C., Valle, J., Latour-Lambert, P., Faure, P., Kzreminski, M.,
Balestrino, D., Haagensen, J. A., Molin, S., Prensier, G. & other
authors (2004). Global impact of mature biofilm lifestyle on
Escherichia coli K-12 gene expression. Mol Microbiol 51, 659–674.
Blattner, F. R., Plunkett, G., III, Bloch, C. A., Perna, N. T., Burland, V.,
Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C. K. & other
Fig. 6. Survival of E. coli biofilm cells following exposure to acid stress (a), base stress (b), oxidative stress (c) and heat stress
(d). For strain designations, see legend to Fig. 5. The value for the untreated control corresponds to the average of the wild-type
biofilm cells without exposure to any stressor and is included for viability comparisons.
M. M. Weber and others
authors (1997). The complete genome sequence of Escherichia coli K-
12. Science 277, 1453–1474.
Costerton, J. W., Cheng, K.-J., Geesey, G. G., Ladd, T. I., Nickel, J. C.,
Dasgupta, M. & Marrie, T. J. (1987). Bacterial biofilms in nature and
disease. Annu Rev Microbiol 41, 435–464.
Davey, M. E. & O’Toole, G. A. (2000). Microbial biofilms: from
ecology to molecular genetics. Microbiol Mol Biol Rev 64, 847–867.
Domka, J., Lee, J. & Wood, T. K. (2006). YliH (BssR) and YceP (BssS)
regulate Escherichia coli K-12 biofilm formation by influencing cell
signaling. Appl Environ Microbiol 72, 2449–2459.
Domka, J., Lee, J., Bansal, T. & Wood, T. K. (2007). Temporal gene-
expression in EscherichiacoliK-12 biofilms. EnvironMicrobiol9, 332–346.
Fabich, A. J., Jones, S. A., Chowdhury, F. Z., Cernosek, A., Anderson, A.,
Smalley, D., McHargue, J. W., Hightower, G. A., Smith, J. T. & other
authors (2008). Comparison of carbon nutrition for pathogenic and
commensal Escherichia coli strains in the mouse intestine. Infect Immun
Finelli, A., Gallant, C. V., Jarvi, K. & Burrows, L. L. (2003). Use of in-
biofilm expression technology to identifygenes involved in Pseudomonas
aeruginosa biofilm development. J Bacteriol 185, 2700–2710.
Graham, L. L. & Beveridge, T. J. (1990). Evaluation of freeze-
substitution and conventional embedding protocols for routine
electron microscopic processing of eubacteria. J Bacteriol 172, 2141–
Hancock, V. & Klemm, P. (2007). Global gene expression profiling of
asymptomatic bacteriuria Escherichia coli during biofilm growth in
human urine. Infect Immun 75, 966–976.
Hernandez, V. J. & Cashel, M. (1995). Changes in conserved region 3
of Escherichia coli s70mediate ppGpp-dependent functions in vivo.
J Mol Biol 252, 536–549.
Herzberg, M., Kaye, I. K., Peti, W. & Wood, T. K. (2006). YdgG (TqsA)
controls biofilm formation in Escherichia coli K-12 through
autoinducer 2 transport. J Bacteriol 188, 587–598.
Ju, Z., Wells, M. C., Heater, S. J. & Walter, R. B. (2007). Multiple tissue
gene expression analyses in Japanese medaka (Oryzias latipes) exposed
to hypoxia. Comp Biochem Physiol C Toxicol Pharmacol 145, 134–144.
Junker, L. M., Peters, J. E. & Hay, A. G. (2006). Global analysis of
candidate genes important for fitness in a competitive biofilm using
DNA-array-based transposon mapping. Microbiology 152, 2233–2245.
Junker, L. M., Toba, F. A. & Hay, A. G. (2007). Transcription in
Escherichia coli PHL628 biofilms. FEMS Microbiol Lett 268, 237–243.
Kang, Y., Durfee, T., Glasner, J. D., Qiu, Y., Frisch, D., Winterberg, K. M.
& Blattner, F. R. (2004). Systematic mutagenesis of the Escherichia coli
genome. J Bacteriol 186, 4921–4930.
Lee, J., Jayaraman, A. & Wood, T. K. (2007a). Indole is an inter-
species biofilm signal mediated by SdiA. BMC Microbiol 7, 42.
Lee, J., Page, R., Garcı ´a-Contreras, R., Palermino, J. M., Zhang, X. S.,
Doshi, O., Wood, T. K. & Peti, W. (2007b). Structure and function of
the Escherichia coli protein YmgB: a protein critical for biofilm
formation and acid-resistance. J Mol Biol 373, 11–26.
Mah, T. F., Pitts, B., Pellock, B., Walker, G. C., Stewart, P. S. &
O’Toole, G. A. (2003). A genetic basis for Pseudomonas aeruginosa
biofilm antibiotic resistance. Nature 426, 306–310.
Matz, C., Bergfeld, T., Rice, S. A. & Kjelleberg, S. (2004).
Microcolonies, quorum sensing and cytotoxicity determine the
survival of Pseudomonas aeruginosa biofilms exposed to protozoan
grazing. Environ Microbiol 6, 218–226.
McEldowney, S. & Fletcher, M. (1986). Variability of the influence of
physicochemical factors affecting bacterial adhesion to polystyrene
substrata. Appl Environ Microbiol 52, 460–465.
McLean, R. J. C., Nickel, J. C., Cheng, K.-J. & Costerton, J. W. (1988).
The ecology and pathogenicity of urease-producing bacteria in the
urinary tract. Crit Rev Microbiol 16, 37–79.
McLean, R. J. C., Fortin, D. & Brown, D. A. (1996). Microbial metal
binding mechanisms and their relation to nuclear waste disposal. Can
J Microbiol 42, 392–400.
McLean, R. J. C., Whiteley, M., Hoskins, B. C., Majors, P. D. & Sharma,
M. M. (1999). Laboratory techniques for studying biofilm growth,
physiology, and gene expression in flowing systems and porous
media. Methods Enzymol 310, 248–264.
Merchant, M. M., Welsh, A. K. & McLean, R. J. C. (2007). Rheinheimera
texasensis sp. nov., a halointolerant freshwater oligotroph. Int J Syst
Evol Microbiol 57, 2376–2380.
Mirza, B. S., Welsh, A. & Hahn, D. (2007). Saprophytic growth of inocu-
lated Frankia sp. in soil microcosms. FEMS Microbiol Ecol 62, 280–289.
Neidhardt, F. C., Bloch, P. L. & Smith, D. F. (1974). Culture medium
for Enterobacteria. J Bacteriol 119, 736–747.
O’Toole, G. A. & Kolter, R. (1998a). Flagellar and twitching motility
are necessary for Pseudomonas aeruginosa biofilm development. Mol
Microbiol 30, 295–304.
O’Toole, G. A. & Kolter, R. (1998b). Initiation of biofilm formation in
Pseudomonas fluorescens WCS365 proceeds via multiple, convergent
signaling pathways: a genetic analysis. Mol Microbiol 28, 449–461.
Pamp, S. J. & Tolker-Nielsen, T. (2007). Multiple roles of biosur-
factants in structural biofilm development by Pseudomonas aerugi-
nosa. J Bacteriol 189, 2531–2539.
Prigent-Combaret, C., Vidal, O., Dorel, C. & Lejeune, P. (1999).
Abiotic surface sensing and biofilm-dependent regulation of gene
expression in Escherichia coli. J Bacteriol 181, 5993–6002.
Ren, D., Bedzyk, L. A., Thomas, S. M., Ye, R. W. & Wood, T. K. (2004).
Gene expression in Escherichia coli biofilms. Appl Microbiol Biotechnol
Rudd, K. E., Humphrey-Smith, I., Wasinger, V. C. & Bairoch, A.
(1998). Low molecular weight proteins: a challenge for post-genomic
research. Electrophoresis 19, 536–544.
Sauer,K., Camper,A.K.,Ehrlich,G.D.,Costerton, J.W.& Davies, D.G.
(2002). Pseudomonas aeruginosa displays multiple phenotypes during
development as a biofilm. J Bacteriol 184, 1140–1154.
Schembri, M. A., Kjærgaard, K. & Klemm, P. (2003). Global gene
expression in Escherichia coli biofilms. Mol Microbiol 48, 253–267.
Semmler, A. B. T., Whitchurch, C. B. & Mattick, J. S. (1999). A
reexamination of twitching motility in Pseudomonas aeruginosa.
Microbiology 145, 2863–2873.
Whiteley, M., Brown, E. & McLean, R. J. C. (1997). An inexpensive
chemostat apparatus for the study of microbial biofilms. J Microbiol
Methods 30, 125–132.
Whiteley, M., Bangera, M. G., Bumgarner, R. E., Parsek, M. R., Teitzel,
G. M., Lory, S. & Greenberg, E. P. (2001a). Gene expression in
Pseudomonas aeruginosa biofilms. Nature 413, 860–864.
Whiteley, M., Ott, J. R., Weaver, E. A. & McLean, R. J. C. (2001b).
Effects of community composition and growth rate on aquifer biofilm
bacteria and their susceptibility to betadine disinfection. Environ
Microbiol 3, 43–52.
Wood, T. K., Gonzalez Barrios, A. F., Herzberg, M. & Lee, J. (2006).
Motility influences biofilm architecture in Escherichia coli. Appl
Microbiol Biotechnol 72, 361–367.
Zhang, X. S., Garcia-Contreras, R. & Wood, T. K. (2007). YcfR (BhsA)
influences Escherichia coli biofilm formation through stress response
and surface hydrophobicity. J Bacteriol 189, 3051–3062.
Edited by: V. Sperandio
Roles of yjfO (bsmA) in E. coli biofilms