Biofilms and Biocomplexity
These intricate matrices can form almost anywhere, and no single
explanation accounts for how they arise and what purposes they serve
Karin Sauer, Alex. H. Rickard, and David G. Davies
“The number of these animalcules in the scurf
of a man’s teeth are so many that I believe they
exceed the number of men in a kingdom.”
Antoni van Leewenhoek in a 1684 report to the
Royal Society of London, considered the first
scientific report on biofilms.
iofilms are receiving attention as
never before, reflecting our increasing
awareness of their impact on health,
the environment, industrial processes,
and natural and manmade materials.
Problems associated with biofilms cost the
United States billions of dollars every year in
B
Summary
• Biofilms are composed of microorganisms attached to surfaces and encased in a hydrated
polymeric matrix containing polysaccharides,
proteins, and nucleic acids.
• Biofilms function in a manner similar to a tissue,
using a primitive circulatory system to pump
fluids and nutrients through channels in the
matrix by changing the ionic strength of the
extracellular milieu, causing periodic contraction of matrix polymers.
• Life in a biofilm imparts protection to insult
from the outside world, with barriers to the
penetration of antimicrobial agents, oxygen,
and nutrients, along with depressed growth
rates and activated adaptive stress responses.
• Biofilm formation is an example of a bacterial
developmental process, albeit one that is distinct from fruiting body or endospore formation.
• Biofilm formation is a complex process requiring the coordinated action of multiple, regulatory proteins, typically including sensor kinases
and response regulators.
energy losses, equipment damage, product contamination, and the management of infections.
Biofilms are implicated in more than 80% of
chronic inflammatory and infectious diseases
caused by bacteria, including otitis media, endocarditis, gastrointestinal ulcers, infections of the
urinary tract, and pulmonary infections in cystic
fibrosis (CF) patients.
So, what are biofilms? In general, biofilms are
composed of microorganisms attached to surfaces and encased in a hydrated polymeric matrix of their own synthesis. The matrix is composed of polysaccharides, proteins, and nucleic
acids which are collectively termed “extracellular polymeric substances” (EPS). The
EPS matrix enables cells in a biofilm to stick
together and is a key element in the development of complex, three-dimensional, attached communities. Water channels are
dispersed throughout biofilms, allowing the
exchange of nutrients, metabolites, and
waste products.
Where do biofilms form? Virtually anywhere. Sites include inorganic natural and
manmade materials above and below
ground, on minerals and metals, including
medical implant materials, and on organic
surfaces such as plant and body tissues.
Biofilm growth surfaces may act as an energy source, a source of organic carbon, or
simply a support material. One common
feature of biofilm environments is that they
are periodically or continuously suffused
with water.
Biofilms Display Specialized
Functions and Properties
Biofilms function in some respects like a
tissue, possessing a primitive circulatory
Karin Sauer is Assistant Professor,
Alex H. Rickard is
Visiting Assistant
Professor, and
David G. Davies is
Associate Professor
in the Department
of Biological Sciences, Binghamton
University (SUNY at
Binghamton), Binghamton, N.Y.
Volume 2, Number 7, 2007 / Microbe Y 347
system. In fact, some investigators contend that
biofilms actively pump fluids through channels
by changing the ionic strength of the extracellular milieu, causing periodic contraction of matrix polymers. Biofilms can be formed by a single
bacterial species, but in nature biofilms typically
consist of assemblages containing many species
of bacteria as well as fungi, algae, yeasts, protozoa, and other microorganisms. For instance,
dental plaque biofilms typically contain more
than 500 bacterial species.
Biofilms are not merely microbial cells that
become stranded at surfaces. We now know that
bacteria in biofilms differ from their planktonic
counterparts in several significant ways. For instance, bacteria grown in a biofilm can be up to
1,500 times more resistant to antibiotics, biocides, and immune chemicals compared to the
same bacteria grown suspended in liquid culture.
No single explanation accounts for how biofilms arise and what advantage they provide to
the resident microorganisms. For example, the
extracellular polymeric matrix protects biofilm
bacteria by acting as a buffer against changes in
the physical environment. The extracellular matrix can also block at least some antimicrobial
agents from entering the biofilm, with the EPS
acting as an ion exchanger, restricting diffusion
of certain compounds into the biofilm from the
surrounding environment. Slow growth within
a biofilm may also lead to increased resistance to
chemical challenge.
Overall, the protective mechanisms at work in
biofilms appear to be distinct from those that are
responsible for antibiotic resistance in planktonic cells. Thus, poor antimicrobial penetration, oxygen and nutrient limitation, slow
growth, and adaptive stress responses constitute
a multilayered defense by biofilms to challenges
from the outside.
Bacteria within biofilms diversify genetically,
even during short-term growth. Such genetic
changes influence multiple traits of bacteria
growing in biofilms, which can be observed even
after these bacteria are removed from a biofilm
context. For instance, biofilm-derived genetic
variants have been observed to exhibit an increased ability to disseminate, increased adhesiveness, increased antimicrobial resistance, and
accelerated biofilm formation. The presence of
these functionally diverse bacteria increases the
ability of a biofilm to adapt to environmental
348 Y Microbe / Volume 2, Number 7, 2007
stresses. Genetic diversity within biofilm clonal
populations is presumed to provide a safeguard
against changing environmental conditions, according to Pradeep Singh at the University of
Washington in Seattle.
Biofilm Formation Can Be Viewed
as a Developmental Process
Biofilm formation, like fruiting body formation
in Myxococcus xanthus or sporulation in Bacillus subtilis, is yet another example of a bacterial
developmental process (Fig. 1). Like other developmental systems, biofilms form following a
series of discrete and well-regulated steps. While
molecular mechanisms differ from species to
species, the broad stages of biofilm development
appear to be conserved among a wide range of
genera. These stages include attachment to a
substrate, the growth and aggregation of cells
into microcolonies, and the maturation and
maintenance of architecture.
The development of biofilm architecture, particularly the spatial arrangement of colonies
within the matrix and the open areas surrounding the colonies, is fundamental to the function
of these complex communities (Microbe, May
2007, p. 231). In the earliest stage, microcolonies form, often in response to intercellular messenger molecules such as acylated homoserine
lactones (AHLs) that are used to detect a surface
or a high density of cells in the local environment, according to David Davies at Binghamton
University in Binghamton, N.Y. As the biofilm
develops, its biomass and thickness are influenced through cross-species bacterial communication via the signal molecule autoinducer 2,
according to Thomas Wood at Texas A & M
University at College Station, Alexander Rickard at Binghamton University, and Paul Kolenbrander at the National Institutes for Health.
Further, rhamnolipid surfactants apparently play
a role in maintaining water channels by influencing cell-to-cell interactions and the attachment of bacterial cells to surfaces.
The extent to which biofilms form and
whether a biofilm is composed of only a few or
hundreds of layers of cells also depends on environmental conditions. The biofilm developmental cycle comes full circle when biofilms disperse
(Fig. 1), allowing cells to spawn new biofilm
communities elsewhere.
For Sauer, Photography and Gardening Provide Sense and Scents outside the Lab
When Karin Sauer left Germany in
2000 for Montana, she had no idea
what she was getting into. “I knew
there were more trout than people
inhabiting Montana, and, according to some maps I found on the
Internet, there were lots of places
with no two-legged creatures,” she
says. “Quite a scary thought, coming from Germany.” Nevertheless,
she packed her bags and left for a
postdoctoral research position at
the Center for Biofilm Engineering
at Montana State University in
Bozeman. It turned out great.
“Living in Montana was sometimes like being on vacation, with
Yellowstone [National Park], great
ski resorts—although I am a terrible skier—and fantastic scenery
nearby,” she says. “It is also the
perfect place to take pictures, one
of my passions.” She also met her
future husband, David, while attending an ASM-sponsored conference on biofilms in Big Sky,
Mont. He had recently been appointed assistant professor at Binghamton University in upstate
New York. In 2003, she became
an assistant professor in the same
department.
“It’s funny how things turn
out,” Sauer says. “I was supposed
to return to Germany and establish my own lab. I already had lab
space and everything—and then I
met David. As you can see, I never
returned to Germany.”
The focus of her research group
is the development of biofilms, or
surface-attached bacterial communities. Her lab focuses on two
microorganisms, Streptococcus
pneumoniae and Pseudomonas
aeruginosa. Her interest in biofilms stems from a summer course
that she took at the Woods Hole
Marine Biology Laboratory (MBL)
in Woods Hole, Mass. during the
year that she completed her Ph.D.
“What fascinated me most was
the ability of bacteria to utilize
insoluble metals for energy generation, forcing the bacteria to a
lifestyle in association to surfaces,” she says. “And so it began—a fascination for biofilms
and surface-associated growth.”
Sauer, 35, was born in Goettingen, in Lower Saxony, but her
home was in Hessen. “I grew up
seeing the border to East Germany from my parents’ kitchen
window,” she recalls. She has two
older brothers— one, a computer
software engineer, and the other,
an employee of the phone company. She is the only scientist in
her extended family.
Sauer says that studying genetically engineered bacteria in high
school biology hooked her on microbiology. “The bacteria were engineered to express a luciferase
gene, and were used in waste water treatment plants to determine
when the water was clean enough
to be released into the river,” she
recalls. “When the water was
clean enough, the bacteria started
to glow or produce light, which
eliminated the need for extensive
water testing. I was so fascinated
by this that it sparked my interest
in microbiology.”
She studied microbiology and
eventually received her doctorate
from Philipps-University in Marburg. She loved Marburg for its
ancient architecture, including a
castle dating from the 11th Century, its river, and its bars. “I suppose that most of my social skills
and beer-drinking habits were
perfected there—I’m kidding,”
she says. “It was a very quaint
town—I love old buildings and
antiques, so I felt right at home.
The only problem was that down-
town was located on a hill.
But the residents figured
out a solution,
an elevator going from uptown to downtown— kind of
fun.”
While her current research was
shaped through her experience at
MBL in Woods Hole, her experimental approaches go back earlier. “While my degree is in microbiology, the lab I worked at was
more or less a biochemistry lab,”
she says. “We purified proteins
and, in part, used molecular biology techniques for downstream
approaches. I still do this. Can’t
get it out of my system.”
Sauer keeps up her German and
continues to do photography, a
hobby she combines with hiking.
“I love it,” she says. “I am not an
expert, but I love capturing the moment, nature, the effect of light,
laughter, a good time. I love landscape pictures, especially in black
and white with the right light. It is
just a shame that it is not possible to
capture the smell with a camera.”
She took up gardening after
moving to Binghamton. “I never
thought I would like it—I certainly never liked it as a child,”
she says. “But I found it brings
balance to my professional life.
Research is slow in producing results and, even then, the accomplishments are mostly on paper.
Gardening, on the other hand,
comes with very visible and fragrant results. It helps me to get
over frustration—and to keep my
head clear.”
Marlene Cimons
Marlene Cimons is a freelance writer
in Bethesda, Md.
Volume 2, Number 7, 2007 / Microbe Y 349
FIGURE 1
The biofilm developmental process in stages. (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and
(v) dispersion.
Looking at biofilm formation as a developmental process, it is not surprising that biofilm
cells differ phenotypically from their planktonic
counterparts in the genes and proteins that they
express. For instance, between 2.9 and 17.1% of
P. aeruginosa genes are differentially expressed
between planktonic cells and biofilms, depending on the growth phase of planktonic cells used
as a reference point, according to Michael Givskov of the Technical University of Denmark,
Lyngby, and his collaborators. Similar findings
are reported for several other species, including
Escherichia coli, Vibrio cholerae, Streptococcus
pneumoniae, Staphylococcus aureus, and Bacillus subtilis.
Meanwhile, Peter Greenberg and his colleagues at the University of Washington found
that approximately 1% of the P. aeruginosa
genome, or approximately 70 genes, show a
change in regulation during the transition from
350 Y Microbe / Volume 2, Number 7, 2007
exponentially growing planktonic cells to 5-day
old biofilms. Their study was conducted at high
cell densities to ensure that quorum sensing was
activated in both planktonic and biofilm cells.
Using in vivo expression technology (IVET) followed by insertional mutational analysis, Lori
Burrows and her collaborators at the University
of Toronto in Canada identified five genes that
are essential for P. aeruginosa to form biofilms
or that affect fitness.
With proteomic analyses, we find that up to
50% of the proteome in P. aeruginosa is modified during biofilm development. Furthermore,
protein expression in biofilms has been shown in
our laboratories to indicate distinct phases in the
developmental process. For instance, specific
early, middle, and late biofilm proteins are detectable in cultures of P. aeruginosa as the biofilm matures. Suites of proteins, which relate to
virulence, antibiotic resistance, and quorum
sensing, are produced progressively. A similar
protein production pattern occurs in Bacillus
cereus, with distinct patterns of proteins appearing in biofilms at different stages of development, according to Volker Brozel and his collaborators at South Dakota State University in
Brookings.
Search for Common Features of Biofilm
Development Proves Challenging
It is intriguing that while numerous research
groups observe genome- or proteome-wide
expression patterns during biofilm formation,
there is limited consensus linking the sets of
genes identified by different laboratories. This
has led some researchers to question the existence of a biofilm phenotype. However, as suggested by George O’Toole of Dartmouth Medical School in Hanover, N.H., bacteria have
more than one way to make a biofilm. Thus, the
availability of a particular carbon source or
other nutrients or a particular environmental
stimulus may trigger alternative developmental
pathways toward the same end point.
While a comprehensive understanding of the
genetic basis of biofilm formation remains elusive, several protein-encoding genes play key
roles, based on mutations that either impair
biofilm formation or alter biofilm structure. For
instance, several proteins play a role in the transition from a planktonic to an attached mode of
growth, including those involved in swimming
and twitching motility, and chemotaxis. However, strains lacking a functional flagellum or
pilus eventually form biofilms if kept in flowing
fluids for an extended time. In such cases, the
structure of the biofilm differs markedly from
that observed for the wild-type strain.
Chemotaxis plays a stage-specific role in biofilm formation—in particular, for the V. cholerae CheY-3 mutant that is defective in forming
monolayers but not biofilms, according to Paula
Watnick and her collaborators at the Tufts-New
England Medical Center in Boston, Mass. Other
mutations that block biofilm formation at various stages include FleR, which controls production of the flagellum, RpoN, membrane proteins
LapA and LapB, and several other gene products.
Like other pathways, biofilm development is
controlled by a number of different regulators.
In P. aeruginosa these regulators include LasR
and RhlR (which are involved in quorum sensing), RpoS, Crc, and PvrR. The protein kinase
PrkC, which is similar to the eukaryotic sensor
Ser/Thr and to the Tyr kinases from Bacillus
subtilis, also appears to regulate biofilms, according to Simone Seror and coworkers at the
Université Paris-Sud, France. Mutants with
prkC deletions have decreased sporulation efficiency and a reduced capacity to form biofilms.
Furthermore, the gene prpC, which encodes a
PPM phosphatase that is cotranscribed with
prkC, is also required for normal biofilm and
endospore formation.
AlgR is a response regulator protein that is
required for synthesizing alginate, a major matrix component of biofilms that form in the
lungs of CF patients. AlgR also appears to play a
role in controlling biofilm formation. For instance, mutations that prevent phosphorylation
of AlgR decrease biofilm maturation without
affecting alginate. Meanwhile, an algR null mutant shows severe defects in biofilm initiation,
suggesting that AlgR regulates additional functions.
The transcriptional regulator MvaT represses
expression of cup genes, which are involved in
the chaperone-usher fimbrial assembly pathway
in P. aeruginosa, according to Alain Filloux and
his colleagues at the Laboratoire d’Ingénierie
des Systèmes Macromoléculaires (LISM) in
Marseille, France. MvaT mutants exhibit enhanced biofilm formation, indicating that various stages of biofilm formation and maturation
are mediated by extracellular appendages, such
as type IV pili and flagella.
In addition, several multicomponent systems
help to control biofilm formation, including the
three-component regulator system SadARS. Deletion in any of the sadARS genes results in a
failure to progress to biofilm maturation in P.
aeruginosa, according to O’Toole at Dartmouth
Medical School.
Similarly, disrupting GacA, the response regulator of the GacA/GacS two-component regulatory system in P. aeruginosa, leads to a 10-fold
reduction in biofilm formation capacity, according to Douglas Storey at the University of Calgary in Alberta, Canada. The GacA/S system
also regulates secondary metabolism, including
exoenzymes, alginate, and siderophore biosynthesis; the system also exerts posttranscriptional
regulatory control of genes within the Gac regulon. Two additional hybrid sensor kinase/
Volume 2, Number 7, 2007 / Microbe Y 351
response regulators, RetS and LadS, signal
through the GacA/S/RsmZ pathway, exerting
opposite effects.
Thus, inactivating RetS in P. aeruginosa attenuates virulence by prematurely activating
genes involved in biofilm formation and coordinate repression of genes required for initial colonization, according to Steven Lory of Harvard
Medical School in Boston, Mass. Meanwhile,
mutants in ladS remain planktonic under conditions in which the parental strain transitions to
biofilm growth, according to Filloux at LISM in
France. Furthermore, the regulators control the
reciprocal expression of genes for type III secretion, biofilm-promoting polysaccharides, and
the small regulatory RNA rsmZ. Thus, LadS
and RetS may mediate a switch between these
bacterial lifestyles via rsmZ. Furthermore, the
findings suggest that biofilm formation may be
regulated via protein phosphorylation, a common modification used in signal transduction in
microorganisms in response to stimuli such as
changes in nutrient concentrations or iron availability.
Even Greater Complexity
May Underlie Biofilm Formation
As complex as the catalogue of factors involved
in controlling biofilm formation has become, it
is apparently far from complete. For example,
the bacterial intracellular signaling molecule
cyclic-di-GMP is implicated as a global intracellular messenger in bacterial biofilm formation—
modulating, among other functions, the production of exopolysaccharides. Furthermore, cdi-GMP is implicated as a signal controlling
biofilm dispersion, the transitioning between
sessile and motile lifestyles. For instance, high
c-di-GMP concentrations in several bacterial
species stimulate biofilm formation and EPS production (and thus, adhesiveness), but suppress
motility. However, low concentrations inhibit
biofilm formation, repress EPS production, and
stimulate swimming and swarming motilities.
This involvement of c-di-GMP in forming or
dispersing biofilm occurs in several bacterial
species, including Salmonella typhimurium, P.
aeruginosa, P. putida, and Shewanella oneiden-
352 Y Microbe / Volume 2, Number 7, 2007
sis. We recently learned that c-di-GMP levels are
regulated in response to environmental cues mediated by the sensory protein BdlA in a signaling
cascade that links sensing, c-di-GMP levels, and
detachment.
However, c-di-GMP appears not to be universal in signaling biofilm developmental processes
and regulating adhesiveness of cells. For example, in Escherichia coli, the RNA-binding protein CsrA, which helps to regulate central carbon flux, apparently plays a role in forming and
dispersing biofilms, according to Tony Romeo
at Emory University School of Medicine in Atlanta, Ga. The effects of CsrA are mediated
through the collective regulation of glycogen
biosynthesis, carbon metabolism, and of flagellum biosynthesis and motility. Further, CsrA
may play a role in producing an adhesin.
In summary, biofilm formation is a complex
process requiring the coordinated action of multiple regulatory proteins, many of them twocomponent regulatory systems, to complete the
developmental cycle. Bacterial signaling plays a
role throughout this cycle, with quorum sensing
signaling molecules affecting biofilm architecture. Meanwhile, c-di-GMP levels play a part in
regulating the transitioning between sessile and
motile lifestyles, probably in response to environmental signaling.
Environmental sensing and the existence of
multiple pathways for the stage-specific progression of biofilm development may explain some
of the differences that researchers studying biofilm genomics and proteomics observe. Further,
biofilm development appears to be different
from bacterial developmental processes such as
sporulation and fruiting body formation. These
developmental processes rely primarily on
sigma factor regulation, whereas biofilm formation apparently does not. To date, no core regulator for biofilm formation has been identified.
Instead, several two-component regulatory systems are essential for biofilm formation, much
as they are involved in eukaryotic cells and
tissues during crucial developmental processes
such as embryogenesis. This fascinating similarity could mean that regulatory signaling cascades found in eukaryotic cells originated
among bacterial cells living within biofilms.
SUGGESTED READING
Boles, B. R., M. Thoendel, and P. K. Singh. 2004. Self-generated diversity produces “insurance effects” in biofilm
communities. Proc. Natl. Acad. Sci. USA 101:16630 –16635.
Davies, D. G., M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, and E. P. Greenberg. 1998. The involvement of
cell-to-cell signals in the development of a bacterial biofilm. Science 280:295–298.
Furukawa, S., S. L. Kuchma, and G. A. O’Toole. 2006. Keeping their options open: acute versus persistent infections. J.
Bacteriol. 188:1211–1217.
Gonzalez Barrios, A. F., R. Zuo, Y. Hashimoto, L. Yang, W. E. Bentley, and T. K. Wood. 2006. Autoinducer 2 controls
biofilm formation in Escherichia coli through a novel motility quorum-sensing regulator (MqsR, B3022). J Bacteriol.
188:305–316.
Kolenbrander, P. E., R. J. Palmer, Jr., A. H. Rickard, N. S. Jakubovics, N. I. Chalmers, and P. I. Diaz. 2006. Bacterial
interactions and successions during plaque development. Periodontol. 2000. 42: 47–79.
O’Toole, G. A., and P. S. Stewart. 2005. Biofilms strike back. Nature Biotechnol. 23:1378 –1379.
Romling, U., M. Gomelsky, and M. Y. Galperin. 2005. C-di-GMP: the dawning of a novel bacterial signalling system. Mol.
Microbiol. 57:629 – 639.
Sauer, K. 2003. The genomics and proteomics of biofilm formation. Genome Biol. 4:219.0 –219.5.
Southey-Pillig, C. J, D. G. Davies, and K. Sauer. 2005. Characterization of temporal protein production in Pseudomonas
aeruginosa biofilms. J. Bacteriol. 187:8114 – 8126.
Stoodley, P., K. Sauer, D. G. Davies, and J. W. Costerton. 2002. Biofilms as complex differentiated communities. Annu. Rev.
Microbiol. 56:187–210.
Food Microbiology
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Volume 2, Number 7, 2007 / Microbe Y 353