Mol Biotechnol (2013) 55:288–297

DOI 10.1007/s12033-013-9700-6

REVIEWS

Influence of Small RNAs on Biofilm Formation Process in Bacteria

Mohammad Ali Ghaz-Jahanian • Fatemeh Khodaparastan •

Aydin Berenjian • Hoda Jafarizadeh-Malmiri

Published online: 6 September 2013

Ó Springer Science+Business Media New York 2013

Abstract Small non-coding RNAs (sRNAs) play a sig- Introduction

nificant role in regulation of bacterial physiological
behaviors. After sensing any environmental cue such as A biofilm is defined as a combination of certain microor-
fluctuation of nutrient concentration, temperature, pH, and ganisms (about 15 % volume) from a single species or a
osmolarity, these sRNAs interfere to transmit these signals community of multiple species and extracellular products
to target regulators and genes. sRNAs have key role in (about 85 %) which adheres to a solid support and forms a
biofilm formation process by base pairing with target thick layer with an external structure [1–3]. Floating bac-
mRNAs or interaction with modulating proteins to both teria, known as planktonic microorganisms, are exist in an
positive and negative regulation mechanisms. There are aqueous environment in suspended form and are pre-
various regulatory systems to characterize the initiation and requisite for a biofilm formation [1]. Small RNAs are
formation of special bacterial biofilms that are mostly important players in biofilm formation [3]. Total RNome of
described as two component systems based on sRNAs a cell consist of two classes of RNAs: messenger RNAs
functions. In this study, regulatory pathways that are (mRNAs) and various types of nonprotein-coding RNAs
important for biofilm formation and genetic responses to (npcRNAs). mRNAs are translated into protein and
environmental stimuli in mature biofilms were evaluated. npcRNAs are involved in cell regulatory functions
Some of the regulatory systems that produce common including chromosome modification, transcription and
types of biofilms such as curli, PGA, cellulose and poly- translation, splicing, developmental timing, cell differen-
saccharides such as alginate, colonic acid, Psl and their tiation, proliferation, apoptosis, and organ development.
involved sRNAs functions were also discussed. The term small RNAs (sRNAs), is often used for short-
bacterial npcRNAs [4]. Based on the regulatory mechanism,
Keywords Small non-coding RNA (sRNA)  the sRNAs can be divided into different groups including
Bacterial biofilm  c-diGMP  Modulating protein  protein activity-modulating sRNAs, cis-encoded base
Regulatory system  Gene expression pairing sRNAs, trans-encoded base pairing sRNAs, and the
recently discovered CRISPR sRNAs (Clustered Regularly
Interspaced Short Palindromic Repeats) [5]. sRNAs have
several important regulatory functions and often act as
environmental signals transmitters when stressful changes
M. A. Ghaz-Jahanian  F. Khodaparastan  occur in cell surrounding. These RNAs help modulate
H. Jafarizadeh-Malmiri (&)
changes in cellular metabolism such as cell density, iron
Department of Chemical Engineering, Sahand University
of Technology, PO Box 51335-1996, Tabriz, Iran starvation, temperature, oxidative stress, or phosphor-sugar
e-mail: [email protected] stress, through down-regulate gene expression, which in
turn optimize the utilization of available nutrients and
A. Berenjian
improve the probability for survival [6]. In bacteria, sRNAs
School of Chemical and Biomolecular Engineering,
The University of Sydney, Sydney, Australia have been shown to activate genes by a variety of direct or
e-mail: [email protected] indirect mechanisms [7].

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sRNAs have impetrative role throughout the bacterial Cis and Trans-Encoded Base Pairing sRNA
biofilm-formation process. In fact, sRNAs initially act
through signaling molecules mainly 30 –50 -cyclic diguanylic Base pairing sRNAs with their target mRNAs are catego-
acid (c-di-GMP) to rapid translate micro-environmental rized as cis or trans based on their location within the
changes into intracellular communication network. This bacterial genome relative to their targets. sRNAs tran-
process governs the bacteria to secrete biofilm matrix by scribed from the DNA strand directly opposite to their
sequestering or activating target RNAs and proteins. The mRNA targets are designated cis-encoded sRNAs and
crucial roles of sRNAs are expression of regulating genes share extensive complementarity to their targets. In con-
to synthesis special bacterial biofilms [1, 2]. The function trast, trans-encoded sRNAs are located elsewhere on the
of sRNAs to regulate biofilms occurs by binding to their genome that function in trans as diffusible molecules, and
target mRNAs or regulatory proteins [5]. Virtually, all share limited complementarily in their base pairing inter-
types of bacteria can form biofilms and this may be the actions (see Fig. 1) [13].
preferred mode of bacterial existence in nature. Therefore, Most of the cis-encoded antisense sRNAs expressed
the objective of this review was to study sRNAs functions from bacteriophage, plasmids, and transposons function to
in bacteria and their roles in several regulatory systems maintain the appropriate copy number of the mobile ele-
which lead to biofilm formation. ment [10].
Theoretically, base pairing between a trans-encoded
sRNA and its target could promote transcription termina-
tion or anti-termination as has been found for some cis-
sRNAs Regulatory Function in Bacteria
encoded sRNAs, or alter mRNA stability through changes
in polyadenylation. In fact, each trans-encoded sRNA
Small non-coding RNA molecules (sRNA) are key regu-
typically base pairs with multiple mRNAs [14].The
lators participating in complex networks, which adapt
capacity for multiple base pairing interactions can be
metabolism in response to rapid environmental changes.
explained by the fact that trans-encoded sRNAs make more
These RNAs also modulate a wide range of physiological
limited contacts with their target mRNAs in discontinuous
responses in bacteria [8]. sRNAs can act to modulate both
patches, rather than extended stretches of perfect comple-
the synthesis of proteins (mRNA transcription, translation,
mentarity, as for cis-encoded antisense sRNAs. The region
and stability) and the activity of specific proteins by
of potential base pairing between trans-encoded sRNAs
binding to them. sRNAs also can simultaneously regulate
and target mRNAs typically encompasses 10–25 nucleo-
multiple mRNA targets and change the pattern of polarity
tides, but in all cases in which it has been examined, only a
within an operon [9]. They achieve these diverse outcomes
core of the nucleotides seem to be critical for regulation. In
via several mechanisms including changes in RNA con-
many cases, the RNA chaperone Hfq is required for trans-
formation, protein binding, base pairing with target RNAs,
encoded sRNA-mediated regulation to facilitate sRNA–
and interactions with DNA [10]. The function of sRNAs in
mRNA interactions due to limited complementarity
the regulation of biofilm formation occurs via two general
between the sRNA and target mRNA [10].
mechanisms: [1] sRNAs acting by base pairing with target
RNAs and [2] protein binding [11].
Hfq Protein as sRNA Chaperones

sRNA Base Pairing with Target mRNA When the mRNA is controlled by a trans-acting sRNA, the
RNA chaperone protein Hfq (host factor for Qb RNA
Most characterized sRNAs-regulate gene expression by replication) is often required to facilitate a stable trans-
base pairing with mRNAs and fall into two broad classes: acting sRNA–mRNA target interaction. Hfq is especially
those having extensive potential for base pairing with their important if the level of complementarity between the
target RNA and those with more limited complementarily
[10]. These sRNAs regulate the translation and stability of
target mRNAs and are functionally analogous to eukaryotic
miRNAs in many respects [12]. Although the two tran-
scripts are encoded in the same region of DNA, they are
transcribed from opposite strands as discrete RNA species.
For few cases in which it has been examined, the initial
interaction between the sRNA and target RNA involves
only limited pairing, although the duplex can, subse-
quently, be extended [10]. Fig. 1 Cis and trans-acting sRNAs on target mRNA [15]

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trans-acting sRNA and the target mRNA is low [15]. of sRNA and proteins can be influenced by several dif-
In vitro experiments suggest that Hfq generally binds an ferent factors and the dissociation can be actively or pas-
A/U-rich single-stranded region often located adjacent to a sively controlled [16].
stem-loop structure, reminiscent of binding sites for the
related Sm (Sec1p/Munc18) and Lsm (Sm-like) proteins in
eukaryotes [16]. There is evidence suggesting Hfq facili- Activation of Biofilm Regulatory System
tates base pairing by increasing annealing rates by stabi-
lizing cognate sRNA–mRNA duplexes or by promoting Environmental Effects on sRNAs
structural remodeling of one of the RNA partners [17–20].
Hfq binds strongly to many small RNAs and is required for A common theme in microbial development involves an
the activity of many of them. Part of this requirement may input of environmental cues that results in an output of an
be a result of the stabilization of at least some of these altered physiological state or behavior [22]. Bacteria have
small RNAs by Hfq (Spot 42; DsrA, RyhB). However, it evolved numerous mechanisms to sense external signals, to
stimulates pairing between small RNAs and their comple- translate them into complex cellular responses and,
mentary targets in vitro, suggesting that it acts as an RNA thereby, to mediate responses to physiological demands
chaperone [9, 21]. [23]. Biofilm formation is regulated in response to envi-
ronmental conditions and cues, although the specifics vary
sRNAs Interaction with Modulating Protein considerably among different species. This complex pro-
cess requires the establishment of cell–cell and cell–surface
Most sRNAs are conjugated with proteins and work attachments which are mediated by proteins, polysaccha-
in vivo, and their interactions (sRNA–protein) can be rides and nucleic acids [24, 25].
divided into two groups. In the first group, the sRNA Biofilm formation can be influenced by the nutritional
provides the specificity and the primary activity to the status and there is evidence that changes in gene expression
RNA–protein partnership. In the second group sRNAs may be required for this process. However, by attachment
interact with proteins which in turn, regulate their activity of bacteria into a surface, additional changes in bacterial
[16]. In order to form normal targets, some of these sRNAs gene expression must occur in order to provide the
act by sequestering proteins, particularly RNA-binding organism for its life on a solid substrate [26]. For example
proteins. However, sRNA binding to proteins also can in E. coli, signals which drive and modulate biofilm life-
make more complex results such as activity modification of style transition are nutrient limitation, low temperature, and
an enzyme (see Fig. 2) [16]. In common cases Protein- cell envelope alterations [27]. Interestingly, Poly-b-1,6-N-
binding sRNAs antagonize and sequester their cognate acetyl-D-glucosamine (PGA) synthesis inversely regulated
regulatory proteins by mimicking the protein-binding with curli expression, which is repressed by high NaCl
sequences found in several mRNAs [11]. concentration and high temperature, which allows the
As clearly observed in Fig. 2, bacterial sRNA binding to bacteria to modify biofilm composition in a changing
proteins can effectively inhibit and/or modify protein environment [27]. The csgA gene encoding the curli
activities. It is proposed that sRNA binding to proteins also adhesion is under control of the EnvZ–OmpR two-com-
can activate or bind two or more proteins. The combination ponent regulatory system important for regulating genes
such as ompC and ompF in response to changes in external
osmolarity. Prigent-Combaret studies has been demon-
strated that biofilm-grown cells in E. coli are exposed to
increased osmolarity, decreased levels of oxygen, and
influenced by cell–cell signaling molecules [28].
Based on environmental conditions, sRNAs have toward
functions in several types of regulatory patterns such as
biofilm development versus motile lifestyle. They can
contribute to the decision whether flagella or biofilm matrix
components are produced and provide an additional regu-
latory layer to the inverse regulation of these components
[26, 27]. Table 1, indicates sRNAs dual functions affected
by environmental cues that regulate the biofilm formation
or motility in E. coli and salmonella [27].
Fig. 2 Types of bacterial sRNA effects on regulatory protein activity It seems that some sRNAs play their regulatory roles
[16] under ‘‘housekeeping’’ or non-stress conditions, while,

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Table 1 Types of sRNA with dual functions: biofilm synthesis versus motility in several environmental conditions (E.Coli and salmonella) [27]
sRNA Targets Transcription Environmental condition Result Type of Description
factors biofilm

ArcZ CsgD, ArcB/A Deplete growth medium Biofilm Curli/ ArcZ is negatively controlled by the ArcB/
RpoS formation cellulose ArcA two component system and indirectly
FlhDC Repressed stimulate biofilm formation in Salmonella
motility
DsrA FlhDC HNS, HdfR Low temperature (25 °C) Repressed Curli/ DsrA indirectly activates flhDC expression
motility cellulose by inhibiting the expression of the flhDC
repressor HdfR
CsgD, Biofilm It directly interferes with the expression of
RpoS formation H-NS since H-NS downregulates rpoS
Mcas FlhDC CRP Nutrient availability is Active PGA McaS activates flagella by direct interaction
suboptim motility with flhDC mRNA
CsgD Cells are in the postexponential
Phase of the growth cycle Biofilm McaS directly inhibits CsgD expression and
temperature = 37 °C formation activates PGA production
Carbon source availability does
not yet limit growth
OmrA/B CsgD, OmpR Low osmolarity Anti OmrA/OmrB indirectly inhibits csgD
RpoS biofilm transcription via the diguanylate cyclase
YdaM
FlhDC High osmolarity Repressed FlhDC, OmrA/B and CsgD are all under
motility complex control of the EnvZ/OmpR two-
component system, which senses changes
in osmolarity
OxyS FlhDC OxyR, Rcs Oxidative stress Repressed OxyS turns down flagella expression by
motility direct interaction and has indirect negative
RpoS Anti effects on rpoS expression and therefore
biofilm possibly on biofilm functions
RprA RpoS, Rcs Cell envelope perturbation Biofilm Colonic The transcription of RprA is activated by the
CsgD formation acid Rcs phosphorelay system, which also
FlhDC Repressed negatively regulates flhDC transcription
motility and colanic acid synthesis

some of them are highly induced upon exposure to stresses diffusion-limited environment, the intracellular signaling
such as high osmolarity (OmrA/B), cell envelope pertur- method has been studied for bacteria [29]. The cell–cell
bation (MicA, RprA) or oxidative stress (OxyS), which in communication systems are called Quorum sensing (QS)
turn, they mainly contribute to controlling their targets and they are known to be involved in a range of important
under these specific conditions. Therefore, biofilm matrix microbial activities including extracellular enzyme bio-
production can be integrated with multiple stress responses synthesis, biosurfactant synthesis, antibiotic biosynthesis,
[26, 28]. For example, in response to severe cell surface biofilm development, EPS (exopolysaccharide) synthesis
stress sensed by the Rcs (regulators of capsule synthesis) and extracellular virulence factors in Gram-negative bac-
system, RprA can induce the general stress response master teria [30]. Two QS processes have been described for
regulator RpoS. At the same time RprA shut downs the bacteria. The autoinducer-1 (AI-1) type is mainly involved
expression of the cell envelope-located and energy-con- in intra-species communication and the autoinducer-2 (AI-2)
suming machinery for the production of biofilm matrix type is associated with inter-species interaction [31].
[27]. These molecules accumulate in the environment in pro-
portion with increasing cell density. When autoinducers
Intracellular Signaling System reach a crucial threshold concentration, the bacteria detect
and respond to them by synchronously altering gene
Intracellular communication between bacteria is generally expression [32].
carried out by bacterial products that are able to diffuse Gram-negative bacteria produce and release AI mole-
away from one cell and enter into another cell. In a cules, which are generally N-actyl homoserine lactone

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(AHL) molecules and their function control the cell-pop- low c-di-GMP concentrations and motility, has been well
ulation density. Bacteria can detect the accumulation of established by over expression of GGDEF and EAL domain
AHL signals [31]. In Gram-positive bacteria, communica- proteins in many bacteria [34]. Small non-coding RNAs
tion is carried out with modified oligopeptides generating have crucial roles in the regulation of bacterial QS. Regu-
the signals and membrane-bound sensor histidine kinases lation by sRNAs rather than by proteins is presumed to be
acting as receptors [31]. beneficial when a rapid response is required. This can be due
In several bacteria the cyclic nucleotide bis-(30 –50 )-cyc- to the short time required to synthesize or degrade sRNAs
lic-di-guanosine monophosphate (c-di-GMP) has emerged as compared with synthesizing and degrading proteins [39].
an important and ubiquitous second messenger generally
plays a role in the regulation of functions associated with the
two different lifestyles adopted by most bacterial species, Regulatory Systems of Various Bacterial Biofilms
that is, the motile planktonic and the sedentary-adhesive
lifestyles. The function of c-di-GMP as a ‘lifestyle-switch Poly-b-1,6-N-Acetyl-D-glucosamine (PGA) Biofilm
regulator’ is employed by different bacterial species in dif-
ferent physiological and ecological contexts [33]. The three PGA is a polysaccharide polymer known to participate in
protein domains involved in the metabolism of c-di-GMP biofilm formation in some gram negative bacteria such as
are named after the most prominent conserved amino acid Staphylococcus aureus, Staphylococcus epidermidis and
motifs found in their active sites, that is, GGDEF, EAL, and E. coli [40].
HD-GYP [33]. The carbon storage regulatory system (Csr) is widely
The regulatory concept of c-di-GMP signaling predicts distributed among eubacteria and has been found to control
the c-di-GMP signaling to be substantially different among a variety of virulence-linked physiological traits [23]. The
cells in a biofilm depending on whether cells move or are global regulator CsrA is a small translational regulatory
embedded within an extracellular matrix. Thus, c-di-GMP protein consisting of 61 amino acids. This protein involves
signaling takes place on a nano-scale determined by the in positive regulation of flagella synthesis, carbon metab-
microenvironment around the cell [34]. In general, func- olism and glycolysis, in negative regulation of biofilm
tions that contribute to biofilm formation, are positively (PGA) formation, glycogen biosynthesis and catabolism,
regulated by c-di-GMP, while motility is downregulated by and in glyconeogenesis in E. coli (see Fig. 4) as well as
c-di-GMP, as can be shown conveniently by overproducing numerous functions in the interactions with animal and
GGDEF domain proteins such as YdaM and YaiC or EAL plant hosts [7, 23].
domain proteins such as MlrA (see Fig. 3) [35, 36]. Despite its global role in bacterial adaptation, only a few
As clearly observed in Fig. 3, in Salmonella typhimu- direct mRNA targets have been identified, including five in
rium high c-di-GMP concentrations stimulated biofilm E. coli. By binding to mRNA leaders and preventing trans-
formation, the production of adhesive surface organelles lation, followed by destabilizing of the transcript, CsrA has
such as cellulose and curli fimbriae, and suppressed been shown to downregulate expression of the glgCAP
motility [37, 38]. These consequences of high c-di-GMP operon, encoding the glycogen synthesis apparatus, the cstA
concentrations on bacterial behavior are most precisely gene, involved in carbon starvation and the pgaABCD operon,
described as locking the cells in a sessile state. In contrast,
low c-di-GMP concentrations inhibit biofilm formation and
the production of adhesive surface organelles and stimulate
swimming and swarming motility. The correlation between
high c-di-GMP concentrations and biofilm formation, and

Fig. 4 Carbon storage regulatory system (Csr) in E. coli include:

CsrA regulatory protein that regulate flagella (motility) synthesis and
represses biofilm formation and CsrB and CsrC sRNAs that activate
Fig. 3 Modulation of c-di-GMP concentration by over expression of biofilm formation (PGA) by sequestering CsrA. Protein CsrD control
GGDEF and EAL domain proteins degradation of CsrB and CsrC [33]

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encoding the biofilm polysaccharide poly-b-1,6-N-acetyl-D- contrast to most of the fimbrial operons, these genes occur
glucosamine (PGA) [23]. Regulation of CsrA activity is throughout several enterobacterial species like Salmonella
mediated in part by the action of the two sRNAs CsrB and spp., E. coli, Shigella spp., Enterobacter spp., Citrobacter
CsrC. CsrB and CsrC RNAs antagonize the activity of CsrA spp., and Raoultella ornithinolytica [26].
by binding and therefore sequestering this protein. Tran- Expression of cellulose and curli fimbriae in S. typhimu-
scription of the Csr sRNAs is controlled by the two-compo- rium is dependent on the transcriptional regulator CsgD. The
nent system BarA–UvrY, thus permitting the integration of csgD gene (curli subunit gene D) encodes for a transcrip-
environmental signals into the Csr signaling network [23, 41]. tional regulator of the LuxR superfamily and has been shown
CsrB and CsrC sRNA levels are controlled by the nutrient to positively control the two extracellular matrix compounds
availability in the growth medium. Degradation of CsrB and (see Fig. 5). CsgD enables the production of curli fimbriae by
CsrC is controlled by CsrD. In nutrient-poor minimal medium transcriptional activation of the csgBAC operon that encodes
CsrB and CsrC sRNA levels were high in contrast to complex the structural genes of curli fimbriae [26, 36].
LB medium, in which csrB and csrC expression was low. This
finding agrees well with the earlier documented observation Cellulose Biofilm
that csrB levels increase when the bacteria enter stationary
phase as the growth medium gets depleted [42]. The search Cellulose production is a widespread phenomenon in
revealed that CsrA is a regulator for several GGDEF/EAL Enterobacteriaceae, including S. typhimurium, S. enterica
proteins, in particular of the two GGDEF proteins YcdT and subsp. Enterica serovar Enteritidis, and commensal and
YdeH. Both proteins produce c-di-GMP in vivo and control pathogenic strains of E. coli, Citrobacter spp. and Enter-
flagella-mediated swimming motility [27, 43]. obacter spp. In these bacteria, cellulose production is
clearly associated with the ability to form a rigid biofilm at
Curli Fimbriae Biofilm the air–liquid interface; however, these characteristics vary
between strains [40].
Curli fimbriae aggregate at the cell surface to form 6- to Cellulose biosynthesis is also positively regulated by
12-nm-diameter structures whose length varies between 0.5 CsgD. Thereby CsgD stimulates the transcription of AdrA
and 1 lm. Curli have been demonstrated to attach to pro- (agfD-dependent regulator), a putative inner membrane
teins of the extracellular matrix such as fibronectin, lami- protein that harbors a cytoplasmic GGDEF domain. AdrA
nin, and plasminogen, thus promoting adhesion of the activates cellulose production on the post-transcriptional
bacteria to different human cells [40]. level either by direct interaction with one or more of the
Genes involved in curli production are clustered in two gene products of the bacterial cellulose synthesis operons
divergently transcribed operons: the csgBAC operon, bcsABZC–bcsEFG, or by production of a cyclic nucleotide
encoding the structural components of curli and csgDEFG which acts as an activator of cellulose biosynthesis [26].
operon, encoding a transcriptional regulator (CsgD) and the Small regulatory RNAs OmrA and OmrB that previously
curli export machinery (CsgE-G) (see Fig. 5) [40]. In known to repress mRNAs of several E. coli porins were
recently discovered to repress csgD [44, 45]. When csgD is not
expressed, the morphotype is a conventional smooth and white
colony which does not produce any extracellular matrix [26].
The CsgD targets were classified into two groups: group
I genes, such as fliE and yhbT, are repressed by CsgD,
while group II genes, including yccT and adrA, are acti-
vated by CsgD. The fliE and fliEFGH operons for flagellum
formation are directly repressed by CsgD, while CsgD
activates the adrA gene, which encodes an enzyme for
synthesis of cyclic di-GMP, a bacterial second messenger,
which in turn inhibits flagellum production and rotation.
Taking these findings together, we propose that the cell
motility for planktonic growth is repressed by CsgD,
thereby promoting the switch to biofilm formation [46].

Psl/Pel Polysaccharide

Fig. 5 Synthesis of two types of biofilms (E. coli): curli and cellulose Extracellular polysaccharides comprise a major component
and their related pathways of the biofilm matrix and play a key role in shaping and

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providing structural support to the biofilm [47]. These Recent studies further characterized the RsmA regulon and
polymers are very diverse and are often involved in the show that the pel and psl genes are among the direct targets
establishment of productive cell-to-cell contacts that con- of RsmA repression [51, 52]. It is now clear that a retS
tribute to the formation of biofilms [48]. mutation results in upregulation of rsmY and rsmZ, which
The structural composition of the Psl (polysaccharide prevents RsmA activity and allows pel and psl gene
synthesis locus) has recently been elucidated and consists expression, while at the same time positively influence the
of a repeating pentasaccharide containing D-mannose, D- genes that involve in the type III secretion system (T3SS)
glucose and L-rhamnose. The assembly of Psl on the cell [50].
surface follows a remarkable helical distribution, and this
organization has been proposed to provide a scaffold for
other matrix components, as well as contributing to cell– Alginate Polysaccharide
cell interaction. Psl has also been proposed to facilitate
attachment to eukaryotic cells [49]. The production of The alginate polysaccharide is a polymer consisting of
exopolysaccharides, such as Pel and Psl, is required for P. mannuronic and guluronic acid residues, and its overpro-
aeruginosa biofilm formation. Acute infections are often duction gives bacterial colonies a characteristic mucoid
characterized by fast growth, motility, cyto-toxicity, and appearance [50]. Alginate production was found to be a
rapid progression of disease. Conversely, chronic infec- hallmark of P. aeruginosa chronic infection in the lungs of
tions are believed to be associated with slower growth, cystic fibrosis (CF) patients to protect bacteria from
biofilm formation, antibiotic resistance, and persistence adverse environments and contribute to biofilm develop-
[50]. ment [38]. However, alginate shapes the structure of bio-
The GacS/GacA has been known for a long time as a films and increases the resistance to antibiotics such as
regulator of virulence and biofilm formation. The response tobramycin [53]. Regulation of alginate biosynthesis has
regulator GacA has only two targets, namely the promoters been extensively investigated and involves a complex
of the small RNAs RsmY and RsmZ, the activity of which regulatory network [50]. In P. aeruginosa biofilms, cells
it enhances. In P. aeruginosa and other bacteria, the role of are embedded in an EPS matrix composed primarily of
these two sRNAs is to titrate the translational repressor alginate. A series of examined the expression of algC, a
RsmA. RsmA prevents translation of a large set of tran- gene required for alginate biosynthesis, using techniques
scripts by binding directly to target mRNAs and blocking that allow the simultaneous monitoring of attachment and
accessibility to the ribosome-binding site (see Fig. 6). algC gene expression within individual cells throughout
biofilm development. These studies showed that algC
expression was activated as early as 15 min after the bac-
terial cell attaches to either a Teflon or glass substratum,
indicating that algC expression was induced by surface
attachment.
Not all cells, however, exhibited induction of algC
expression upon attachment. Cells that do not undergo algC
up-regulation are less able to remain attached to the surface
relative to cells in which expression is activated. Thus,
algC functions are not required for initial cell-to-surface
attachment but may be important to maintain attachment.
Furthermore, increase expression of the algC gene corre-
sponds to an increase in alginate production, indicating that
synthesis of alginate is also initiated after attachment [28,
54, 55]. The conversion to a mucoid phenotype is generally
a result of mutations in mucA, an anti-sigma factor that
Fig. 6 Signaling cascade that converges on the small RNAs RsmY
leads to increased expression of algT, an alternative sigma
and RsmZ, which act by sequestering the translational repressor
RsmA. RsmA reciprocally regulates factors involved in acute factor that controls alginate biosynthesis. Garrett et al. [56]
infection (motility and the T3SS) and chronic infection (Pel, Psl). study indicates that induction of the algT regulon results in
The RetS/LadS/GacS sensors appear to influence the activity of the decreased expression of the fliC gene, which encodes fla-
GacA response regulator and subsequent levels of sRNAs. HptB
gellin, the structural subunit of flagellum. Thus, induction
(histidine phosphotransfer) module is an integral part of the protein,
whereas hybrid sensors require an independent Hpt protein to transmit of algT results in increased alginate synthesis and a coor-
their signal. BfiS/R (biofilm initiation) is sensor kinases [50] dinate decrease in flagellum synthesis [28].

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Table 2 Bacterial biofilms and their regulatory systems

Type of Bacterial species Target Incoded sRNA Functional mechanism Reference
biofilm protein/ gene(s)/
mRNA operon(s)

Cellulose S. typhimurium, E. coli CsgD, bcsABZC– BcsA Synthesis is positively regulated by CsgD [26]
AdrA bcsEFG,
adrA
Curli fimbriae Salmonella spp., E. coli CsgD, csgDEFG– OmrA, OmrA and OmrB repress csgD. In the case [26, 44,
Shigella spp., Enterobacter GlyA csgBAC, OmeB of YdaM mRNA, RprA base pairs 59]
spp., Citrobacter spp., YdaM glyA RprA directly downstream of the translational
Raoultella ornithinolytica start codon for adhesive curli fimbriae
Colanic acid S. typhimurium, E. coli RpoS cpsABCDE RprA The Rcs system inversely regulate flagella [27, 60]
Klebsiella pneumoniae RcsA, expression and late biofilm functions
Erwinia spp. RcsB
Poly-b-1,6-N- E. coli, Vibrio cholerae CsrA, glgCAP, CsrB, The non-coding RNAs CsrB and CsrC [42]
acetyl-D- CsrD pgaABCD CsrC activate biofilm formation process by
glucosamine sequestering CsrA
(PGA)
Psl/Pel Pseudomonas aeruginosa RsmA pel, psl RsmY, sRNAs act by sequestering the repressor [50]
RsmZ, RsmA. RsmA reciprocally regulates
RsmZ factors involved in acute infection
(motility) and chronic infection (Pel, Psl
biofilms)
Alginate Pseudomonas aeruginosa MucA algC, algT GacS, algC gene expression biofilm development. [28]
GacA Induction of the algT regulon resulted in
decreased expression of the fliC gene,
which encodes flagellin

Colanic Acid Conclusion

Colanic acid is a negatively charged polymer of glucose, Biofilm refer to biological deposits on any surface. Biofilms
galactose, fucose, and glucuronic acid that forms a pro- consist of both microbes and their extracellular products,
tective capsule around the bacterial cell under specific usually polysaccharides. There are a number of mechanisms
growth and environmental [30]. Colanic acid synthesis by which numbers of microbial species are able to come into
involves 19 genes located in the same cluster, named wca closer contact with a surface, attach firmly to it, promote
(formerly known as cps) depends on the three-component cell–cell interactions and grow as a complex structure. The
phosphorelay system RcsC/RcsD/RcsB and requires an ability of many bacteria to adhere to surfaces and to form
auxiliary positive transcription regulator RcsA [40] which biofilms has major implications in a variety of industries. The
can numerous stimuli that include cell envelope perturba- formation of a biofilm is determined not only by the nature of
tions, high osmolarity, desiccation, low temperature and the attachment surface, but also by the characteristics of the
growth on surfaces, play a role in biofilm maturation [57]. bacterial cell and by environmental factors. Bacteria adapt to
In part by cooperating with various other transcription environmental changes using extracellular organelles that
factors, the phosphorylated response regulator RcsB not improve their chances of survival. sRNAs plays an important
only activates the genes involved in colanic acid, but also role in the formation of bacterial biofilms. sRNAs act in core
stimulates the expression of the sRNA RprA and many regulatory pathways of biofilm formation, such as those
other target genes. In addition, RcsB inhibits flhDC regulating motility and matrix production. sRNAs contribute
expression and thereby exerts a negative effect on motility. in regulatory systems by sequestering, antagonizing, or
RpoS sigma factor modulates the Rcs system by control- activating regulatory target proteins such as CsrA and CsgD
ling the expression of the small protein YmgB, which in response to environmental stresses, or by directly affecting
stimulates activity of the Rcs system [58]. In general, the the synthesis of proteins promoting or disfavoring the for-
Rcs system seems to inversely regulate flagella expression mation of biofilms. These sRNAs may interact with proteins
and late biofilm functions [27]. Table 2, explains types of such as Hfq to ultimately control the stability and processing
biofilms in various bacteria and their regulatory compo- of target transcripts. They can both positively and negatively
nents to formation, include sRNAs, target mRNAs, pro- modulate transcription, translation, mRNA stability, and
teins, and specific genes or operons in related systems. DNA maintenance or silencing.

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