Although exopolysaccharides (EPSs) are a large component of bacterial biofilms, their contribution to biofilm structure and function has been examined for only a few organisms. In each of these cases EPS has been shown to be required for cellular attachment to abiotic surfaces. Here, we undertook a genetic approach to examine the potential role of colanic acid, an EPS of Escherichia coli K-12, in biofilm formation. Strains either proficient or deficient in colanic acid production were grown and allowed to adhere to abiotic surfaces and were then examined both macroscopically and microscopically. Surprisingly, we found that colanic acid production is not required for surface attachment. Rather, colanic acid is critical for the formation of the complex three-dimensional structure and depth of E. coli biofilms.Bacterial biofilms have been described as sessile bacterial communities that live attached to each other and to surfaces (2,3,4,5,10). In natural settings, many bacterial species live predominantly in these communities, with a smaller portion of the bacterial population subsisting as free-swimming (planktonic) organisms (10). In addition to their abundance in natural environments, biofilms also impinge significantly upon our industrialized world. For example, bacterial biofilms can form on catheters and prostheses and thereby cause persistent, antibiotic-resistant infections (5, 12). Biofilms can also clog pipes (1) and contaminate food in industrial settings (21). However, biofilms can also have beneficial functions, for example, by acting as biocontrol agents by preventing fungal infections in certain plants (9). Given the preponderance of biofilm communities in nature as well as their medical and industrial impact, it is clearly important to understand the molecular mechanisms that govern both the formation and dissolution of these sessile communities.The three-dimensional architecture of a number of singlespecies bacterial biofilms has been previously described (5, 8). The two most generalizable features of these biofilms are microcolonies, composed of cells surrounded by large amounts of exopolysaccharide (EPS), and water-filled channels, which have been hypothesized to promote the influx of nutrients and the efflux of waste products.Previous work with Pseudomonas aeruginosa and with Escherichia coli has shown that EPS (alginate and colanic acid, respectively) synthesis is induced upon attachment of the bacteria to a surface (6,7,17). However, these results have not revealed the role(s) that EPS plays in biofilm formation. Studies with the gram-negative organisms Shewanella putrefaciens and Vibrio cholerae and the gram-positive organism Staphylococcus epidermidis revealed that EPS is required for initial attachment to surfaces (15, 20; D. Newman and R. Kolter, unpublished data). Here, we describe the role of EPS in E. coli biofilm formation and note that this role is dramatically different than that described for S. putrefaciens, V. cholerae, and S. epidermidis.Isolation of an E. coli strain defective in...

DegP is a heat-shock inducible periplasmic protease in Escherichia coli. Unlike the cytoplasmic heat shock proteins, DegP is not transcriptionally regulated by the classical heat shock regulon coordinated by or32.Rather, the degP gene is transcriptionally regulated by an alternate heat shock or factor, orE. Previous studies have demonstrated a signal transduction pathway that monitors the amount of outer-membrane proteins in the bacterial envelope and modulates degP levels in response to this extracytoplasmic parameter. To analyze the transcriptional regulation of degP, we examined mutations that altered transcription of a degP-lacZ operon fusion. Gain-of-function mutations in cpxA, which specifies a two-component sensor protein, stimulate transcription from degP. Defined null mutations in cpxA or the gene encoding its cognate response regulator, cpxR, decrease transcription from degP. These null mutations also prevent transcriptional induction of degP in response to overexpression of a gene specifying an envelope lipoprotein. Cpx-mediated transcription of degP is partially dependent on the activity of Eor E, suggesting that the Cpx pathway functions in concert with Eor E and perhaps other RNA polymerases to drive transcription of degP.[Key Words: Heat shock; (rE; receptor kinase; lipoprotein; response regulator] Received November 23, 1994; revised version accepted January 12, 1995.The heat shock, or stress, proteins are a ubiquitous set of proteins whose synthesis is induced in response to environmental insults such as abrupt temperature elevation. It is thought that during times of stress these proteins maintain viability of the cell by degrading proteins that have been irreversibly inactivated and by promoting the renaturation/activation of reversibly inactivated proteins. Thus, heat shock proteins are often proteases or molecular chaperones (Gething and Sambrook 1992;Bukau 1993;Craig et al. 1993). Although heat shock proteins are required during times of stress, many of them also perform important functions in unstressed cells. For example, in Escherichia coli the molecular chaperones are thought to assist in protein folding (Zeilstra-Ryalls et al. 1991;Gething and Sambrook 1992), whereas proteases such as Lon serve post-translational regulatory roles (Gottesman 1989;Goldberg 1992}. Because heat shock proteins perform such fundamental functions, it is not surprising that they are found in a variety of subcellular compartments in both prokaryotic and eukaryotic cells (Deshaies et al. 1988;Strauch and Beckwith 1988; Craig et al. 1989;Rose et al. 1989). Interestingly, the regulation of stress proteins found in one compartment is often coordinated independently of stress proteins within other compartments (Strauch and Beckwith 1988;Strauch et al. 1989;Mori et al. 1993). For example, stresses that specifically perturb the bacterial envelope in E. coli increase the synthesis of the periplasmic protease DegP (Lipinska et al. 1990), whereas the synthesis of cytoplasmic stress proteins remains unaffected (Mecsas et a...

The assembly of interactive protein subunits into extracellular structures, such as pilus fibers in the Enterobacteriaceae, is dependent on the activity of PapD-like periplasmic chaperones. The ability of PapD to undergo a β zippering interaction with the hydrophobic C-terminus of pilus subunits facilitates their folding and release from the cytoplasmic membrane into the periplasm. In the absence of the chaperone, subunits remained tethered to the membrane and were driven off-pathway via non-productive interactions. These offpathway reactions were detrimental to cell growth; wild-type growth was restored by co-expression of PapD. Subunit misfolding in the absence of PapD was sensed by two parallel pathways: the Cpx twocomponent signaling system and the σE modulatory pathway.

Transcription of the agn43 locus, which specifies an outer membrane protein of Escherichia coli, is regulated in a phase‐variable fashion by the OxyR–DNA binding protein and Dam methylase. Despite its well‐characterized regulation, the function of Ag43 has remained elusive until now. Previous studies indicated that Ag43 mediates autoaggregation of certain strains of E. coli in liquid culture. Given this phenotype, we examined the role of Ag43 in biofilm formation. Here, we report that Ag43 contributes to E. coli biofilm formation in glucose‐minimal medium, but not in Luria–Bertani broth. In addition, we show that flagellar‐mediated motility is required for biofilm formation in both rich and minimal environments. Altogether, our results suggest that E. coli uses both common and specific gene sets for the development of biofilms under various growth conditions.

The LamB-LacZ-PhoA tripartite fusion protein is secreted to the periplasm, where it exerts a toxicity of unknown origin during high-level synthesis in the presence of the inducer maltose, a phenotype referred to as maltose sensitivity. We selected multicopy suppressors of this toxicity that allow growth of the tripartite fusion strains in the presence of maltose. Mapping and subclone analysis of the suppressor locus identified a previously uncharacterized chromosomal region at 4.7 min that is responsible for suppression. DNA sequence analysis revealed a new gene with the potential to code for a protein of 236 amino acids with a predicted molecular mass of 25,829 Da. The gene product contains an amino-terminal signal sequence to direct the protein for secretion and a consensus lipoprotein modification sequence. As predicted from the sequence, the suppressor protein is labeled with [ 3 H]palmitate and is localized to the outer membrane. Accordingly, the gene has been named nlpE (for new lipoprotein E). Increased expression of NlpE suppresses the maltose sensitivity of tripartite fusion strains and also the extracytoplasmic toxicities conferred by a mutant outer membrane protein, LamBA23D. Suppression occurs by activation of the Cpx two-component signal transduction pathway. This pathway controls the expression of the periplasmic protease DegP and other factors that can combat certain types of extracytoplasmic stress.