In response to certain environmental signals, bacteria will differentiate from an independent free-living mode of growth and take up an interdependent surface-attached existence. These surface-attached microbial communities are known as biofilms. In flowing systems where nutrients are available, biofilms can develop into elaborate three-dimensional structures. The development of biofilm architecture, particularly the spatial arrangement of colonies within the matrix and the open areas surrounding the colonies, is thought to be fundamental to the function of these complex communities. Here we report a new role for rhamnolipid surfactants produced by the opportunistic pathogen Pseudomonas aeruginosa in the maintenance of biofilm architecture. Biofilms produced by mutants deficient in rhamnolipid synthesis do not maintain the noncolonized channels surrounding macrocolonies. We provide evidence that surfactants may be able to maintain open channels by affecting cell-cell interactions and the attachment of bacterial cells to surfaces. The induced synthesis of rhamnolipids during the later stages of biofilm development (when cell density is high) implies an active mechanism whereby the bacteria exploit intercellular interaction and communication to actively maintain these channels. We propose that the maintenance of biofilm architecture represents a previously unrecognized step in the development of these microbial communities.Although bacteria are commonly viewed as solitary life forms, these organisms are more typically colonial creatures. In their natural settings, bacteria persist within microbial communities, where they exploit elaborate systems of intercellular interaction and communication to adjust to changing environmental parameters. Moreover, biofilm formation has also been linked to the emergence of a variety of opportunistic human pathogens (5). For example, organisms such as Staphylococcus epidermidis and Pseudomonas aeruginosa form biofilms on implants and dead or living tissue, thereby contributing to a variety of persistent infections.Thinking about bacterial populations as connected organisms capable of concerted multicellular activities has provided researchers with novel insights into microbial biology. The key to such multicellular behavior lies in the ability of each individual cell to sense and respond to information from nearby cells, and this behavior requires a certain population size or quorum of cells. The development of biofilms is a process that involves both a quorum of cells and multicellular behavior (6). Single-species biofilms are of particular interest due to their clinical importance and the monospecies biofilms formed by P. aeruginosa has become a prominent model for studying this aspect of microbial biology.The complex structure of microbial biofilms has only recently been determined. Detailed analysis by scanning confocal laser microscopy has shown that biofilms of P. aeruginosa formed on solid surfaces and exposed to a continuous flow of fresh nutrients are open, highly hydrate...

Pseudomonas aeruginosa is capable of twitching, swimming, and swarming motility. The latter form of translocation occurs on semisolid surfaces, requires functional flagella and biosurfactant production, and results in complex motility patterns. From the point of inoculation, bacteria migrate as defined groups, referred to as tendrils, moving in a coordinated manner capable of sensing and responding to other groups of cells. We were able to show that P. aeruginosa produces extracellular factors capable of modulating tendril movement, and genetic analysis revealed that modulation of these movements was dependent on rhamnolipid biosynthesis. An rhlB mutant (deficient in mono-and dirhamnolipid production) and an rhlC mutant (deficient in dirhamnolipid production) exhibited altered swarming patterns characterized by irregularly shaped tendrils. In addition, agar supplemented with rhamnolipid-containing spent supernatant inhibited wild-type (WT) swarming, whereas agar supplemented with spent supernatant from mutants that do not make rhamnolipids had no effect on WT P. aeruginosa swarming. Addition of purified rhamnolipids to swarming medium also inhibited swarming motility of the WT strain. We also show that a sadB mutant does not sense and/or respond to other groups of swarming cells and this mutant was capable of swarming on media supplemented with rhamnolipid-containing spent supernatant or purified rhamnolipids. The abilities to produce and respond to rhamnolipids in the context of group behavior are discussed.

A tool kit of vectors was designed to manipulate and express genes from a wide range of gram-negative species by using in vivo recombination. Saccharomyces cerevisiae can use its native recombination proteins to combine several amplicons in a single transformation step with high efficiency. We show that this technology is particularly useful for vector design. Shuttle, suicide, and expression vectors useful in a diverse group of bacteria are described and utilized. This report describes the use of these vectors to mutate clpX and clpP of the opportunistic pathogen Pseudomonas aeruginosa and to explore their roles in biofilm formation and surface motility. Complementation of the rhamnolipid biosynthetic gene rhlB is also described. Expression vectors are used for controlled expression of genes in two pseudomonad species. To demonstrate the facility of building complicated constructs with this technique, the recombination of four PCR-generated amplicons in a single step at >80% efficiency into one of these vectors is shown. These tools can be used for genetic studies of pseudomonads and many other gram-negative bacteria.Cloning using Saccharomyces cerevisiae homologous recombination is a powerful technique in which PCR primers with stretches of homologous DNA are used to target recombination of an amplicon with a vector (9,21,25,27). Cloning of an amplicon with yeast recombination into an intact vector can be done without restriction enzymes, but this requires the use of a selectable or counterselectable marker. The formation of a gap or double-stranded break in the vector by restriction enzyme digestion allows selective cloning of unmarked amplicons (29). An amplicon can seamlessly replace any part of the DNA in a vector with no need for enzyme sites at the junctions of the recombined pieces of DNA, as long as the yeast replication machinery is not replaced and the ends of the amplicons (or other DNA with homologous ends) to be added to a vector are on either side of the digest site (gap) in the vector. Therefore, multiple unmarked pieces of DNA can efficiently be assembled together in a single step using this method. In addition to fusions, directed mutations, restriction sites, and other changes can be designed into the primers. This technique is particularly useful for building vectors and complex constructs.The basic concept of cloning with gap repair is as follows. A vector able to replicate in yeast with a selective marker, such as URA3, is digested (gapped) with a restriction enzyme. Primers that amplify the region of DNA that one wants to insert into the vector are designed, containing sequences of homology to the yeast-replicating plasmid (30 to 40 bp in length). One or more targeted amplicons and the gapped vector are simultaneously introduced into yeast cells. A subset of these cells recombines the amplicon(s) and vector via Saccharomyces cerevisiae native recombination enzymes, generating stable circularized plasmids. Linearized or gapped vectors are not stable in yeast, while vectors that circularize...

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methylaccepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.Pseudomonas aeruginosa is an important model organism for the study of gram-negative biofilm development, yet little is known about the molecular mechanisms underlying the initial events leading to the surface interactions that characterize the early steps in bacterial biofilm formation. Microscopic observations (23,26,40,51) and genetic analyses (2) revealed two sequential events that lead to stable surface interactions. First, a bacterial cell pole contacts the surface in a process referred to as reversible attachment. This is a relatively unstable interaction, as reversibly attached bacteria can readily return to a planktonic existence. The second event is a transition from the polar association to one that is mediated by the long axis of the cell body, referred to as irreversible attachment. In P. aeruginosa, the only mutation known to block the transition from reversible to irreversible attachment is in the sadB gene (2).Another key aspect of biofilm formation by P. aeruginosa is the production of an extracellular matrix. In pseudomonads, this matrix is thought to be comprised of exopolysaccharides (EPS), DNA, and protein (19). The biofilm matrix has typically been credited with structuring the mature biofilm (4). Studies have identified the pel and psl loci as two sets of genes predicted to be involved in the production of the polysaccharide component of the matrix required for biofilm maturation by P. aeruginosa on abiotic surfaces, although only the pel gene cluster is found in P. aeruginosa strain PA14 (7,8,15,27), the focus of study in this report. Interestingly, recent studies suggest that the pel locus also plays a role in early biofilm formation. A pel mutant of P. aeruginosa PAK shows a strong attachment defect in a strain lacking type IV pili (48) and P. aeruginosa PAO1 with a mutation in the psl locus has a block in biofilm initiat...

This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The ⌬ldh ⌬pta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.