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...

Pseudomonas aeruginosa is a ubiquitous bacterium capable of twitching, swimming, and swarming motility. In this study, we present evidence that P. aeruginosa has two flagellar stators, conserved in all pseudomonads as well as some other gram-negative bacteria. Either stator is sufficient for swimming, but both are necessary for swarming motility under most of the conditions tested, suggesting that these two stators may have different roles in these two types of motility.

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The Rcs two-component pathway is involved in the regulation of capsule production in Escherichia coli. RcsC is predicted to be the sensor component of this two-component pathway, and in this study we present the first genetic data that support the role of RcsC as a hybrid sensor kinase.

A mini-Tn5Cm insertion has been identified that significantly reduced the amount of an extracellular activating signal for a lacZ fusion (cma37::lacZ) in Providencia stuartii. The transposon insertion was located immediately upstream of an open reading frame encoding a putative CysE ortholog. The CysE enzyme, serine acetyltransferase, catalyzes the conversion of serine to O-acetyl-L-serine (OAS). This activating signal was also produced by Escherichia coli, and production was abolished in a strain containing a null allele of cysE. Products of the CysE enzyme (OAS, N-acetyl-L-serine [NAS], O-acetyl-L-threonine, and N-acetyl-L-threonine) were individually tested for the ability to activate cma37::lacZ. Only OAS was capable of activating the cma37::lacZ fusion. The ability of OAS to activate the cma37::lacZ fusion was abolished by pretreatment at pH 8.5, which converts OAS to NAS. However, the activity of the native signal in conditioned medium was not decreased by treatment at pH 8.5. In contrast, conditioned medium prepared from cells grown at pH 8.5 exhibited a 4-to 10-fold-higher activity, relative to pH 6.0. Additional genes regulated by the CysE-dependent signal and OAS were identified in P. stuartii and E. coli. The response to the extracellular signal in E. coli was dependent on CysB, a positive activator that requires NAS as a coactivator. In E. coli, a cysE mutant formed biofilms at an accelerated rate compared to the wild type, suggesting a physiological role for this extracellular signal.Cell-to-cell communication in bacteria is mediated by a wide variety of distinct chemical signals, and a number of reviews on this subject have been compiled (11,12,17,22). In gramnegative bacteria, these signals include the N-acylhomoserine lactones, autoinducer 2 (AI-2), quinolones, cyclic dipeptides, and the tryptophan derivative indole (2,12,26,32,38). In gram-positive bacteria, peptides are the primary mode of communication (11,17,22). Although peptide-mediated signals have not been isolated from gram-negative bacteria, recent data suggest that such systems are present (13,29,30).Several extracellular signals have been identified in Escherichia coli. Indole, a product of the tryptophanase-mediated catabolism of tryptophan, is capable of activating the astD, tnaB, and gabT genes (38). Studies using a tnaA mutant unable to produce indole have indicated that a second signal is required for full activation of the astD and gabT genes (38). A second signal in E. coli, the AI-2 furanone signal, has been shown by microarray analysis to control the expression of a large number of genes in E. coli (8,34). Additional genes in E. coli that are regulated by undefined extracellular signals include rpoS, sdiA, cysK, and ftsQAZ (1,14,23,33,37).The cysteine regulon in E. coli comprises a set of genes involved in the biosynthesis of cysteine from L-serine (18,19). These genes are positively controlled by the CysB activator in concert with the molecule N-acetyl-L-serine (NAS) (19). The production of NAS occurs in two steps. First, se...

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DjlA is a bitopic inner membrane protein, which belongs to the DnaJ co-chaperone family in Escherichia coli. Overproduction of DjlA leads to the synthesis of colanic acid, resulting in mucoidy, via the activation of the two-component regulatory system RcsC/B that controls the cps (capsular polysaccharide) operon. This induction requires both the co-chaperone activity of DjlA, in cooperation with DnaK and GrpE, and its unique transmembrane (TM) domain. Here, we show that the TM segment of DjlA acts as a dimerisation domain: when fused to the N-terminal DNAbinding domain of the lambda cI repressor protein, it can substitute for the native C-terminal dimerisation domain of cI, thus generating an active cI repressor. Replacing the TM domain of DjlA by other TM domains, with or without dimerising capacity, revealed that dimerisation is not sufficient for the induction of cps expression, indicating an additional sequence-or structurally specific role for the TM domain. Finally, the conserved glycines present in the TM domain of DjlA are essential for the induction of mucoidy, but not for dimerisation.

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