Overproduction of the exopolysaccharide alginate causes mucoid conversion in Pseudomonas aeruginosa and is a poor prognosticator in cystic fibrosis. The ECF factor AlgU and its cognate antifactor MucA are two principal regulators of alginate production. Here, we report the identification of three positive regulators of alginate biosynthesis: PA4033 (designated mucE), PA3649 (designated mucP), and algW. MucE, a small protein (9.5 kDa), was identified as part of a global mariner transposon screen for new regulators of alginate production. A transposon located in its promoter caused the overexpression of MucE and mucoid conversion in P. aeruginosa strains PAO1 and PA14. Accumulation of MucE in the envelope resulted in increased AlgU activity and reduced MucA levels. Three critical amino acid residues at the C terminus of MucE (WVF) were required for mucoid conversion via two predicted proteases AlgW (DegS) and MucP (RseP/YaeL). Moreover, as in Escherichia coli, the PDZ domain of AlgW was required for signal transduction. These results suggest that AlgU is regulated similarly to E. coli E except that the amino acid triad signals from MucE and other envelope proteins that activate AlgW are slightly different from those activating DegS.alginate ͉ anti-factor ͉ PDZ domain ͉ protease cascade ͉ signal specificity

We report the construction of a series of Escherichia-Pseudomonas broad-host-range expression vectors utilizing the P BAD promoter and the araC regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads.Gene cloning, disruption, deletion, complementation analysis, and allelic exchange are central to prokaryotic molecular genetics. In Pseudomonas aeruginosa, Schweizer and colleagues developed the pUCP family of general-purpose vectors for cloning and gene expression (24, 29) based on the well-characterized pUC18/19 vectors (32) and the cryptic mini-plasmid pRO1614 (19). Other promoters are also in routine use, such as the tac (4, 6), T7 (28), and araBAD promoter-based (8, 11) vectors for regulated expression in Escherichia coli and many other bacterial species (e.g., see references 2, 18, and 25). In E. coli, AraC represses the araBAD promoter (P BAD ) and the expression of a cloned gene is induced by the addition of L-arabinose. Pseudomonas researchers have used the inducible properties of the araC regulator and the P BAD promoter cassette for the controlled gene expression by integrating the araC-P BAD -specific transcription fusion into the chromosome by using a suicide vector or an integration-proficient vector (1,3,13,17,30,31). In the present study, we modified the existing EscherichiaPseudomonas shuttle vectors pUCP20T, -26, -28T, and -30T by replacing the lac promoter with the araC-P BAD cassette to allow conditional expression in pseudomonads and other bacteria, e.g., Burkholderia spp.Construction and features of pHERD vectors. Functional genetic analysis requires vectors capable of conditional expression. The P BAD promoter has been used for gene expression extensively in E. coli and some in P. aeruginosa and Burkholderia spp. (12,27,31). We first constructed three shuttle vectors, pHERD20T, -28T, and -30T (Fig. 1), based on EscherichiaPseudomonas shuttle vectors pUCP20T, pUCP28T, and pUCP30T (29) and the commercial expression vector pBAD/ Thio-TOPO (Invitrogen). The 368-bp fragment of the pUCP vectors spanning two restriction sites, AflII and EcoRI, was replaced with the araC-P BAD fragment (1.3 kb), produced via PCR with pBAD/Thio-TOPO as the template and primers pBAD-F and pBAD-R (Table 1). The PCR product was purified and directly digested with AflII and EcoRI, and the two fragments were ligated into the pUCP vectors, creating pHERD20T (Fig. 1). The EcoRI/AflII regions of these vectors were sequenced to confirm that no mutations were introduced during the cloning pro-

Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters P algU and P algD . Deletion of alternative factor RpoN ( 54 ) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1.

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BackgroundBacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR.ResultsRpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains.ConclusionIn S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0357-0) contains supplementary material, which is available to authorized users.

Bacterial floc formation plays a central role in the activated sludge (AS) process, which has been widely utilized for sewage and wastewater treatment. The formation of AS flocs has long been known to require exopolysaccharide biosynthesis. This study demonstrates an additional requirement for a PEP-CTERM protein in Zoogloea resiniphila, a dominant AS bacterium harboring a large exopolysaccharide biosynthesis gene cluster. Two members of a wide-spread family of high copy number-per-genome PEP-CTERM genes, transcriptionally regulated by the RpoN sigma factor and accessory PrsK-PrsR two-component system and at least one of these, pepA, must be expressed for Zoogloea to build the floc structures that allow gravitational sludge settling and recycling. Without PrsK or PrsR, Zoogloea cells were planktonic rather than flocculated and secreted exopolysaccharides were released into the growth broth in soluble form. Overexpression of PepA could circumvent the requirement of rpoN, prsK and prsR for the floc-forming phenotype by fixing the exopolysaccharides to bacterial cells. However, overexpression of PepA, which underwent post-translational modifications, could not rescue the long-rod morphology of the rpoN mutant. Consistently, PEP-CTERM genes and exopolysaccharide biosynthesis gene cluster are present in the genome of the floc-forming Nitrospira comammox and Mitsuaria strain as well as many other AS bacteria.