A large family of anti‐activators accompanying XylS/AraC family regulatory proteins

AraC Negative Regulators (ANR) suppress virulence genes by directly down‐regulating AraC/XylS members in Gram‐negative bacteria. In this study, we sought to investigate the distribution and molecular mechanisms of regulatory function for ANRs among different bacterial pathogens. We identified more than 200 ANRs distributed in diverse clinically important gram negative pathogens, including Vibrio spp., Salmonella spp., Shigella spp., Yersinia spp., Citrobacter spp., enterotoxigenic (ETEC) and enteroaggregative E. coli (EAEC), and members of the Pasteurellaceae. By employing a bacterial two hybrid system, pull down assays and surface plasmon resonance (SPR) analysis, we demonstrate that Aar (AggR‐activated regulator), a prototype member of the ANR family in EAEC, binds with high affinity to the central linker domain of AraC‐like member AggR. ANR‐AggR binding disrupted AggR dimerization and prevented AggR‐DNA binding. ANR homologs of Vibrio cholerae, Citrobacter rodentium, Salmonella enterica and ETEC were capable of complementing Aar activity by repressing aggR expression in EAEC strain 042. ANR homologs of ETEC and Vibrio cholerae bound to AggR as well as to other members of the AraC family, including Rns and ToxT. The predicted proteins of all ANR members exhibit three highly conserved predicted α‐helices. Site‐directed mutagenesis studies suggest that at least predicted α‐helices 2 and 3 are required for Aar activity. In sum, our data strongly suggest that members of the novel ANR family act by directly binding to their cognate AraC partners.     

Identification of GntR as regulator of the glucose metabolism in Pseudomonas aeruginosa

In contrast to Escherichia coli, glucose metabolism in pseudomonads occurs exclusively through the Entner‐Doudoroff (ED) pathway. This pathway, as well as the three routes to generate the initial ED pathway substrate, 6‐phosphogluconate, is regulated by the PtxS, HexR and GtrS/GltR systems. With GntR (PA2320) we report here the identification of an additional regulator in Pseudomonas aeruginosa PAO1. GntR repressed its own expression as well as that of the GntP gluconate permease. In contrast to PtxS and GtrS/GltR, GntR did not modulate expression of the toxA gene encoding the exotoxin A virulence factor. GntR was found to bind to promoters P_gntR and P_gntP and the consensus sequence of its operator was defined as 5′‐AC‐N‐AAG‐N‐TAGCGCT‐3′. Both operator sites overlapped with the RNA polymerase binding site and we show that GntR employs an effector mediated de‐repression mechanism. The release of promoter bound GntR is induced by gluconate and 6‐phosphogluconate that bind with similar apparent affinities to the GntR/DNA complex. GntR and PtxS are paralogous and may have evolved from a common ancestor. The concerted action of four regulatory systems in the regulation of glucose metabolism in Pseudomonas can be considered as a model to understand complex regulatory circuits in bacteria.     

The xylA promoter of Bacillus megaterium mediates constitutive gene expression in Escherichia coli

In the present study, we demonstrate that the Escherichia coli–Bacillus megaterium shuttle vector pHIS1522 can be used as a versatile expression vector. Recombinant genes under the control of the xylA promoter are constitutively expressed at a high level in E. coli strains, whereas their expression is strongly induced by the addition of xylose in B. megaterium. The utilization of D ‐xylose is known to be dependent on the xylAB genes in a number of bacteria. For B. megaterium a XylA‐based expression system was established that allows tightly regulated and highly efficient heterologous gene expression. The open reading frame (ORF) of the fluorescent protein turboRFP was cloned under the control of the xylA promoter of B. megaterium in the shuttle vector pHIS1522. Unexpectedly, tRFP expression was not only observed in B. megaterium, but also in E. coli. Based on fluorescence measurements and Western blot analysis, expression was comparable or slightly higher compared with the commonly used pET vectors. Therefore, pHIS1522 can be used as a versatile expression vector in both, B. megaterium and E. coli.     

Type VI secretion modulates quorum sensing and stress response in Vibrio anguillarum

Type VI protein secretion systems (T6SS) are essential for virulence of several Gram‐negative bacteria. In this study, we identified a T6SS in Vibrio anguillarum, a marine bacterium that causes a hemorrhagic septicemia in fish. A partial operon vtsA‐H (v ibrio t ype s ix secretion) was sequenced and shown to encode eight proteins. VtsE‐H are signature proteins found in other T6SSs, while VtsA‐D are not associated with T6SS studied so far. In‐frame deletions were made in each gene. Secretion of a haemolysin‐co‐regulated‐like protein (Hcp), a protein secreted by all studied T6SSs, was decreased in VtsE‐H. Unexpectedly, VtsA, VtsC and VtsD activated while VtsB and VtsE‐H repressed hcp expression. The T6SS proteins also regulated expression of two extracellular proteases, EmpA and PrtV, but inversely to Hcp expression. This regulation was indirect as T6S positively regulated expression of the stress‐response regulator RpoS and the quorum‐sensing regulator VanT, which positively regulate protease expression. Moreover, VtsA‐H proteins were not needed for virulence but did play a role in various stress responses. Thus, these data characterize a new role for T6S in the ecology of bacteria and we hypothesize this role to be a signal sensing mechanism that modulates the expression of regulators of the general stress response.     

Escherichia coli W as a new platform strain for the enhanced production of L-valine by systems metabolic engineering

A less frequently employed Escherichia coli strain W, yet possessing useful metabolic characteristics such as less acetic acid production and high L ‐valine tolerance, was metabolically engineered for the production of L ‐valine. The ilvA gene was deleted to make more pyruvate, a key precursor for L ‐valine, available for enhanced L ‐valine biosynthesis. The lacI gene was deleted to allow constitutive expression of genes under the tac or trc promoter. The ilvBN^mut genes encoding feedback‐resistant acetohydroxy acid synthase (AHAS) I and the L ‐valine biosynthetic ilvCED genes encoding acetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, and branched chain amino acid aminotransferase, respectively, were amplified by plasmid‐based overexpression. The global regulator Lrp and L ‐valine exporter YgaZH were also amplified by plasmid‐based overexpression. The engineered E. coli W (ΔlacI ΔilvA) strain overexpressing the ilvBN^mut, ilvCED, ygaZH, and lrp genes was able to produce an impressively high concentration of 60.7 g/L L ‐valine by fed‐batch culture in 29.5 h, resulting in a high volumetric productivity of 2.06 g/L/h. The most notable finding is that there was no other byproduct produced during L ‐valine production. The results obtained in this study suggest that E. coli W can be a good alternative to Corynebacterium glutamicum and E. coli K‐12, which have so far been the most efficient L ‐valine producer. Furthermore, it is expected that various bioproducts including other amino acids might be more efficiently produced by this revisited platform strain of E. coli. Bioeng. 2011; 108:1140–1147. © 2010 Wiley Periodicals, Inc.