Pathogenic bacteria frequently cloak themselves with a capsular polysaccharide layer. Escherichia coli group 1 capsules are formed from repeat-unit polysaccharides with molecular weights exceeding 100 kDa. The export of such a large polar molecule across the hydrophobic outer membrane in Gram-negative bacteria presents a formidable challenge, given that the permeability barrier of the membrane must be maintained. We describe the 2.26 Å structure of Wza, an integral outer membrane protein, that is essential for capsule export. Wza is an octamer, with a composite molecular weight of 340 kDa, and it forms an "amphora"-like structure. The protein has a large central cavity 100 Å long and 30 Å wide. The transmembrane region is a novel α-helical barrel, and is linked to three additional novel periplasmic domains, marking Wza as the representative of a new class of membrane protein.Although Wza is open to the extracellular environment, a flexible loop in the periplasmic region occludes the cavity and may regulate the opening of the channel. The structure defines the route taken by the capsular polymer as it exits the cell, using the structural data we propose a mechanism for the translocation of the large polar capsular polysaccharide.

Zinc-finger domain transcriptional regulators regulate a myriad of functions in eukaryotes. Interestingly, ancestral versions (MucR) from Alpha-proteobacteria control bacterial virulence/symbiosis. Whether virulence regulators can also control cell cycle transcription is unknown. Here we report that MucR proteins implement a hitherto elusive primordial S→G1 transcriptional switch. After charting G1-specific promoters in the cell cycle model Caulobacter crescentus by comparative ChIP-seq, we use one such promoter as genetic proxy to unearth two MucR paralogs, MucR1/2, as constituents of a quadripartite and homeostatic regulatory module directing the S→G1 transcriptional switch. Surprisingly, MucR orthologues that regulate virulence and symbiosis gene transcription in Brucella, Agrobacterium or Sinorhizobium support this S→G1 switch in Caulobacter. Pan-genomic ChIP-seq analyses in Sinorhizobium and Caulobacter show that this module indeed targets orthologous genes. We propose that MucR proteins and possibly other virulence regulators primarily control bacterial cell cycle (G1-phase) transcription, rendering expression of target (virulence) genes periodic and in tune with the cell cycle.

Recently we described the isolation of spontaneous bacteriophage K139-resistant Vibrio cholerae O1 El Tor mutants. In this study, we identified phage-resistant isolates with intact O antigen but altered core oligosaccharide which were also affected in galactose catabolism; this strains have mutations in the galU gene. We inactivated another gal gene, galE, and the mutant was also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharide (LPS). Both gal mutants as well as a rough LPS (R-LPS) mutant were investigated for the ability to colonize the mouse small intestine. The galU and R-LPS mutants, but not the galE mutant, were defective in colonization, a phenotype also associated with O-antigen-negative mutants. By investigating several parameters in vitro, we could show that galU and R-LPS mutants were more sensitive to short-chain organic acids, cationic antimicrobial peptides, the complement system, and bile salts as well as other hydrophobic agents, indicating that their outer membrane no longer provides an effective barrier function. O-antigen-negative strains were found to be sensitive to complement and cationic peptides, but they displayed significant resistance to bile salts and short-chain organic acids. Furthermore, we found that galU and galE are essential for the formation of a biofilm in a spontaneous phageresistant rugose variant, suggesting that the synthesis of UDP-galactose via UDP-glucose is necessary for biosynthesis of the exopolysaccharide. In addition, we provide evidence that the production of exopolysaccharide limits the access of phage K139 to its receptor, the O antigen. In conclusion, our results indicate involvement of galU in V. cholerae virulence, correlated with the observed change in LPS structure, and a role for galU and galE in environmental survival of V. cholerae.The causative agent of the intestinal disease cholera is Vibrio cholerae, a gram-negative motile bacterium. Of the more than 150 known serogroups, only the noncapsulated O1 and the encapsulated O139 serogroup have been found to be associated with epidemic cholera. Epidemic O139 strains are related to and were derived from O1 El Tor strains after genetic alterations of the O-antigen biosynthesis gene cluster (16). The ongoing seventh pandemic, which began in 1961, is caused by O1 El Tor strains (3). V. cholerae is a natural inhabitant of aquatic ecosystems and is known to attach to environmental surfaces such as plants, filamentous green algae, zooplankton, crustaceans, or insects (8). Recently, V. cholerae O1 El Tor was found to form a three-dimensional biofilm on abiotic surfaces (70). Biofilm formation may be important in the life cycle of pathogenic V. cholerae strains, because they reside within natural aquatic habitats during interepidemic periods. O1 El Tor strains are also able to switch to a rugose colony phenotype. This morphology correlates with the constitutively production of an exopolysaccharide allowing biofilm formation on abiotic surfaces (65...

IntroductionWhen O 2 delivery is impaired, the resulting hypoxia activates homeostatic mechanisms at the systemic and cellular level. 1 This response involves changes in gene expression mediated by hypoxiainducible factor-1 (HIF-1), the master transcription factor of oxygen-regulated genes. HIF-1 is a heterodimeric protein comprising the oxygen-sensitive ␣-subunit (HIF-1␣, or the more cell-typespecifically expressed HIF-2␣) and the oxygen-insensitive ␤-subunit. 2 Oxygen-regulated gene expression involves binding of HIF to cis-regulatory hypoxia response elements (HREs) of HIF target genes such as erythropoietin or vascular endothelial growth factor. 3 The molecular basis for the hypoxia-induced stability and activity of HIF-1␣ and HIF-2␣ is the O 2 -dependent hydroxylation of distinct prolyl residues. [4][5][6] A family of oxygen-, iron-and 2-oxoglutarate-dependent prolyl-4-hydroxylases has been described recently to hydroxylate the oxygen-labile ␣ subunits of HIF-1 and HIF-2. 5,7,8 This family consists of 3 members called prolyl-4-hydroxylase domain (PHD) 1, PHD2, PHD3, or HIF prolyl hydroxylase (HPH) 3, HPH2, and HPH1, respectively. 4,5 Following prolyl-4-hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL) recognizes HIF␣ subunits and targets them for rapid ubiquitination and proteasomal degradation. 9 Binding of pVHL strictly requires prior modification of human HIF-1␣ and HIF-2␣ by prolyl-4-hydroxylation at prolines 402 and 564 or prolines 405 or 531, respectively. 10,11 Limited oxygen supply prevents HIF␣ hydroxylation and degradation. 12 In addition to protein stability, oxygen-dependent C-terminal asparagine hydroxylation of HIF␣ by factor-inhibiting HIF (FIH) prevents transcriptional cofactor recruitment, thereby fine-tuning HIF-1 activity after a further decrease in oxygen availability. 13 Most interestingly, in addition to HIF␣, ankyrin repeats present in IB and NF-B family members have recently been described to be hydroxylated by FIH, demonstrating that hydroxylation is not restricted to the HIF signaling pathway. 14 Besides similarities in the hydroxylation reaction in vitro, the 3 PHDs differ in their ability to hydroxylate HIF-1␣ in vivo and in their organ-specific expression pattern. [15][16][17] Moreover, the phenotypes of PHD knock-out mice demonstrate divergent roles of the 3 PHDs during embryonic development. 18 These data indicate that under physiologic conditions, PHD1, PHD2, and PHD3 mediate different, probably even HIF-independent, oxygen-regulated signal transduction pathways.By searching for novel targets of PHD3 using yeast 2-hybrid technology, we identified activating transcription factor-4 (ATF-4) as a novel interaction partner, and we found that PHD3 confers oxygen-dependent ATF-4 protein stability in a pVHL-independent manner. ATF-4-deficient mice are severely anemic during fetal development, apparently because of an impairment in definitive hematopoiesis. 19 In addition, overexpression of A...