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Protein O-mannosyltransferases (PMTs) add mannose to serine/threonine (S/T)-rich proteins in the endoplasmic reticulum, facilitating proper folding and trafficking through the secretory pathway. These enzymes share a conserved architecture that includes a large transmembrane domain housing the catalytic pocket and a lumenal β-trefoil-folded MIR domain. Although S/T-rich regions in acceptor proteins are generally disordered, it remains unclear how PMTs selectively target these regions over other intrinsically disordered sequences. Here, using cryo-EM and X-ray crystallography, we demonstrate that the Saccharomyces cerevisiae Pmt4 dimer recognizes an S/T-rich peptide at two distinct sites. A groove above the catalytic pocket in the transmembrane domain binds the mannose-accepting S/T site, while the lumenal MIR domain engages an S/T-X-S/T motif. Notably, the substrate peptide is simultaneously bound by the catalytic pocket of one Pmt4 protomer and the MIR domain of the other, revealing an unexpected cooperative dual substrate recognition mechanism. This mechanism likely underpins the invariant dimeric architecture observed in all PMT family members. The yeast Pmt4 is a membrane embedded protein Omannosyltransferase. Du et al found that Pmt4 assembles a homodimer and detects the coincidence of the S/T acceptor site and an adjacent S/T-X-S/T motif in a model substrate, ensuring modification only to the S/T-rich regions of acceptor substrates.

Uropathogenic Escherichia coli assemble surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili facilitate bacterial colonization of the kidney and pyelonephritis. P pili are assembled through the conserved chaperone-usher pathway. Much of the structural and functional understanding of the chaperone-usher pathway has been gained through investigations of type 1 pili, which promote binding to the bladder and cystitis. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, and difficulties isolating stable P pilus assembly intermediates. Here, we circumvent these hindrances and determine cryo-electron microscopy structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates, revealing processive steps in P pilus biogenesis and capturing new conformational dynamics of the usher assembly machine. Escherichia coli form pili structures in order to initiate infection of the urinary tract. Here, Thanassi et al., have solved the structures of pili assembly intermediates and provided insights into their biogenesis and assembly.

Background Rhodosporidium toruloides is a β-carotenoid accumulating, oleaginous yeast that has great biotechnological potential. The lack of reliable and efficient genetic manipulation tools have been a major hurdle blocking its adoption as a biotechnology platform. Results We report for the first time the development of a highly efficient targeted gene deletion method in R. toruloides ATCC 10657 via Agrobacterium tumefaciens -mediated transformation. To further improve targeting frequency, the KU70 and KU80 homologs in R. toruloides were isolated and characterized in detail. A KU70 -deficient mutant (∆ku70e) generated with the hygromycin selection cassette removed by the Cre- loxP recombination system showed a dramatically improved targeted gene deletion frequency, with over 90% of the transformants being true knockouts when homology sequence length of at least 1 kb was used. Successful gene targeting could be made with homologous flanking sequences as short as 100 bp in the ∆ku70e strain. KU70 deficiency did not perturb cell growth although an elevated sensitivity to DNA mutagenic agents was observed. Compared to the other well-known oleaginous yeast, Yarrowia lipolytica, R. toruloides KU70/KU80 genes contain much higher density of introns and are the most GC-rich KU70/KU80 genes reported. Conclusions The KU70 -deficient mutant generated herein was effective in improving gene deletion frequency and allowed shorter homology sequences to be used for gene targeting. It retained the key oleaginous and fast growing features of R. toruloides . The strain should facilitate both fundamental and applied studies in this important yeast, with the approaches taken here likely to be applicable in other species in subphylum Pucciniomycotina.

Background Rhodotorula toruloides is an outstanding producer of lipids and carotenoids. Currently, information on the key metabolic pathways and their molecular basis of regulation remains scarce, severely limiting efforts to engineer it as an industrial host. Results We have adapted Agrobacterium tumefaciens -mediated transformation (ATMT) as a gene-tagging tool for the identification of novel genes in R. toruloides . Multiple factors affecting transformation efficiency in several species in the Pucciniomycotina subphylum were optimized. The Agrobacterium transfer DNA (T-DNA) showed predominantly single-copy chromosomal integrations in R. toruloides , which were trackable by high efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). To demonstrate the application of random T-DNA insertions for strain improvement and gene hunting, 3 T-DNA insertional libraries were screened against cerulenin, nile red and tetrazolium violet respectively, resulting in the identification of 22 mutants with obvious phenotypes in fatty acid or lipid metabolism. Similarly, 5 carotenoid biosynthetic mutants were obtained through visual screening of the transformants. To further validate the gene tagging strategy, one of the carotenoid production mutants, RAM5, was analyzed in detail. The mutant had a T-DNA inserted at the putative phytoene desaturase gene CAR1 . Deletion of CAR1 by homologous recombination led to a phenotype similar to RAM5 and it could be genetically complemented by re-introduction of the wild-type CAR1 genome sequence. Conclusions T-DNA insertional mutagenesis is an efficient forward genetic tool for gene discovery in R. toruloides and related oleaginous yeast species. It is also valuable for metabolic engineering in these hosts. Further analysis of the 27 mutants identified in this study should augment our knowledge of the lipid and carotenoid biosynthesis, which may be exploited for oil and isoprenoid metabolic engineering.

Ustilago scitaminea is the causal agent of sugar-cane smut disease. There is, however, no genetic transformation method for it. Here we report the development of an efficient mutagenesis method based on Agrobacterium tumefaciens-mediated transformation. To improve transformation efficiency, a range of conditions, including the codon-usage preference of the selection marker gene, promoters and the culture conditions for transformation were optimized. A strong promoter to drive marker gene expression, optimized codon usage of selection marker gene, controlled water content and pH of co-culture medium were critical factors affecting transformation efficiency. Our findings provide a useful tool for genetic analysis of this important plant pathogen.

Screening humanized antibodies from a human Fab phage display library is an effective and quick method to obtain beta-amyloid oligomers. Thus, the present study prepared amyloid-beta 42 oli- gomers and constructed a have human Fab phage display library based on blood samples from six healthy people. After three rounds of biopanning in vitro, a human single-domain antibody that spe- cifically recognized amyloid-beta 42 oligomers was identified. Western blot and enzyme-linked im- munosorbent assay demonstrated this antibody bound specifically to human amyloid-beta 42 tetramer and nonamer, but not the monomer or high molecular weight oligomers. This study suc- cessfully constructed a human phage display library and screened a single-domain antibody that specifically recognized amyloid-beta 42 oligomers.

Pathogenic bacteria such as Escherichia coli assemble surface structures termed pili, or fimbriae, to mediate binding to host-cell receptors 1 . Type 1 pili are assembled via the conserved chaperone–usher pathway 2 – 5 . The outer-membrane usher FimD recruits pilus subunits bound by the chaperone FimC via the periplasmic N-terminal domain of the usher. Subunit translocation through the β-barrel channel of the usher occurs at the two C-terminal domains (which we label CTD1 and CTD2) of this protein. How the chaperone–subunit complex bound to the N-terminal domain is handed over to the C-terminal domains, as well as the timing of subunit polymerization into the growing pilus, have previously been unclear. Here we use cryo-electron microscopy to capture a pilus assembly intermediate (FimD–FimC–FimF–FimG–FimH) in a conformation in which FimD is in the process of handing over the chaperone-bound end of the growing pilus to the C-terminal domains. In this structure, FimF has already polymerized with FimG, and the N-terminal domain of FimD swings over to bind CTD2; the N-terminal domain maintains contact with FimC–FimF, while at the same time permitting access to the C-terminal domains. FimD has an intrinsically disordered N-terminal tail that precedes the N-terminal domain. This N-terminal tail folds into a helical motif upon recruiting the FimC-subunit complex, but reorganizes into a loop to bind CTD2 during handover. Because both the N-terminal and C-terminal domains of FimD are bound to the end of the growing pilus, the structure further suggests a mechanism for stabilizing the assembly intermediate to prevent the pilus fibre diffusing away during the incorporation of thousands of subunits. The structure of a pilus assembly intermediate reveals the timing of subunit polymerization and how chaperone–subunit complexes are transferred from N-terminal to C-terminal domains of the usher in the formation of bacterial pili.

The oleaginous yeast Rhodosporidium toruloides , which belongs to the Pucciniomycotina subphylum in the Basidiomycota , has attracted strong interest in the biofuel community recently due to its ability to accumulate more than 60% of dry biomass as lipid under high-density fermentation. A 3,543-nucleotide (nt) DNA fragment of the glyceraldehyde-3-phosphate dehydrogenase gene ( GPD1 ) was isolated from R. toruloides ATCC 10657 and characterized in details. The 1,038-nt mRNA derived from seven exons encodes an open reading frame (ORF) of 345 amino acids that shows high identity (80%) to the Ustilago maydis homolog. Notably, the ORF is composed of codons strongly biased towards cytosine at the Wobble position. GPD1 is transcriptionally regulated by temperature shock, osmotic stress, and carbon source. Nested deletion analysis of the GPD1 promoter by GFP reporter assay revealed that two regions, −975 to −1,270 and −1,270 to −1,429, upstream from the translational start site of GPD1 were important for responses to various stress stimuli. Interestingly, a 176-bp short fragment maintained 42.2% promoter activity of the 795-bp version in U. maydis whereas it was reduced to 17.4% in R. toruloides . The GPD1 promoter drove strong expression of a codon-optimized enhanced green fluorescent protein gene (Rt GFP ) and a codon-optimized hygromycin phosphotransferase gene ( hpt-3 ), which was critical for Agrobacterium tumefaciens -mediated transformation in R. toruloides .

Pili are adhesive organelles of Gram-negative bacteria used to initiate an infection in the host. Uropathogenic Escherichia coli has two types of adhesive organelles: the type 1 (Fim) and the P (Pap) pili, which are assembled by the chaperone–usher (CU) pathway. Usher is an outer membrane protein that consists of a β-barrel domain in the membrane, flanked by an amino terminal domain (NTD) and two carboxyl terminal domains (CTD1 and CTD2) at the periplasmic side, and a plug domain that blocks the β-barrel in the resting apo state. Pilus biogenesis is a multistep process. Earlier studies have demonstrated that the usher NTD functions to recruit the chaperone–subunit complex, and the CTD1 and CTD2 facilitate subunit polymerization and protein secretion. However, how the chaperone–subunit complex is recruited by NTD and transferred from the NTD to the CTDs, and when the subunit is polymerized into the growing pilus, remain largely unanswered. By using protein engineering and single-particle cryo–electron microscopy, we captured a FimD–pilus tip assembly intermediate comprising FimDCFGH in a configuration in which the usher FimD is in the process of handing over FimC–FimF from its NTD to the CTDs. The pilus subunit polymerization occurs before subunit handover and reveals the mechanisms of subunit handover from FimD NTD to CTDs. We also found the cyclic conformational changes of the N-terminal tail which play crucial roles in the multi-step assembly process. In the second part of my thesis research, we captured three P pilus assembly intermediates in which the PapC usher is fully activated and is in the process ofrecruiting, polymerizing, and secreting P pilus tip subunits. Our cryo-EM structural analysis reveals new conformational dynamics of the usher and shows the movement of CTD2 to facilitate the NTD-to-CTD chaperone–subunit transfer process.

ABSTRACT Uropathogenic Escherichia coli (UPEC) assemble hair-like surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili mediate the adherence of UPEC to the kidney epithelium, facilitating bacterial colonization and pyelonephritis1. P pili are assembled through the conserved chaperone-usher (CU) pathway2-4. In this pathway, a dedicated chaperone facilitates the folding of nascent pilus subunits in the periplasm and an integral outer membrane (OM) protein termed the usher provides the assembly platform and secretion channel for the pilus fiber. Much of the structural and functional understanding of the CU pathway has been gained through investigations of type 1 pili, which promote UPEC binding to the bladder epithelium and the development of cystitis5. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, as well as difficulties in isolating stable P pilus assembly intermediates from bacteria. Here, we have devised a method to circumvent these hindrances and determined cryo-EM structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates. These structures show processive steps in P pilus biogenesis, reveal differences between P and type 1 pili, and capture new conformational dynamics of the usher assembly machine. Competing Interest Statement The authors have declared no competing interest.