Sequence and origin of the Streptomyces intergenetic-conjugation helper plasmid pUZ8002.

Conjugation of plasmids from Escherichia coli is essential for the genetic manipulation of Streptomyces spp. To facilitate intergeneric conjugation from E. coli to Streptomyces the conjugative machinery required for genetic transfer is usually provided by the non-transferable helper plasmid, pUZ8002. Here we present the complete nucleotide sequence of pUZ8002, describe the previously undocumented creation process, and provide details of the sequence relative to the parental pUZ8 plasmid and another previously published pUZ8002 sequence.

Salmonella enterica serovar Typhimurium STM1266 encodes a regulator of curli biofilm formation: the brfS gene.

The major biofilm pathway in Salmonella enterica serovar Typhimurium involves specific growth conditions that induce the csgA gene whose product forms surface curli fibers that mediate biofilm formation. We have found that the previously uncharacterized STM1266 gene in S. Typhimurium plays a role in regulating biofilm formation via the curli pathway. S. Typhimurium ΔSTM1266 strains display a biofilm defect, and overexpression of STM1266 results in enhanced biofilm formation. STM1266 deletion resulted in lowered csgA expression using promoter-reporter ß-galactosidase assays, and csgA and csgD deletions abrogate the effects of STM1266 overexpression on biofilm formation while deletion of bcsA (encoding an essential enzyme for cellulose formation) has no effect. In a mouse infection model, the ΔSTM1266 strain displayed results similar to those seen for previously reported ΔcsgA strains. The STM1266 gene is predicted to encode a DNA-binding transcriptional regulator of the MerR family and is homologous to the Escherichia coli BluR regulator protein. We respectfully propose to ascribe the name brfS (biofilm regulator for Salmonella Typhimurium) to the STM1266 gene.

Role of Hypoxia in the Control of the Cell Cycle.

The cell cycle is an important cellular process whereby the cell attempts to replicate its genome in an error-free manner. As such, mechanisms must exist for the cell cycle to respond to stress signals such as those elicited by hypoxia or reduced oxygen availability. This review focuses on the role of transcriptional and post-transcriptional mechanisms initiated in hypoxia that interface with cell cycle control. In addition, we discuss how the cell cycle can alter the hypoxia response. Overall, the cellular response to hypoxia and the cell cycle are linked through a variety of mechanisms, allowing cells to respond to hypoxia in a manner that ensures survival and minimal errors throughout cell division.

Manipulating microcompartment operons to study mechanism and function.

The gene systems that encode functional bacterial microcompartments (BMCs) are typically comprised of between 10-23 genes, often in a contiguous operon. BMC genes can be studied as whole native operons or as subsets of genes that form structures for specific applications. Recent examples of such studies highlight the flexible modular nature of BMC operons/genes and the options that exist to harness their functions via manipulation at the DNA level. This work also demonstrates the transfer and functional expression of BMC operons/genes across bacterial species. Recombineering, DNA synthesis technology, and advanced cloning techniques have all been applied in creative ways to study the nature of BMC mechanism and function.

Phenotypes, transcriptome, and novel biofilm formation associated with the ydcI gene.

The ydcI gene has previously been shown to encode a DNA-binding protein involved with acid stress resistance and induced biofilm formation in a strain of Salmonella enterica serovar Typhimurium. In addition, characterisation of the ydcI gene in Escherichia coli and other bacteria demonstrated strikingly different tolerance for induced ydcI expression across Gram negative species. In this report, we investigated the conservation of these phenotypes across multiple strains of S. Typhimurium and E. coli, and we used RNA Seq to identify the transcriptome of the ΔydcI mutant compared to WT in S. Typhimurium and E. coli (to establish the YdcI regulon in each species). We constructed deletion mutants in each species based on the RNA Seq results and tested these mutants for the relevant ydcI-related phenotypes. Though no evidence for a role in these phenotypes was found via the RNA Seq deletion mutants, we found that the ydcI-induced biofilm in S. Typhimurium is formed independently of the major biofilm genes csgA and bcsA indicating a potentially novel type of biofilm formation.

Oxygen-sensing mechanisms in cells.

The importance of oxygen for the survival of multicellular and aerobic organisms is well established and documented. Over the years, increased knowledge of its use for bioenergetics has placed oxygen at the centre of research on mitochondria and ATP-generating processes. Understanding the molecular mechanisms governing cellular oxygen sensing and response has allowed for the discovery of novel pathways oxygen is involved in, culminating with the award of the Nobel Prize for Medicine and Physiology in 2019 to the pioneers of this field, Greg Semenza, Peter Ratcliffe and William Kaelin. However, it is now beginning to be appreciated that oxygen can be a signalling molecule involved in a vast array of molecular processes, most of which impinge on gene expression control. This review will focus on the knowns and unknowns of oxygen as a signalling molecule, highlighting the role of 2-oxoglutarate-dependent dioxygenases as central players in the cellular response to deviations in oxygen tension.

The cloned SPI-1 type 3 secretion system can be functionally expressed outside Salmonella backgrounds.

Due to its potential for use in bacterial engineering applications, we previously cloned the SPI-1 type 3 secretion system (T3SS) genes from the genome of Salmonella enterica serovar Typhimurium strain LT2. We have documented that this clone, while functionally expressed in S. Typhimurium strains, displays a severe expression defect in other Gram negative backgrounds including Escherichia coli. To address this issue, we compared SPI-1 DNA sequence across different backgrounds, fully sequenced the original SPI-1 clone, and cloned SPI-1 from other S. Typhimurium strains. In this process, we were able to successfully obtain SPI-1 clones that are functionally expressed in E. coli indicating the first such result for a full-length SP-1 T3SS clone. We discovered that the original cloning technique using a DNA homology-based capture method was the root of the expression defect and that the FRT-Capture technique is preferable over the homology-based method. This result paves the way for future studies and applications using cloned SPI-1 and other T3SS in non-Salmonella bacterial backgrounds.

Author Correction: Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors induce autophagy and have a protective effect in an in-vitro ischaemia model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors induce autophagy and have a protective effect in an in-vitro ischaemia model.

This study compared effects of five hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD) inhibitors on PC12 cells and primary rat neurons following oxygen-glucose deprivation (OGD). At 100 µM, the PHD inhibitors did not cause cytotoxicity and apoptosis. MTT activity was only significantly reduced by FG4592 or Bayer 85-3934 in PC12 cells. The PHD inhibitors at 100 µM significantly increased the LC3-II/LC3-I expression ratio and downregulated p62 in PC12 cells, so did FG4592 (30 µM) and DMOG (100 µM) in neurons. HIF-1α was stabilised in PC12 cells by all the PHD inhibitors at 100 µM except for DMOG, which stabilised HIF-1α at 1 and 2 mM. In primary neurons, HIF-1α was stabilised by FG4592 (30 µM) and DMOG (100 µM). Pretreatment with the PHD inhibitors 24 hours followed by 24 hour reoxygenation prior to 6 hours OGD (0.3% O2) significantly reduced LDH release and increased MTT activity compared to vehicle (1% DMSO) pretreatment. In conclusion, the PHD inhibitors stabilise HIF-1α in normoxia, induce autophagy, and protect cells from a subsequent OGD insult. The new class of PHD inhibitors (FG4592, FG2216, GSK1278863, Bay85-3934) have the higher potency than DMOG. The interplay between autophagy, HIF stabilisation and neuroprotection in ischaemic stroke merits further investigation.

Celebrating 75 years.

INTRODUCTION: Following the introduction of shock wave lithotripsy (SWL), ureteroscopy (URS), and percutaneous nephrolithotomy (PCNL), the subspecialty of endourology was born in the late 1970s. The purpose of this study was to report milestones in Canadian endourology, highlighting Canada's contributions to the field. METHODS: A review of the literature was performed from the late 1970s to the present. The literature review included bibliographic and digital resources. Additionally, records and recollections by various individuals were used, including some who were directly involved. RESULTS: Endourology was born in Canada when SWL, URS, and PCNL emerged as minimally invasive treatment options for stones in the early to mid-1980s. According to our research, the first PCNL was performed at the University of Toronto in 1981. Dr. Joachim Burhenne, a Harvard-trained radiologist from Germany, first used extracorporeal SWL in Canada at the University of British Columbia (UBC) for the treatment of biliary stones. Treatment for urinary tract stones followed at UBC and Dalhousie University. The first worldwide use of the holmium laser for lithotripsy of urinary tract calculi took place at the University of Western Ontario. Other endourology milestones in Canada include the formation of the Canadian Endourology Group and the emergence of the Endourological Society-accredited fellowship programs at the University of Toronto and Western University in the 1990s. Canada hosted the 21st and 35th World Congress of Endourology and Shock Wave Lithotripsy annual meeting in Montreal and Vancouver, respectively. CONCLUSIONS: Canadian urologists have led many advances in SWL, URS, and PCNL over the past four decades and, for a relatively small community, have made significant contributions to the field. Through the training of the next generation of endourologists at Canadian institutions, the future of endourology in Canada is bright.

Differentially Marked IncP-1ß R751 Plasmids for Cloning via Recombineering and Conjugation.

We demonstrate here for the first time the use of an IncP-1ß plasmid, R751, as a gene capture vehicle for recombineering/conjugation strategies to clone large segments of bacterial genomes (20 - 100 + Kb). We designed R751 derivatives containing alternative markers for greater flexibility when using the R751 vehicle across different bacteria. These markers are removable if desired as part of the cloning procedure (with no extra steps needed). We demonstrated utility via cloning of 38 and 22 kb genomic segments from Salmonella enterica serovar Typhimurium and Escherichia coli, respectively. The plasmids expand the options available for use in recombineering/conjugation-based cloning applications.We demonstrate here for the first time the use of an IncP-1ß plasmid, R751, as a gene capture vehicle for recombineering/conjugation strategies to clone large segments of bacterial genomes (20 ­ 100 + Kb). We designed R751 derivatives containing alternative markers for greater flexibility when using the R751 vehicle across different bacteria. These markers are removable if desired as part of the cloning procedure (with no extra steps needed). We demonstrated utility via cloning of 38 and 22 kb genomic segments from Salmonella enterica serovar Typhimurium and Escherichia coli, respectively. The plasmids expand the options available for use in recombineering/conjugation-based cloning applications.

Hypoxia induces rapid changes to histone methylation and reprograms chromatin.

Oxygen is essential for the life of most multicellular organisms. Cells possess enzymes called molecular dioxygenases that depend on oxygen for activity. A subclass of molecular dioxygenases is the histone demethylase enzymes, which are characterized by the presence of a Jumanji-C (JmjC) domain. Hypoxia can alter chromatin, but whether this is a direct effect on JmjC-histone demethylases or due to other mechanisms is unknown. Here, we report that hypoxia induces a rapid and hypoxia-inducible factor-independent induction of histone methylation in a range of human cultured cells. Genomic locations of histone-3 lysine-4 trimethylation (H3K4me3) and H3K36me3 after a brief exposure of cultured cells to hypoxia predict the cell's transcriptional response several hours later. We show that inactivation of one of the JmjC-containing enzymes, lysine demethylase 5A (KDM5A), mimics hypoxia-induced cellular responses. These results demonstrate that oxygen sensing by chromatin occurs via JmjC-histone demethylase inhibition.

Tissue engineering toward temporomandibular joint disc regeneration.

Treatments for temporomandibular joint (TMJ) disc thinning and perforation, conditions prevalent in TMJ pathologies, are palliative but not reparative. To address this, scaffold-free tissue-engineered implants were created using allogeneic, passaged costal chondrocytes. A combination of compressive and bioactive stimulation regimens produced implants with mechanical properties akin to those of the native disc. Efficacy in repairing disc thinning was examined in minipigs. Compared to empty controls, treatment with tissue-engineered implants restored disc integrity by inducing 4.4 times more complete defect closure, formed 3.4-fold stiffer repair tissue, and promoted 3.2-fold stiffer intralaminar fusion. The osteoarthritis score (indicative of degenerative changes) of the untreated group was 3.0-fold of the implant-treated group. This tissue engineering strategy paves the way for developing tissue-engineered implants as clinical treatments for TMJ disc thinning.

Transfer and analysis of Salmonella pdu genes in a range of Gram-negative bacteria demonstrate exogenous microcompartment expression across a variety of species.

Bacterial microcompartments (MCPs) are protein organelles that typically house toxic or volatile reaction intermediates involved in metabolic pathways. Engineering bacteria to express exogenous MCPs will allow these cells to gain useful functions involving molecule compartmentalization. We cloned a 38 kb region from the Salmonella enterica serovar Typhimurium genome containing the pdu 1,2 propanediol (1,2 PD) utilization and cob/cbi genes using the FRT-Capture strategy to clone and transfer large genomic segments. We transferred this clone to a range of Gram-negative bacteria and found the clone to be functional for 1,2 PD metabolism in a variety of species including S. Typhimurium Δpdu, Escherichia coli, Salmonella bongori, Klebsiella pneumoniae, Cronobacter sakazakii, Serratia marcescens, and different Pseudomonas species. We successfully isolated MCPs expressed from the clone from several, but not all, of these strains, and we observed this utilizing a range of different media and in the absence of protease inhibitor. We also present a mini-prep protocol that allows rapid, small-scale screening of strains for MCP production. To date, this is the first analysis of cloned, exogenous microcompartment expression across several different Gram-negative backgrounds and provides a foundation for MCP use in a variety of bacterial species using a full, intact clone.

A Survey of Sleep Medicine Physician Perceptions on the Surgical Treatment of Obstructive Sleep Apnea.

PURPOSE: Surgical treatment for obstructive sleep apnea (OSA) varies by specialty. Our survey sought to answer 3 principal questions: 1) To which surgical specialists are sleep physicians referring patients for upper airway surgery? 2) Which surgical treatment do sleep specialists find to be most effective in treating OSA? 3) Do sleep medicine physicians believe that maxillomandibular advancement (MMA) is worthwhile to patients who are surgical candidates? MATERIALS AND METHODS: We formulated a cross-sectional survey. The study sample was obtained by identifying all practices that advertised as sleep medicine specialists in Houston, Texas, by using Internet searches. Physicians who treated children were excluded. Seventy-nine surveys were hand delivered to offices in the greater Houston area; the survey included 6 questions to determine referral and surgical preferences for OSA. Variable responses included years in practice, specialty, and a comments section. A 10-point Likert scale was used to assess sleep medicine physicians' referral patterns and perceptions regarding surgical treatment of OSA. Numerical data were analyzed by calculating mean values and by dividing responses into "disagree" (<5), "neutral" (5), and "agree" (>5). RESULTS: Twenty-six surveys were returned. More sleep medicine physicians referred patients to ear, nose, and throat surgeons (52%) than to oral and maxillofacial surgeons (20%). MMA was viewed as the most effective surgery (72%), followed by "none" (16%), "other" (8%), and uvulopalatopharyngoplasty (4%). More respondents viewed the benefits versus risks as favorable for MMA (44%) than for uvulopalatopharyngoplasty (29%). CONCLUSIONS: The results of this survey show that sleep medicine physicians in the greater Houston area view MMA as the most favorable and effective surgical option for treating OSA. Although MMA was most often referred for, more respondents refer patients to ear, nose, and throat surgeons than to oral and maxillofacial surgeons for surgical management of OSA. Years in practice displayed no correlation in referral patterns or preference for type of OSA surgery.

The Bacterial iprA Gene Is Conserved across Enterobacteriaceae, Is Involved in Oxidative Stress Resistance, and Influences Gene Expression in Salmonella enterica Serovar Typhimurium.

UNLABELLED: The iprA gene (formerly known as yaiV or STM0374) is located in a two-gene operon in the Salmonella enterica serovar Typhimurium genome and is associated with altered expression during spaceflight and rotating-wall-vessel culture conditions that increase virulence. However, iprA is uncharacterized in the literature. In this report, we present the first targeted characterization of this gene, which revealed that iprA is highly conserved across Enterobacteriaceae We found that S Typhimurium, Escherichia coli, and Enterobacter cloacae ΔiprA mutant strains display a multi-log-fold increase in oxidative stress resistance that is complemented using a plasmid-borne wild-type (WT) copy of the S Typhimurium iprA gene. This observation was also associated with increased catalase activity, increased S Typhimurium survival in macrophages, and partial dependence on the katE gene and full dependence on the rpoS gene. Our results indicate that IprA protein activity is sensitive to deletion of the N- and C-terminal 10 amino acids, while a region that includes amino acids 56 to 80 is dispensable for activity. RNA sequencing (RNA-Seq) analysis revealed several genes altered in expression in the S Typhimurium ΔiprA mutant strain compared to the WT, including those involved in fimbria formation, spvABCD-mediated virulence, ethanolamine utilization, the phosphotransferase system (PTS) transport, and flagellin phase switching from FlgB to FliC (likely a stochastic event) and several genes of hypothetical or putative function. IMPORTANCE: Overall, this work reveals that the conserved iprA gene measurably influences bacterial biology and highlights the pool of currently uncharacterized genes that are conserved across bacterial genomes. These genes represent potentially useful targets for bacterial engineering, vaccine design, and other possible applications.

Transfer of the cloned Salmonella SPI-1 type III secretion system and characterization of its expression mechanisms in Gram negative bacteria in comparison with cloned SPI-2.

Cloned type III secretion systems have much potential to be used for bacterial engineering purposes involving protein secretion and substrate translocation directly into eukaryotic cells. We have previously cloned the SPI-1 and SPI-2 type III systems from the Salmonella enterica serovar Typhimurium genome using plasmid R995 which can conveniently capture large genomic segments for transfer between bacterial strains. However, though expressed and functional in Salmonella strains, cloned SPI-1 was previously observed to have a serious expression defect in other Gram negative bacteria including Escherichia coli. Here we show that cloned SPI-1 expression and secretion can be detected in the secretion preps from E. coli and Citrobacter indicating the first observation of non-Salmonella SPI-1 expression. We describe a compatible plasmid system to introduce engineered SPI-1 substrates into cloned SPI-1 strains. However, a SPI-1 translocation defect is still observed in E. coli, and we show that this is likely due to a defect in SipB expression/secretion in this species. In addition, we also examined the requirement for the hilA and ssrAB regulators in the expression of cloned SPI-1 and SPI-2, respectively. We found a strict requirement for hilA for full cloned SPI-1 expression and secretion. However, though we found that ssrAB is required for full cloned SPI-2 expression in a range of media across different bacteria, it is not required for cloned SPI-2 expression in MgM8 inducing media in S. Typhimurium. This suggests that under SPI-2 inducing conditions in S. Typhimurium, other factors can substitute for loss of ssrAB in cloned SPI-2 expression. The results provide key foundational information for the future use of these cloned systems in bacteria.