Structural elucidation of the O-antigen polysaccharide from Escherichia coli O125ac and biosynthetic aspects thereof

Enteropathogenic Escherichia coli O125, the cause of infectious diarrheal disease, is comprised of two serogroups, viz., O125ab and O125ac, which display the aggregative adherence pattern with epithelial cells. Herein, the structure of the O-antigen polysaccharide from E. coli O125ac:H6 has been elucidated. Sugar analysis revealed the presence of fucose, mannose, galactose and N-acetyl-galactosamine as major components. Unassigned H-1 and C-13 NMR data from one- and two-dimensional NMR experiments of the O125ac O-antigen in conjunction with sugar components were used as input to the CASPER program, which can determine polysaccharide structure in a fully automated way, and resulted in the following branched pentasaccharide structure of the repeating unit: -> 4)[beta-d-Galp-(1 -> 3)]-beta-d-GalpNAc-(1 -> 2)-alpha-d-Manp-(1 -> 3)-alpha-l-Fucp-(1 -> 3)-alpha-d-GalpNAc-(1 ->, where the side chain is denoted by square brackets. The proposed O-antigen structure was confirmed by H-1 and C-13 NMR chemical shift assignments and determination of interresidue connectivities. Based on this structure, that of the O125ab O-antigen, which consists of hexasaccharide repeating units with an additional glucosyl group, was possible to establish in a semi-automated fashion by CASPER. The putative existence of gnu and gne in the gene clusters of the O125 serogroups is manifested by N-acetyl-d-galactosamine residues as the initial sugar residue of the biological repeating unit as well as within the repeating unit. The close similarity between O-antigen structures is consistent with the presence of two subgroups in the E. coli O125 serogroup

Sigma E controls biogenesis of the antisense RNA MicA

Adaptation stress responses in the Gram-negative bacterium Escherichia coli and its relatives involve a growing list of small regulatory RNAs (sRNAs). Previous work by us and others showed that the antisense RNA MicA downregulates the synthesis of the outer membrane protein OmpA upon entry into stationary phase. This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay. In this article, we investigate the transcriptional regulation of the micA gene. Induction of MicA is dependent on the alarmone ppGpp, suggestive of alternative σ factor involvement, yet MicA accumulates in the absence of the general stress/stationary phase σ(S). We identified stress conditions that induce high MicA levels even during exponential growth—a phase in which MicA levels are low (ethanol, hyperosmolarity and heat shock). Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor σ(E) is activated. The strict dependence of micA transcription on σ(E) is supported by three observations. Induced overexpression of σ(E) increases micA transcription, an ΔrpoE mutant displays undetectable MicA levels and the micA promoter has the consensus σ(E) signature. Thus, MicA is part of the σ(E) regulon and downregulates its target gene, ompA, probably to alleviate membrane stress

Diet change affects intestinal microbiota restoration and improves vertical sleeve gastrectomy outcome in diet-induced obese rats

Purpose: Obesity, a worldwide health problem, is linked to an abnormal gut microbiota and is currently most efectively treated by bariatric surgery. Our aim was to characterize the microbiota of high-fat fed Sprague-Dawley rats when subjected to bariatric surgery (i.e., vertical sleeve gastrectomy) and posterior refeeding with either a high-fat or control diet. We hypothesized that bariatric surgery followed by the control diet was more efective in reverting the microbiota modifcations caused by the high-fat diet when compared to either of the two factors alone. Methods: Using next-generation sequencing of ribosomal RNA amplicons, we analyzed and compared the composition of the cecal microbiota after vertical sleeve gastrectomy with control groups representing non-operated rats, control fed, high-fat fed, and post-operative diet-switched animals. Rats were fed either a high-fat or control low-fat diet and were separated into three comparison groups after eight weeks comprising no surgery, sham surgery, and vertical sleeve gastrectomy. Half of the rats were then moved from the HFD to the control diet. Using next-generation sequencing of ribosomal RNA amplicons, we analyzed the composition of the cecal microbiota of rats allocated to the vertical sleeve gastrectomy group and compared it to that of the non-surgical, control fed, high-fat fed, and post-operative diet-switched groups. Additionally, we correlated diferent biological parameters with the genera exhibiting the highest variation in abundance between the groups. Results: The high-fat diet was the strongest driver of altered taxonomic composition, relative microbial abundance, and diversity in the cecum. These efects were partially reversed in the diet-switched cohort, especially when combined with sleeve gastrectomy, resulting in increased diversity and shifting relative abundances. Several highly-afected genera were correlated with obesity-related parameters. Conclusions: The dysbiotic state caused by high-fat diet was improved by the change to the lower fat, higher fber control diet. Bariatric surgery contributed signifcantly and additively to the diet in restoring microbiome diversity and complexity. These results highlight the importance of dietary intervention following bariatric surgery for improved restoration of cecal diversity, as neither surgery nor change of diet alone had the same efects as when combined

Elucidation of the O-antigen structure of Escherichia coli O93 and characterization of its biosynthetic genes

The structure of the O-antigen from the international reference strain Escherichia coli O93:-:H16 has been determined. A nonrandom modal chain-length distribution was observed for the lipopolysaccharide, a pattern which is typical when long O-specific polysaccharides are expressed. By a combination of (i) bioinformatics information on the gene cluster related to O-antigen synthesis including putative function on glycosyl transferases, (ii) the magnitude of NMR coupling constants of anomeric protons, and (iii) unassigned 2D H-1, C-13-HSQC, and H-1,H-1-TOCSY NMR spectra it was possible to efficiently elucidate the structure of the carbohydrate polymer in an automated fashion using the computer program CASPER. The polysaccharide also carries O-acetyl groups and their locations were determined by 2D NMR experiments showing that similar to 1/2 of the population was 2,6-di-O-acetylated, similar to 1/4 was 2-O-acetylated, whereas similar to 1/4 did not carry O-acetyl group(s) in the 3-O-substituted mannosyl residue of the repeating unit. The structure of the tetrasaccharide repeating unit of the O-antigen is given by: -> 2)-beta-D-Manp-(1 -> 3)-beta-D-Manp2Ac6Ac-(1 -> 4)-beta-D-GlcpA-(1 -> 3)-alpha-D-GlcpNAc-(1 ->, which should also be the biological repeating unit and it shares structural elements with capsular polysaccharides from E. coli K84 and K50. The structure of the acidic O-specific polysaccharide from Cellulophaga baltica strain NN015840(T) differs to that of the O-antigen from E. coli O93 by lacking the O-acetyl group at O6 of the O-acetylated mannosyl residue

Target prediction and a statistical sampling algorithm for RNA-RNA interaction

It has been proven that the accessibility of the target sites has a critical influence for miRNA and siRNA. In this paper, we present a program, rip2.0, not only the energetically most favorable targets site based on the hybrid-probability, but also a statistical sampling structure to illustrate the statistical characterization and representation of the Boltzmann ensemble of RNA-RNA interaction structures. The outputs are retrieved via backtracing an improved dynamic programming solution for the partition function based on the approach of Huang et al. (Bioinformatics). The $O(N^6)$ time and $O(N^4)$ space algorithm is implemented in C (available from \url{http://www.combinatorics.cn/cbpc/rip2.html})Comment: 7 pages, 10 figure

Detection and isolation of airborne SARS-CoV-2 in a hospital setting

Transmission mechanisms for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are incompletely understood. In particular, aerosol transmission remains unclear, with viral detection in air and demonstration of its infection potential being actively investigated. To this end, we employed a novel electrostatic collector to sample air from rooms occupied by COVID-19 patients in a major Swedish hospital. Electrostatic air sampling in conjunction with extraction-free, reverse-transcriptase polymerase chain reaction (hid-RT-PCR) enabled detection of SARS-CoV-2 in air from patient rooms (9/22; 41%) and adjoining anterooms (10/22; 45%). Detection with hid-RT-PCR was concomitant with viral RNA presence on the surface of exhaust ventilation channels in patients and anterooms more than 2 m from the COVID-19 patient. Importantly, it was possible to detect active SARS-CoV-2 particles from room air, with a total of 496 plaque-forming units (PFUs) being isolated, establishing the presence of infectious, airborne SARS-CoV-2 in rooms occupied by COVID-19 patients. Our results support circulation of SARS-CoV-2 via aerosols and urge the revision of existing infection control frameworks to include airborne transmission

A new Vibrio cholerae sRNA modulates colonization and affects release of outer membrane vesicles

We discovered a new small non-coding RNA (sRNA) gene, vrrA of Vibrio cholerae O1 strain A1552. A vrrA mutant overproduces OmpA porin, and we demonstrate that the 140 nt VrrA RNA represses ompA translation by base-pairing with the 5′ region of the mRNA. The RNA chaperone Hfq is not stringently required for VrrA action, but expression of the vrrA gene requires the membrane stress sigma factor, σE, suggesting that VrrA acts on ompA in response to periplasmic protein folding stress. We also observed that OmpA levels inversely correlated with the number of outer membrane vesicles (OMVs), and that VrrA increased OMV production comparable to loss of OmpA. VrrA is the first sRNA known to control OMV formation. Moreover, a vrrA mutant showed a fivefold increased ability to colonize the intestines of infant mice as compared with the wild type. There was increased expression of the main colonization factor of V. cholerae, the toxin co-regulated pili, in the vrrA mutant as monitored by immunoblot detection of the TcpA protein. VrrA overproduction caused a distinct reduction in the TcpA protein level. Our findings suggest that VrrA contributes to bacterial fitness in certain stressful environments, and modulates infection of the host intestinal tract

Translational activation of rpoS mRNA by the non-coding RNA DsrA and Hfq does not require ribosome binding

At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, σS, involves the small regulatory RNA (sRNA) DsrA and the RNA chaperone Hfq. The Hfq-mediated DsrA-rpoS interaction relieves an intramolecular secondary structure that impedes ribosome access to the rpoS ribosome binding site. In addition, DsrA/rpoS duplex formation creates an RNase III cleavage site within the duplex. Previous biochemical studies suggested that DsrA and Hfq associate with the 30S ribosomal subunit protein S1, which implied a role for the ribosome in sRNA-mediated post-transcriptional regulation. Here, we show by ribosome profiling that Hfq partitions with the cytoplasmic fraction rather than with 30S subunits. Besides, by employing immunological techniques, no evidence for a physical interaction between Hfq and S1 was obtained. Similarly, in vitro studies did not reveal a direct interaction between DsrA and S1. By employing a ribosome binding deficient rpoS mRNA, and by using the RNase III clevage in the DsrA/rpoS duplex as a diagnostic marker, we provide in vivo evidence that the Hfq-mediated DsrA/rpoS interaction, and consequently the structural changes in rpoS mRNA precede ribosome binding. These data suggest a simple mechanistic model in which translational activation by DsrA provides a translationally competent rpoS mRNA to which 30S subunits can readily bind

Characterization of the role of ribonucleases in Salmonella small RNA decay

In pathogenic bacteria, a large number of sRNAs coordinate adaptation to stress and expression of virulence genes. To better understand the turnover of regulatory sRNAs in the model pathogen, Salmonella typhimurium, we have constructed mutants for several ribonucleases (RNase E, RNase G, RNase III, PNPase) and Poly(A) Polymerase I. The expression profiles of four sRNAs conserved among many enterobacteria, CsrB, CsrC, MicA and SraL, were analysed and the processing and stability of these sRNAs was studied in the constructed strains. The degradosome was a common feature involved in the turnover of these four sRNAs. PAPI-mediated polyadenylation was the major factor governing SraL degradation. RNase III was revealed to strongly affect MicA decay. PNPase was shown to be important in the decay of these four sRNAs. The stability of CsrB and CsrC seemed to be independent of the RNA chaperone, Hfq, whereas the decay of SraL and MicA was Hfq-dependent. Taken together, the results of this study provide initial insight into the mechanisms of sRNA decay in Salmonella, and indicate specific contributions of the RNA decay machinery components to the turnover of individual sRNAs