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

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

Characterization of the public transit air microbiome and resistome reveals geographical specificity

BackgroundThe public transit is a built environment with high occupant density across the globe, and identifying factors shaping public transit air microbiomes will help design strategies to minimize the transmission of pathogens. However, the majority of microbiome works dedicated to the public transit air are limited to amplicon sequencing, and our knowledge regarding the functional potentials and the repertoire of resistance genes (i.e. resistome) is limited. Furthermore, current air microbiome investigations on public transit systems are focused on single cities, and a multi-city assessment of the public transit air microbiome will allow a greater understanding of whether and how broad environmental, building, and anthropogenic factors shape the public transit air microbiome in an international scale. Therefore, in this study, the public transit air microbiomes and resistomes of six cities across three continents (Denver, Hong Kong, London, New York City, Oslo, Stockholm) were characterized.ResultsCity was the sole factor associated with public transit air microbiome differences, with diverse taxa identified as drivers for geography-associated functional potentials, concomitant with geographical differences in species- and strain-level inferred growth profiles. Related bacterial strains differed among cities in genes encoding resistance, transposase, and other functions. Sourcetracking estimated that human skin, soil, and wastewater were major presumptive resistome sources of public transit air, and adjacent public transit surfaces may also be considered presumptive sources. Large proportions of detected resistance genes were co-located with mobile genetic elements including plasmids. Biosynthetic gene clusters and city-unique coding sequences were found in the metagenome-assembled genomes.ConclusionsOverall, geographical specificity transcends multiple aspects of the public transit air microbiome, and future efforts on a global scale are warranted to increase our understanding of factors shaping the microbiome of this unique built environment

Evaluation of Bacteria and Fungi DNA Abundance in Human Tissues

Whereas targeted and shotgun sequencing approaches are both powerful in allowing the study of tissue-associated microbiota, the human: microorganism abundance ratios in tissues of interest will ultimately determine the most suitable sequencing approach. In addition, it is possible that the knowledge of the relative abundance of bacteria and fungi during a treatment course or in pathological conditions can be relevant in many medical conditions. Here, we present a qPCR-targeted approach to determine the absolute and relative amounts of bacteria and fungi and demonstrate their relative DNA abundance in nine different human tissue types for a total of 87 samples. In these tissues, fungi genomes are more abundant in stool and skin samples but have much lower levels in other tissues. Bacteria genomes prevail in stool, skin, oral swabs, saliva, and gastric fluids. These findings were confirmed by shotgun sequencing for stool and gastric fluids. This approach may contribute to a more comprehensive view of the human microbiota in targeted studies for assessing the abundance levels of microorganisms during disease treatment/progression and to indicate the most informative methods for studying microbial composition (shotgun versus targeted sequencing) for various samples types

The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium inaugural meeting report

The Metagenomics and Metadesign of the Subways and Urban Biomes (MetaSUB) International Consortium is a novel, interdisciplinary initiative comprised of experts across many fields, including genomics, data analysis, engineering, public health, and architecture. The ultimate goal of the MetaSUB Consortium is to improve city utilization and planning through the detection, measurement, and design of metagenomics within urban environments. Although continual measures occur for temperature, air pressure, weather, and human activity, including longitudinal, cross-kingdom ecosystem dynamics can alter and improve the design of cities. The MetaSUB Consortium is aiding these efforts by developing and testing metagenomic methods and standards, including optimized methods for sample collection, DNA/RNA isolation, taxa characterization, and data visualization. The data produced by the consortium can aid city planners, public health officials, and architectural designers. In addition, the study will continue to lead to the discovery of new species, global maps of antimicrobial resistance (AMR) markers, and novel biosynthetic gene clusters (BGCs). Finally, we note that engineered metagenomic ecosystems can help enable more responsive, safer, and quantified cities

Transcriptional and Post-Transcriptional Regulation of the Escherichia coli luxS mRNA; Involvement of the sRNA MicA

Background: The small RNA (sRNA) MicA has been shown to post-transcriptionally regulate translation of the outer membrane protein A (OmpA) in Escherichia coli. It uses an antisense mechanism to down-regulate OmpA protein synthesis and induce mRNA degradation. MicA is genomically localized between the coding regions of the gshA and luxS genes and is divergently transcribed from its neighbours. Transcription of the luxS gene which originates within or upstream of the MicA sequence would thus be complementary to the sRNA. LuxS regulation is as yet unclear. Methodology/Principal Findings: In this report, I show that the luxS mRNA exists as three long (major) transcripts of sizes that suggest just such interaction. The sRNA MicA’s expression affects the abundance of each of these luxS transcripts. The involvement of the ribonuclease, RNase III in the accumulation of the shortest transcript is demonstrated. When MicA accumulates during growth, or is induced to be over-expressed, the cleaved mRNA species is observed to increase in intensity. Using primer extension and 59-RACE experiments in combination with sRNA overexpression plasmids, I identify the exact origin of two of the three luxS transcripts, one of which is seen to result from a previously unidentified s S dependent promoter. Conclusions/Significance: The presented data provides strong evidence that MicA functions in cis and in trans, targeting both luxS mRNA as well as the previously established ompA and phoP regulation. The proposed luxS regulation by MicA would be in tandem with another sRNA CyaR, shown recently to be involved in inhibiting translation of the luxS mRNA. Regulation of luxS expression is additionally shown to occur on a transcriptional level via s S with variable transcript levels in different growth phases unlike what was previously assumed. This is the first known case of an sRNA in E. coli which targets both in cis (luxS mRNA) and in trans (ompA and phoP mRNAs)

Functional characterization of the small antisense RNA MicA in Escherichia coli

The Escherichia coli small RNA (sRNA) MicA was identified recently in a genomewide search for sRNAs. It is encoded between the genes gshA and luxS in E. coli and its close relatives. The function of sRNAs in bacteria is generally believed to be in maintenance of homeostasis via stress-induced modulation of gene expression. Our studies on MicA have been aimed at attributing function(s) to this molecule. We carried out high throughput assays aimed at identifying genes that are differentially regulated upon knocking out or overexpressing MicA. Among the protein candidates identified was the outer membrane protein, OmpA. Subsequent analysis allowed us to show this regulation to be antisense in nature with MicA binding within the translation initiation region of ompA mRNA. Furthermore, blocking the ribosome from loading caused a translational decoupling that instigates degradation of the mRNA. The regulation was apparent in early stationary phase and seen to be dependent on the RNA chaperone Hfq. We went on to characterize the regulation of MicA, looking at its own transcription. Testing various stress conditions, we were able to identify putative promoter elements that we confirmed using transcriptional fusions. The results showed MicA to be dependent on the extracytoplasmic function ECF sigma E (σE) and could not detect MicA in mutants deleted for this factor. Lastly, we identified an additional target for MicA being the adjacently encoded luxS mRNA. The LuxS protein is essential for the synthesis of the quorum sensing AI-2 molecule. Transcription of the luxS mRNA is commences within the gshA gene, on the other side of MicA coding region. We were able to show that MicA interacts with luxS mRNA and is recognized by RNase III which processes this complex leading to a shorter luxS mRNA isoform. The significance of this processing event is as yet undetermined. Our data elucidated a new promoter driving transcription of luxS, and we demonstrated this promoter to be stationary phase responsive. In summary, the work presented here characterizes the sRNA MicA as a dual regulatory sRNA molecule, moonlighting between its cis-encoded target and its trans-encoded target.