Maltose uptake and its regulation in Bacillus subtilis

Extracts prepared from cultures of Bacillus subtilis, grown on maltose as the sole carbon source, lacked maltose phosphotransferase system activity. There was, however, evidence for a maltose phosphorylase activity, and such extracts also possessed both glucokinase and glucose phosphotransferase system activities. Maltose was accumulated by whole cells of B. subtilis by an energy-dependent mechanism. This uptake was sensitive to the effects of uncouplers, suggesting a role for the proton-motive force in maltose transport. Accumulation of maltose was inhibited in the presence of glucose, and there was no accumulation of maltose by a strain carrying the ptsI6 null-mutation. A strain carrying the temperature-sensitive ptsI1 mutation accumulated maltose normally at 37 degrees C but, in contrast to the wild-type, was devoid of maltose transport activity at 47 degrees C. The results indicate a role for the phosphotransferase system in the regulation of maltose transport activity in this organism.     

Probing the stability of the SpCas9–DNA complex after cleavage

CRISPR–Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.     

Observed Antibody Space: A diverse database of cleaned,annotated, and translated unpaired and paired antibody sequences

The antibody repertoires of individuals and groups have been used to explore disease states, understand vaccine responses, and drive therapeutic development. The arrival of B‐cell receptor repertoire sequencing has enabled researchers to get a snapshot of these antibody repertoires, and as more data are generated, increasingly in‐depth studies are possible. However, most publicly available data only exist as raw FASTQ files, making the data hard to access, process, and compare. The Observed Antibody Space (OAS) database was created in 2018 to offer clean, annotated, and translated repertoire data. In this paper, we describe an update to OAS that has been driven by the increasing volume of data and the appearance of paired (VH/VL) sequence data. OAS is now accessible via a new web server, with standardized search parameters and a new sequence‐based search option. The new database provides both nucleotides and amino acids for every sequence, with additional sequence annotations to make the data Minimal Information about Adaptive Immune Receptor Repertoire compliant, and comments on potential problems with the sequence. OAS now contains 25 new studies, including severe acute respiratory syndrome coronavirus 2 data and paired sequencing data. The new database is accessible at http://opig.stats.ox.ac.uk/webapps/oas/, and all data are freely available for download.     

Correlation of probiotic Lactobacillus salivarius growth phase with its cell wall-associated proteome

Lactobacillus salivarius subsp. salivarius UCC118 is a probiotic bacterium that was originally isolated from human intestinal tissues and was subsequently shown in a pilot study to alleviate symptoms associated with mild-moderate Crohn's disease. Strain UCC118 can adhere to animal and human intestinal tissue, and to both healthy and inflamed ulcerative colitis mucosa, irrespective of location in the gut. In this study, an enzymatic technique has been combined with proteomic analysis to correlate bacterial growth phase with the presence of factors present in the cell wall of the bacterium. Using PAGE electrophoresis, it was determined that progression from lag to log to stationary growth phases in vitro correlated with increasing prominence of an 84kD protein associated with in vitro adherence ability. Isolated proteins from the 84kD band region were further separated by two-dimensional electrophoresis, resolving this band into 20 individual protein spots at differing isoelectric points. The protein moieties were excised, trypsin digested and subjected to tandem mass spectrometry. The observed proteins are analogous to those reported to be associated with the Listeria monocytogenes cell-wall proteome, and include DnaK, Ef-Ts and pyruvate kinase. These data suggest that at least some of the beneficial attributes of probiotic lactobacilli, and in particular this strain, may be due to nonpathogenic mimicry of pathogens and potentially be mediated through a form of attenuated virulence.     

Crystal protein synthesis is dependent on early sporulation gene expression in Bacillus sphaericus

Insertional mutations in the spo0A and spoIIAC genes of Bacillus sphaericus 2362 were prepared by conjugation with Escherichia coli using a suicide plasmid containing cloned portions of the target genes. The mutants resembled their Bacillus subtilis counterparts phenotypically and were devoid of crystal proteins as determined by electron microscopy, SDS-PAGE and Western blots. The mutants had greatly reduced toxicity to anopheline mosquito larvae compared to the parental strain. We conclude that crystal protein synthesis in this bacterium is dependent on expression of early sporulation genes.     

Oospore production of Pythium artotrogus

The addition of cholesterol, b-sitosterol, stigmasterol and compost infusion to corn meal agar induced marked increases (four-fold) in oospore production, with only a small stimulation of growth rate. Progesterone stimulated oospore production, but inhibited growth. Estrone had no effect on reproduction, but inhibited growth. Testosterone greatly inhibited both growth and reproduction. The optimum concentration of cholesterol for maximum oospore production was 20–100 mg per liter. Growth and oospore production occurred over the initial pH range of 4.5–8.5, and over the temperature range of 12–38°C. Growth occurred at 9°, but no oospores formed. Cholesterol increased the temperature tolerance of the mycelium. Nystatin had no effect on growth or reproduction at 15 mg per liter; filipin and fungichromin completely prevented, and amphotericin B strongly inhibited oospore production at this concentration. Fungichromin was the most inhibitory to growth, filipin was intermediate and amphotericin B only slightly inhibitory. Cholesterol (15 mg/liter) annulled the growth inhibition, but not the inhibition of reproduction. Fluorodeoxyuridine and bromodeoxyuridine inhibited growth and oospore production. Deoxyadenosine had no effect. These experiments indicate that the metabolic processes involved in mycelium synthesis are markedly different from the processes involved in sexual reproduction since they were affected differently by the sterols, antibiotics, and the thymidine analogues.

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Zusammenfassung Der Zusatz von Cholesterol, beta Sitosterol, Stigmasterol und Kompost Infusion zu Maismehlagar verursachte eine vierfache Zunahme der Oosporenbildung, mit nur einer geringen Stimulierung des Wachstumanteiles. Progesterone stimulierte die Oosporenbildung, verhinderte jedoch das Wachstum. Estrone hat keine Wirkung an Reproduktion, aber verhinderte das Wachstum. Testosterone verhinderte wesentlich sowohl das Wachstum als auch die Reproduktion. Die optimale Konzentration von Cholesterol war für die höchste Oosporenbildung 20–100 mg pro Liter. Wachstum und Oosporenbildung erfolgte über die ursprüngliche pH-Weite von 4.5–8.5, und über die Temperaturweite von 12°–38°C. Wachstum erfolgte bei 9°C, aber keine Oosporenbildung. Cholesterol vergrößerte die Temperaturtoleranz des Myceliums. Nystatin hat keinen Einfluß an Wachstum oder Reproduktion bei 15 mg pro Liter: Filipin und Fungichromin verhinderte völlig und Amphotericin B sehr stark die Produktion bei dieser Konzentration. Fungichromin B Zeigte nur eine geringe Wachstum, Filipin war in der Mitte und Amphotericin B zeigte nur eine geringe Verhinderung. Cholesterol (15 mg/liter) vernichtete die Wachstumverhinderung jedoch nicht die Reproduktion. Fluorodeoxyuridine und Bromodeoxyuridine verhinderten Wachstum und Oosporenbildung. Deoxyadenosine hatte keine Wirkung. Diese Versuche zeigen, daß die metabolischen Prozesse, die in der Synthese des Myzeliums mitwirken, wesentlich verschieden von den Prozessen sind, die in Sexual-reproduktion mitwirken, da sie verschiedentlich von Sterols, Antibiotiks und von den Thymidine-analogen beeinflußt worden sind.

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Contribution No. 26 from the Botany Section, The Department of Biology.     

Depth-Dependent Differences in Community Structure of the Human Colonic Microbiota in Health

Objective

The aims of this study were to develop techniques for spatial microbial assessment in humans and to establish colonic luminal and mucosal spatial ecology, encompassing longitudinal and cross-sectional axes.

Design

A microbiological protected specimen brush was used in conjunction with a biopsy forceps to sample the colon in nine healthy volunteers undergoing colonoscopy. Terminal Restriction Fragment Length Polymorphism analysis was used to determine the major variables in the spatial organization of the colonic microbiota.

Results

Protected Specimen Brush sampling retrieved region-specific, uncontaminated samples that were enriched for bacterial DNA and depleted in human DNA when compared to biopsy samples. Terminal Restriction Fragment Length Polymorphism analysis revealed a segmentation of bacterial communities between the luminal brush and biopsy-associated ecological niches with little variability across the longitudinal axis of the colon and reduced diversity in brush samples.

Conclusion

These results support the concept of a microbiota with little longitudinal variability but with some degree of segregation between luminal and mucosal communities.     

Prediction of Preterm Deliveries from EHG Signals Using Machine Learning

There has been some improvement in the treatment of preterm infants, which has helped to increase their chance of survival. However, the rate of premature births is still globally increasing. As a result, this group of infants are most at risk of developing severe medical conditions that can affect the respiratory, gastrointestinal, immune, central nervous, auditory and visual systems. In extreme cases, this can also lead to long-term conditions, such as cerebral palsy, mental retardation, learning difficulties, including poor health and growth. In the US alone, the societal and economic cost of preterm births, in 2005, was estimated to be $26.2 billion, per annum. In the UK, this value was close to £2.95 billion, in 2009. Many believe that a better understanding of why preterm births occur, and a strategic focus on prevention, will help to improve the health of children and reduce healthcare costs. At present, most methods of preterm birth prediction are subjective. However, a strong body of evidence suggests the analysis of uterine electrical signals (Electrohysterography), could provide a viable way of diagnosing true labour and predict preterm deliveries. Most Electrohysterography studies focus on true labour detection during the final seven days, before labour. The challenge is to utilise Electrohysterography techniques to predict preterm delivery earlier in the pregnancy. This paper explores this idea further and presents a supervised machine learning approach that classifies term and preterm records, using an open source dataset containing 300 records (38 preterm and 262 term). The synthetic minority oversampling technique is used to oversample the minority preterm class, and cross validation techniques, are used to evaluate the dataset against other similar studies. Our approach shows an improvement on existing studies with 96% sensitivity, 90% specificity, and a 95% area under the curve value with 8% global error using the polynomial classifier.