Protoplast fusion in Bacillus species produces frequent,unbiased, genome-wide homologous recombination

In eukaryotes, fine-scale maps of meiotic recombination events have greatly advanced our understanding of the factors that affect genomic variation patterns and evolution of traits. However, in bacteria that lack natural systems for sexual reproduction, unbiased characterization of recombination landscapes has remained challenging due to variable rates of genetic exchange and influence of natural selection. Here, to overcome these limitations and to gain a genome-wide view on recombination, we crossed Bacillus strains with different genetic distances using protoplast fusion. The offspring displayed complex inheritance patterns with one of the parents consistently contributing the major part of the chromosome backbone and multiple unselected fragments originating from the second parent. Our results demonstrate that this bias was in part due to the action of restriction–modification systems, whereas genome features like GC content and local nucleotide identity did not affect distribution of recombination events around the chromosome. Furthermore, we found that recombination occurred uniformly across the genome without concentration into hotspots. Notably, our results show that species-level genetic distance did not affect genome-wide recombination. This study provides a new insight into the dynamics of recombination in bacteria and a platform for studying recombination patterns in diverse bacterial species.     

Measuring the unknown: An estimator and simulation study for assessing case reporting during epidemics

The fraction of cases reported, known as ‘reporting’, is a key performance indicator in an outbreak response, and an essential factor to consider when modelling epidemics and assessing their impact on populations. Unfortunately, its estimation is inherently difficult, as it relates to the part of an epidemic which is, by definition, not observed. We introduce a simple statistical method for estimating reporting, initially developed for the response to Ebola in Eastern Democratic Republic of the Congo (DRC), 2018–2020. This approach uses transmission chain data typically gathered through case investigation and contact tracing, and uses the proportion of investigated cases with a known, reported infector as a proxy for reporting. Using simulated epidemics, we study how this method performs for different outbreak sizes and reporting levels. Results suggest that our method has low bias, reasonable precision, and despite sub-optimal coverage, usually provides estimates within close range (5–10%) of the true value. Being fast and simple, this method could be useful for estimating reporting in real-time in settings where person-to-person transmission is the main driver of the epidemic, and where case investigation is routinely performed as part of surveillance and contact tracing activities.     

RELATIONSHIP BETWEEN CELL CYCLE AND LIGHT‐LIMITED GROWTH RATE IN OCEANIC PROCHLOROCOCCUS (MIT9312) AND SYNECHOCOCCUS (WH8103) (CYANOBACTERIA)1

The relationships between growth rate, cell‐cycle parameters, and cell size were examined in two unicellular cyanobacteria representative of open‐ocean environments: Prochlorococcus (strain MIT9312) and Synechococcus (strain WH8103). Chromosome replication time, C, was constrained to a fairly narrow range of values (～4–6 h) in both species and did not appear to vary with growth rate. In contrast, the pre‐ and post‐DNA replication periods, B and D, respectively, decreased with increasing growth rate from maxima of ～30 and 10–20 h to minima of ～4–6 and 2–3 h, respectively. The combined duration of the chromosome replication and postreplication periods (C+D), a quantity often used in the estimation of Prochlorococcus in situ growth rates, varied ～2.4‐fold over the range of growth rates examined. This finding suggests that assumptions of invariant C+D may adversely influence Prochlorococcus growth rate estimates. In both strains, cell mass was the greatest in slowly growing cells and decreased 2‐ to 3‐fold over the range of growth rates examined here. Estimated cell mass at the start of replication appeared to decrease with increasing growth rate, indicating that the initiation of chromosome replication in Prochlorococcus and Synechococcus is not a simple function of cell biomass, as suggested previously. Taken together, our results reflect a notable degree of similarity between oceanic Synechococcus and Prochlorococcus strains with respect to their growth‐rate‐specific cell‐cycle characteristics.     

Diversity of anaerobic gut fungal populations analysed using ribosomal ITS1 sequences in faeces of wild and domesticated herbivores

Gut fungal-specific PCR primers have been used to selectively amplify the ITS1 region of gut fungal rDNA recovered from faeces of domestic and wild animals to investigate population diversity. Two different gel-based methods are described for separating populations of gut fungal rDNA amplicons, namely (1) denaturing gradient gel electrophoresis (DGGE) and (2) separation according to small size differences using Spreadex, a proprietary matrix for electrophoresis. Gut fungal populations were characterised by analysis of rDNA in faeces of seventeen domesticated and ten wild herbivores. Sequences derived from these gel-based characterisations were analysed and classified using a hidden Markov model-based fingerprint matching algorithm. Faecal samples contained a broad spectrum of fungi and sequences from five of the six recognised genera were identified, including Cyllamyces, the most recently described gut fungal genus, which was found to be widely distributed in the samples. Furthermore, four other novel groupings of gut fungal sequences were identified that did not cluster with sequences from any of the previously described genera. Both gel- and sequence- based profiles for gut fungal populations suggested a lack of geographical restriction on occurrence of any individual fungal type.     

Protein kinases display minimal interpositional dependence on substrate sequence: potential implications for the evolution of signalling networks

Characterization of in vitro substrates of protein kinases by peptide library screening provides a wealth of information on the substrate specificity of kinases for amino acids at particular positions relative to the site of phosphorylation, but provides no information concerning interdependence among positions. High-throughput techniques have recently made it feasible to identify large numbers of in vivo kinase substrates. We used data from experiments on the kinases ATM/ATR and CDK1, and curated CK2 substrates to evaluate the prevalence of interactions between substrate positions within a motif and the utility of these interactions in predicting kinase substrates. Among these data, evidence of interpositional sequence dependencies is strikingly rare, and what dependency exists does little to aid in the prediction of novel kinase substrates. Significant increases in the ability of models to predict kinase-substrate specificity beyond position-independent models must come largely from inclusion of elements of biological and cellular context, rather than further analysis of substrate sequences alone. Our results suggest that, evolutionarily, kinase substrate fitness exists in a smooth energetic landscape. Taken with results from others indicating that phosphopeptide-binding domains do exhibit interpositional dependence, our data suggest that incorporation of new substrate molecules into phospho-signalling networks may be rate-limited by the evolution of suitability for binding by phosphopeptide-binding domains.     

Biology of the Galapagos shark,Carcharhinus galapagensis, in Hawai'i

Synopsis Catch records from the Hawai'i Cooperative Shark Research and Control Program, which operated in Hawai'i from 1967–1969, were examined and data on the Galapagos shark,Carcharhinus galapagensis were analyzed. A total of 304 Galapagos sharks was caught, predominantly with longlines. More female sharks were caught than males, and the catch was skewed geographically. On the island of O'ahu the highest catch rates occurred along the north and south coasts. High catch rates also occurred near points of land, where longshore currents converge. Average depth of capture was greater for juveniles (45.1 m) and mature males (60.2 m), than for subadults (38.8 m) and mature female sharks (34.2 m). Males appear to reach maturity between 205 and 239 cm total length, and females between 215 and 245 cm. Litter size ranged from 4 to 16 pups, with an average of 8.7. In Hawaiian waters Galapagos sharks are born at just over 80 cm total length. Mating and parturition apparently occur early in the year, and gestation is estimated to be about 12 months. Stomach contents consisted mainly of teleosts and benthic prey, and ontogenetic changes in diet occurred as sharks increased in size. Sharks consumed a smaller proportion of teleosts and more elasmobranchs with increasing size. Dietary diversity also increased with increasing size of shark.     

Phosphorylation on tyrosine-15 of p34(Cdc2) by ErbB2 inhibits p34(Cdc2) activation and is involved in resistance to taxol-induced apoptosis

ErbB2 overexpression confers resistance to taxol-induced apoptosis by inhibiting p34(Cdc2) activation. One mechanism is via ErbB2-mediated upregulation of p21(Cip1), which inhibits Cdc2. Here, we report that the inhibitory phosphorylation on Cdc2 tyrosine (Y)15 (Cdc2-Y15-p) is elevated in ErbB2-overexpressing breast cancer cells and primary tumors. ErbB2 binds to and colocalizes with cyclin B-Cdc2 complexes and phosphorylates Cdc2-Y15. The ErbB2 kinase domain is sufficient to directly phosphorylate Cdc2-Y15. Increased Cdc2-Y15-p in ErbB2-overexpressing cells corresponds with delayed M phase entry. Expressing a nonphosphorylatable mutant of Cdc2 renders cells more sensitive to taxol-induced apoptosis. Thus, ErbB2 membrane RTK can confer resistance to taxol-induced apoptosis by directly phosphorylating Cdc2.     

Simple isolation of functional RNA from woody stems of gymnosperms

Stems of woody conifers contain high levels of polysaccharides and phenolic compounds that complicate the isolation of functional RNA from this highly lignified tissue. These difficulties were overcome by pulverizing the frozen tissue with a stainless-steel mortar and by effectively sequestering interfering phenolic compounds with vinylpyrrolidone polymers, thereby minimizing damage to nucleic acids during extraction. RNases were inhibited by aurintricarboxylic acid, and gelatinous polysaccharides were removed by inclusion of a 10% ethanol precipitation step. RNA was then isolated by precipitation with 33% isopropanol and ultracentrifugation onto a cushion of 5.7 M CsCl. Yields of 10 to 20 μg RNA/g FW were obtained from woody stems of several different gymnosperm species, including grand fir (Abies grandis), lodgepole pine (Pinus contorta), loblolly pine (Pinus taeda), Douglas fir (Pseudotsuga menziesii) and Pacific yew (Taxus brevifolia). The high quality of the RNA obtained was determined by UV-spectrophotometry, denaturing agarose gel electrophoresis, andin-vitro translation, and this material was used to construct cDNA libraries.     

A 96-well DNase I footprinting screen for drug–DNA interactions

The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive ‘one compound–one line’ scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.     

Ubiquitin‐specific protease‐like 1 (USPL1) is a SUMO isopeptidase with essential,non‐catalytic functions

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity‐based search with the suicide inhibitor haemagglutinin (HA)‐SUMO‐vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin‐specific protease‐like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l—an essential but distant zebrafish homologue of USPL1—also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low‐abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.