The Alarmone (p)ppGpp Regulates Primer Extension by Bacterial Primase

Primase is an essential component of the DNA replication machinery, responsible for synthesizing RNA primers that initiate leading and lagging strand DNA synthesis. Bacterial primase activity can be regulated by the starvation-inducible nucleotide (p)ppGpp. This regulation contributes to a timely inhibition of DNA replication upon amino acid starvation in the Gram-positive bacterium Bacillus subtilis. Here, we characterize the effect of (p)ppGpp on B. subtilis DnaG primase activity in vitro. Using a single-nucleotide resolution primase assay, we dissected the effect of ppGpp on the initiation, extension, and fidelity of B. subtilis primase. We found that ppGpp has a mild effect on initiation, but strongly inhibits primer extension and reduces primase processivity, promoting termination of primer extension. High (p)ppGpp concentration, together with low GTP concentration, additively inhibit primase activity. This explains the strong inhibition of replication elongation during starvation which induces high levels of (p)ppGpp and depletion of GTP in B. subtilis. Finally, we found that lowering GTP concentration results in mismatches in primer base pairing that allow priming readthrough, and that ppGpp reduces readthrough to protect priming fidelity. These results highlight the importance of (p)ppGpp in protecting replisome integrity and genome stability in fluctuating nucleotide concentrations upon onset of environmental stress.     

Association of the colistin resistance gene mcr-1 with faecal pollution in water environments in Hanoi,Vietnam

Colistin is one of the antibiotics of last resort for human health. However, the dissemination of the plasmid-mediated colistin resistance gene mcr-1 is of great concern globally. In the One Health framework, the environment is an important component for managing antimicrobial resistance. However, little information is available concerning the prevalence of mcr-1 in water environments. We aimed to reveal the prevalence of mcr-1 in different water environments in Hanoi, Vietnam. Quantitative PCR was applied to detect mcr-1 in four urban drainages receiving untreated domestic wastewater, three rivers, five lakes and two groundwater samples. Urban drainages contained higher concentrations of mcr-1, suggesting that urban residents carry the gene. The class 1 integron-integrase gene was identified as a good surrogate of antibiotic resistance genes including mcr-1. A significant correlation was found between the levels of mcr-1 and the human-specific cross-assembly phage, which is an indicator of human faecal pollution. These results indicated that the primary source of mcr-1 in urban water environments is human faeces, which is consistent with the fact that most domestic wastewater is untreated in Hanoi. The control of untreated wastewater is critical for alleviating the spread of mcr-1 in water environments in Vietnam.     

Targeted amplification and MinION nanopore sequencing of key azole and echinocandin resistance determinants of clinically relevant Candida spp. from blood culture bottles

The objective of the study was to evaluate the use of targeted multiplex Nanopore MinION amplicon re-sequencing of key Candida spp. from blood culture bottles to identify azole and echinocandin resistance associated SNPs. Targeted PCR amplification of azole (ERG11 and ERG3) and echinocandin (FKS) resistance-associated loci was performed on positive blood culture media. Sequencing was performed using MinION nanopore device with R9.4.1 Flow Cells. Twenty-eight spiked blood cultures (ATCC strains and clinical isolates) and 12 prospectively collected positive blood cultures with candidaemia were included. Isolate species included Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Candida auris. SNPs that were identified on ERG and FKS genes using Snippy tool and CLC Genomic Workbench were correlated with phenotypic testing by broth microdilution (YeastOne™ Sensititre). Illumina whole-genome-sequencing and Sanger-sequencing were also performed as confirmatory testing of the mutations identified from nanopore sequencing data. There was a perfect agreement of the resistance-associated mutations detected by MinION-nanopore-sequencing compared to phenotypic testing for acquired resistance (16 with azole resistance; 3 with echinocandin resistance), and perfect concordance of the nanopore sequence mutations to Illumina and Sanger data. Mutations with no known association with phenotypic drug resistance and novel mutations were also detected.     

Avoidance and suppression of plant defenses by herbivores and pathogens

Plants are nutritious and hence herbivores and phytopathogens have specialized to attack and consume them. In turn, plants have evolved adaptations to detect and withstand these attacks. Such adaptations we call ‘defenses’ and they can operate either directly between the plant and the plant consumer or indirectly i.e. when taking effect via other organisms such as predators and parasitoids of herbivores. Plant defenses put selection pressure on plant-consumers and, as a result, herbivores and pathogens have evolved counter-adaptations to avoid, resist, or manipulate plant defenses. Here we review how plant consumers have adapted to cope with plant defenses and we will put special emphasis on the phenomenon of suppression of plant defenses.     

Resistance to the tyrosine kinase inhibitor axitinib is associated with increased glucose metabolism in pancreatic adenocarcinoma

Alterations in energy (glucose) metabolism are key events in the development and progression of cancer. In pancreatic adenocarcinoma (PDAC) cells, we investigated changes in glucose metabolism induced by resistance to the receptor tyrosine kinase inhibitor (RTKI) axitinib. Here, we show that human cell lines and mouse PDAC cell lines obtained from the spontaneous pancreatic cancer mouse model (Kras^G12DPdx1-cre) were sensitive to axitinib. The anti-proliferative effect was due to a G2/M block resulting in loss of 70–75% cell viability in the most sensitive PDAC cell line. However, a surviving sub-population showed a 2- to 3-fold increase in [C-14]deoxyglucose ([C-14]DG) uptake. This was sustained in axitinib-resistant cell lines, which were derived from parental PDAC. In addition to the axitinib-induced increase in [C-14]DG uptake, we observed a translocation of glucose transporter-1 (Glut-1) transporters from cytosolic pools to the cell surface membrane and a 2-fold increase in glycolysis rates measured by the extracellular acidification rate (ECAR). We demonstrated an axitinib-induced increase in phosphorylated Protein Kinase B (pAkt) and by blocking pAkt with a phosphatidylinositol-3 kinase (PI3K) inhibitor we reversed the Glut-1 translocation and restored sensitivity to axitinib treatment. Combination treatment with both axitinib and Akt inhibitor in parental pancreatic cell line resulted in a decrease in cell viability beyond that conferred by single therapy alone. Our study shows that PDAC resistance to axitinib results in increased glucose metabolism mediated by activated Akt. Combining axitinib and an Akt inhibitor may improve treatment in PDAC.     

A world of cytochrome P450s

The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450.     

House flies delay fungal infection by fevering: at a cost

1. Many ectothermic species have evolved the ability to invoke a ‘behavioural fever’ when infected with a pathogen. The relative costs and benefits of this response, however, have rarely been quantified. 2. The aim of this study was investigate the nature and consequences of behavioural fever in the house fly, Musca domestica L., in response to infection with a possible biocontrol agent, the fungal entomopathogen, Beauveria bassiana (Balsamo) Vuillemin. 3. It was found that infected flies preferred higher temperatures and allocated more effort to thermoregulation than uninfected flies. Flies could not overcome infection but the altered thermal behaviour allowed infected flies to extend their survival and to lay more eggs relative to infected flies maintained under constant conditions. However, flies allowed to fever had lower egg viability suggesting a possible cost. 4. Under the present experimental conditions, the putative costs and benefits fever balanced one another resulting in no net change in fitness. Fever did not, therefore, limit the control potential of the fungus. We discuss whether the costs and benefits of behavioural fever might differ in other ecological contexts.     

Transposition-mediated transcriptional overexpression as a mechanism of insecticide resistance

It has been proposed that amplification of genes for esterase that provide resistance to insecticides may originate from transposition events. To test this hypothesis, we have constructed a minigene coding for a soluble acetylcholinesterase under the control of a nontissue-specific promoter (hsp70). When introduced into Drosophila, the gene is expressed in all tissues and the extra acetylcholinesterase produced confers a low level of insecticide resistance (twofold). The minigene was mobilized by crossing the initial transformant with a strain providing a source of P-element transposase. After 34 generations of exposure to the organophosphate parathion, we obtained a strain with a higher resistance (fivefold). This strain had only one extra Ace gene, which overexpressed acetylcholinesterase. Thus, following transposition, resistance resulted from the overexpression of a single copy of the gene and not from gene amplification. Received: 9 August 1996 / Accepted: 27 May 1997