Reiling, S. | Search bioRxiv

bioRxiv2023

The five homologous CiaR-controlled Ccn sRNAs of Streptococcus pneumoniae modulate Zn-resistance.

Zinc is a vital transition metal for Streptococcus pneumoniae, but is deadly at high concentrations. In S. pneumoniae, elevated intracellular free Zn levels result in mis-metallation of key Mn-dependent metabolic and superoxide detoxifying enzymes resulting in Zn intoxication. Here, we report our identification and characterization of the function of the five homologous, CiaRH-regulated Ccn sRNAs in controlling S. pneumoniae virulence and metal homeostasis. We show that deletion of all five ccn genes (ccnA, ccnB, ccnC, ccnD, and ccnE) from S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4) causes Zn hypersensitivity and an attenuation of virulence in a murine invasive pneumonia model. We provide evidence that bioavailable Zn disproportionately increases in S. pneumoniae strains lacking the five ccn genes. Consistent with a response to Zn intoxication or relatively high intracellular free Zn levels, expression of genes encoding the CzcD Zn exporter and the Mn-independent ribonucleotide reductase, NrdD-NrdG, were increased in the {Delta}ccnABCDE mutant relative to its isogenic ccn+ parent strain. The growth inhibition by Zn that occurs as the result of loss of the ccn genes is rescued by supplementation with Mn or OxyraseTM, a reagent that removes dissolved oxygen. Lastly, we found that the Zn-dependent growth inhibition of the {Delta}ccnABCDE strain was not altered by deletion of sodA, whereas the ccn+{Delta}sodA strain phenocopied the {Delta}ccnABCDE strain. Overall, our results indicate that the Ccn sRNAs have a crucial role in preventing Zn intoxication in S. pneumoniae. AUTHOR SUMMARYZn and Mn are essential micronutrients for many bacteria, including Streptococcus pneumoniae. While Zn performs vital structural or catalytic roles in certain proteins, in excess, Zn can inhibit Mn uptake by S. pneumoniae and displace, but not functionally replace Mn from key enzymes including superoxide dismutase A (SodA). Here, we show that the Ccn small regulatory RNAs promote S. pneumoniae resistance to Zn intoxication. Furthermore, we demonstrate that these small regulatory RNAs modulate the ability of S. pneumoniae to cause invasive pneumonia. Altogether, these findings reveal a new layer of regulation of S. pneumoniae Zn homeostasis and suggest that there are factors in addition to known transporters that modulate intracellular, bioavailable Zn levels.

microbiology↗

bioRxiv2018

MinION re-sequencing of Giardia genomes and de novo assembly of a new Giardia isolate

BackgroundGenomes of the parasite Giardia duodenalis are relatively small for eukaryotic genomes, yet there are only six publicly available. Difficulties in assembling the tetraploid G. duodenalis genome from short read sequencing data likely contribute to this lack of genomic information. We sequenced three isolates of G. duodenalis (AWB, BGS, and beaver) on the Oxford Nanopore Technologies MinION whose long reads have the potential to address genomic areas that are problematic for short reads.\n\nResultsUsing a hybrid approach that combines MinION long reads and Illumina short reads to take advantage of the continuity of the long reads and the accuracy of the short reads we generated reference quality genomes for each isolate. The genomes for two of the isolates were evaluated against the available reference genomes for comparison. The third genome for which there is no previous data was then assembled. The long reads were used to find structural variants in each isolate to examine heterozygosity. Consistent with previous findings based on SNPs, Giardia BGS was found to be considerably more heterozygous than the other isolates that are from Assemblage A. We also find an enrichment of variant-specific surface proteins in some of the structural variant regions.\n\nConclusionsOur results show that the MinION can be used to generate reference quality genomes in Giardia and further be used to identify structural variant regions that are an important source of genetic variation not previously examined in these parasites.

genomics↗