Browsing by Author "Silander OK"

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    A simple screen to identify promoters conferring high levels of phenotypic noise.
    (PUBLIC LIBRARY SCIENCE, 2008-12) Freed NE; Silander OK; Stecher B; Böhm A; Hardt W-D; Ackermann M
    Genetically identical populations of unicellular organisms often show marked variation in some phenotypic traits. To investigate the molecular causes and possible biological functions of this phenotypic noise, it would be useful to have a method to identify genes whose expression varies stochastically on a certain time scale. Here, we developed such a method and used it for identifying genes with high levels of phenotypic noise in Salmonella enterica ssp. I serovar Typhimurium (S. Typhimurium). We created a genomic plasmid library fused to a green fluorescent protein (GFP) reporter and subjected replicate populations harboring this library to fluctuating selection for GFP expression using fluorescent-activated cell sorting (FACS). After seven rounds of fluctuating selection, the populations were strongly enriched for promoters that showed a high amount of noise in gene expression. Our results indicate that the activity of some promoters of S. Typhimurium varies on such a short time scale that these promoters can absorb rapid fluctuations in the direction of selection, as imposed during our experiment. The genomic fragments that conferred the highest levels of phenotypic variation were promoters controlling the synthesis of flagella, which are associated with virulence and host-pathogen interactions. This confirms earlier reports that phenotypic noise may play a role in pathogenesis and indicates that these promoters have among the highest levels of noise in the S. Typhimurium genome. This approach can be applied to many other bacterial and eukaryotic systems as a simple method for identifying genes with noisy expression.
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    Bacterial lipopolysaccharide modulates immune response in the colorectal tumor microenvironment.
    (Nature Portfolio, 2023-08-23) Sulit AK; Daigneault M; Allen-Vercoe E; Silander OK; Hock B; McKenzie J; Pearson J; Frizelle FA; Schmeier S; Purcell R
    Immune responses can have opposing effects in colorectal cancer (CRC), the balance of which may determine whether a cancer regresses, progresses, or potentially metastasizes. These effects are evident in CRC consensus molecular subtypes (CMS) where both CMS1 and CMS4 contain immune infiltrates yet have opposing prognoses. The microbiome has previously been associated with CRC and immune response in CRC but has largely been ignored in the CRC subtype discussion. We used CMS subtyping on surgical resections from patients and aimed to determine the contributions of the microbiome to the pleiotropic effects evident in immune-infiltrated subtypes. We integrated host gene-expression and meta-transcriptomic data to determine the link between immune characteristics and microbiome contributions in these subtypes and identified lipopolysaccharide (LPS) binding as a potential functional mechanism. We identified candidate bacteria with LPS properties that could affect immune response, and tested the effects of their LPS on cytokine production of peripheral blood mononuclear cells (PBMCs). We focused on Fusobacterium periodonticum and Bacteroides fragilis in CMS1, and Porphyromonas asaccharolytica in CMS4. Treatment of PBMCs with LPS isolated from these bacteria showed that F. periodonticum stimulates cytokine production in PBMCs while both B. fragilis and P. asaccharolytica had an inhibitory effect. Furthermore, LPS from the latter two species can inhibit the immunogenic properties of F. periodonticum LPS when co-incubated with PBMCs. We propose that different microbes in the CRC tumor microenvironment can alter the local immune activity, with important implications for prognosis and treatment response.
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    Complete genome sequences of cluster A Mycobacteriophages BobSwaget, Fred313, KADY, Lokk, MyraDee, Stagni, and StepMih
    (American Society for Microbiology, 26/10/2017) Butela KA; Hendrickson HL; Silander OK; Freed N; Kagey J; et al.
    Seven mycobacteriophages from distinct geographical locations were isolated, using Mycobacterium smegmatis mc(2)155 as the host, and then purified and sequenced. All of the genomes are related to cluster A mycobacteriophages, BobSwaget and Lokk in subcluster A2; Fred313, KADY, Stagni, and StepMih in subcluster A3; and MyraDee in subcluster A18, the first phage to be assigned to that subcluster.
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    Efficiency of the synthetic self-splicing RiboJ ribozyme is robust to cis- and trans-changes in genetic background
    (John Wiley and Sons, Ltd, 2021-08-24) Vlková M; Morampalli BR; Silander OK
    The expanding knowledge of the variety of synthetic genetic elements has enabled the construction of new and more efficient genetic circuits and yielded novel insights into molecular mechanisms. However, context dependence, in which interactions between cis- or trans-genetic elements affect the behavior of these elements, can reduce their general applicability or predictability. Genetic insulators, which mitigate unintended context-dependent cis-interactions, have been used to address this issue. One of the most commonly used genetic insulators is a self-splicing ribozyme called RiboJ, which can be used to decouple upstream 5' UTR in mRNA from downstream sequences (e.g., open reading frames). Despite its general use as an insulator, there has been no systematic study quantifying the efficiency of RiboJ splicing or whether this autocatalytic activity is robust to trans- and cis-genetic context. Here, we determine the robustness of RiboJ splicing in the genetic context of six widely divergent E. coli strains. We also check for possible cis-effects by assessing two SNP versions close to the catalytic site of RiboJ. We show that mRNA molecules containing RiboJ are rapidly spliced even during rapid exponential growth and high levels of gene expression, with a mean efficiency of 98%. We also show that neither the cis- nor trans-genetic context has a significant impact on RiboJ activity, suggesting this element is robust to both cis- and trans-genetic changes.
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    Growth condition-dependent differences in methylation imply transiently differentiated DNA methylation states in Escherichia coli
    (Oxford University Press on behalf of the Genetics Society of America, 2023-02) Breckell GL; Silander OK
    DNA methylation in bacteria frequently serves as a simple immune system, allowing recognition of DNA from foreign sources, such as phages or selfish genetic elements. However, DNA methylation also affects other cell phenotypes in a heritable manner (i.e. epigenetically). While there are several examples of methylation affecting transcription in an epigenetic manner in highly localized contexts, it is not well-established how frequently methylation serves a more general epigenetic function over larger genomic scales. To address this question, here we use Oxford Nanopore sequencing to profile DNA modification marks in three natural isolates of Escherichia coli. We first identify the DNA sequence motifs targeted by the methyltransferases in each strain. We then quantify the frequency of methylation at each of these motifs across the entire genome in different growth conditions. We find that motifs in specific regions of the genome consistently exhibit high or low levels of methylation. Furthermore, we show that there are replicable and consistent differences in methylated regions across different growth conditions. This suggests that during growth, E. coli transiently differentiate into distinct methylation states that depend on the growth state, raising the possibility that measuring DNA methylation alone can be used to infer bacterial growth states without additional information such as transcriptome or proteome data. These results show the utility of using Oxford Nanopore sequencing as an economic means to infer DNA methylation status. They also provide new insights into the dynamics of methylation during bacterial growth and provide evidence of differentiated cell states, a transient analog to what is observed in the differentiation of cell types in multicellular organisms.
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    Long-read sequencing reveals atypical mitochondrial genome structure in a New Zealand marine isopod
    (The Royal Society, 2022-01-12) Pearman WS; Wells SJ; Dale J; Silander OK; Freed NE
    Most animal mitochondrial genomes are small, circular and structurally conserved. However, recent work indicates that diverse taxa possess unusual mitochondrial genomes. In Isopoda, species in multiple lineages have atypical and rearranged mitochondrial genomes. However, more species of this speciose taxon need to be evaluated to understand the evolutionary origins of atypical mitochondrial genomes in this group. In this study, we report the presence of an atypical mitochondrial structure in the New Zealand endemic marine isopod, Isocladus armatus. Data from long- and short-read DNA sequencing suggest that I. armatus has two mitochondrial chromosomes. The first chromosome consists of two mitochondrial genomes that have been inverted and fused together in a circular form, and the second chromosome consists of a single mitochondrial genome in a linearized form. This atypical mitochondrial structure has been detected in other isopod lineages, and our data from an additional divergent isopod lineage (Sphaeromatidae) lends support to the hypothesis that atypical structure evolved early in the evolution of Isopoda. Additionally, we find that an asymmetrical site previously observed across many species within Isopoda is absent in I. armatus, but confirm the presence of two asymmetrical sites recently reported in two other isopod species.
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    Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding
    (Oxford University Press, 2020-07-18) Freed NE; Vlková M; Faisal MB; Silander OK
    Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (∼5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.