David Bernick | University of California, Santa Cruz (original) (raw)

Papers by David Bernick

American journal of proctology, gastroenterology & colon & rectal surgery, 1981

Pyrobaculum oguniense TE7 is an aerobic hyperthermophilic crenarchaeon isolated from a hot spring... more Pyrobaculum oguniense TE7 is an aerobic hyperthermophilic crenarchaeon isolated from a hot spring in Japan. Here we describe its main chromosome of 2,436,033 bp, with three large-scale inversions and an extra-chromosomal element of 16,887 bp. We have annotated 2,800 protein-coding genes and 145 RNA genes in this genome, including nine H/ACA-like small RNA, 83 predicted C/D box small RNA, and 47 transfer RNA genes. Comparative anal-yses with the closest known relative, the anaerobe Pyrobaculum arsenaticum from Italy, re-veals unexpectedly high synteny and nucleotide identity between these two geographically distant species. Deep sequencing of a mixture of genomic DNA from multiple cells has illu-minated some of the genome dynamics potentially shared with other species in this genus.

In the Eukarya and Archaea, small RNA-guided pseudouridine modification is believed to be an esse... more In the Eukarya and Archaea, small RNA-guided pseudouridine modification is believed to be an essential step in ribosomal RNA maturation. While readily modeled and identified by computational methods in eukaryotic species, these guide RNAs have not been found in most archaeal genomes. Using high-throughput transcriptome sequencing and comparative genomics, we have identified ten novel small RNA families that appear to function as H/ACA pseudouridylation guide sRNAs, yet surprisingly lack several expected canonical features. The new RNA genes are transcribed and highly conserved across at least six species in the archaeal hyperthermophilic genus Pyrobaculum. The sRNAs exhibit a single hairpin structure interrupted by a conserved kinkturn motif, yet only two of ten families contain the complete canonical structure found in all other H/ACA sRNAs. Half of the sRNAs lack the conserved 39-terminal ACA sequence, and many contain only a single 39 guide region rather than the canonical 59 and...

The Limnospira genus is a recently established clade that is economically important due to its wo... more The Limnospira genus is a recently established clade that is economically important due to its worldwide use in biotechnology and agriculture. This genus includes organisms that were reclassified from Arthrospira, which are commercially marketed as “Spirulina.” Limnospira are photoautotrophic organisms that are widely used for research in nutrition, medicine, bioremediation, and biomanufacturing. Despite its widespread use, there is no closed genome for the Limnospira genus, and no reference genome for the type strain, Limnospira fusiformis. In this work, the L. fusiformis genome was sequenced using Oxford Nanopore Technologies MinION and assembled using only ultra-long reads (>35 kb). This assembly was polished with Illumina MiSeq reads sourced from an axenic L. fusiformis culture; axenicity was verified via microscopy and rDNA analysis. Ultra-long read sequencing resulted in a 6.42 Mb closed genome assembled as a single contig with no plasmid. Phylogenetic analysis placed L. fu...

Archaea use C/D box sno-like RNAs (sRNAs) to guide precise 2'- O -methyl modification of ribo... more Archaea use C/D box sno-like RNAs (sRNAs) to guide precise 2'- O -methyl modification of ribosomal and transfer RNAs. Although C/D box sRNAs are the most numerous archaeal small RNA class, most genomes have incomplete sRNA gene annotation because reliable, completely automated detection methods are unavailable. To study archaeal C/D box sRNA structure, function and evolution, we collected, predicted, and curated a comprehensive set of these sRNAs from six species within the crenarchaeal hyperthermophilic genus Pyrobaculum . We used high-throughput small RNA sequencing data, computational methods, and comparative genomics to curate 526 Pyrobaculum C/D box sRNAs and organized them into 110 families based on conservation of their guide sequences. Our comprehensive analysis provides a detailed narrative of C/D box sRNA evolutionary history, implications of target conservation on ribosome maturation, and sRNA impact on genome architecture. We find that in some cases the overlap of C/...

mBio, 2017

Many bacterial genomes are highly variable but nonetheless are typically published as a single as... more Many bacterial genomes are highly variable but nonetheless are typically published as a single assembled genome. Experiments tracking bacterial genome evolution have not looked at the variation present at a given point in time. Here, we analyzed the mouse-passaged Helicobacter pylori strain SS1 and its parent PMSS1 to assess intra- and intergenomic variability. Using high sequence coverage depth and experimental validation, we detected extensive genome plasticity within these H. pylori isolates, including movement of the transposable element IS 607 , large and small inversions, multiple single nucleotide polymorphisms, and variation in cagA copy number. The cagA gene was found as 1 to 4 tandem copies located off the cag island in both SS1 and PMSS1; this copy number variation correlated with protein expression. To gain insight into the changes that occurred during mouse adaptation, we also compared SS1 and PMSS1 and observed 46 differences that were distinct from the within-genome v...

Dedication x Acknowledgments xi 5 Conclusion 91 Appendices 92 A Paired-end mapping of Pyrobaculum... more Dedication x Acknowledgments xi 5 Conclusion 91 Appendices 92 A Paired-end mapping of Pyrobaculum oguniense 93 B Small RNA transcripts associated with protein coding regions 95 Bibliography 102

Frontiers in bioengineering and biotechnology, 2015

Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both t... more Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which ...

PLoS Pathogens, 2014

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not... more Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR inframe deletion mutant (DiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis DiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.

Medical & Biological Engineering & Computing, 1979

American journal of proctology, gastroenterology & colon & rectal surgery, 1981

Pyrobaculum oguniense TE7 is an aerobic hyperthermophilic crenarchaeon isolated from a hot spring... more Pyrobaculum oguniense TE7 is an aerobic hyperthermophilic crenarchaeon isolated from a hot spring in Japan. Here we describe its main chromosome of 2,436,033 bp, with three large-scale inversions and an extra-chromosomal element of 16,887 bp. We have annotated 2,800 protein-coding genes and 145 RNA genes in this genome, including nine H/ACA-like small RNA, 83 predicted C/D box small RNA, and 47 transfer RNA genes. Comparative anal-yses with the closest known relative, the anaerobe Pyrobaculum arsenaticum from Italy, re-veals unexpectedly high synteny and nucleotide identity between these two geographically distant species. Deep sequencing of a mixture of genomic DNA from multiple cells has illu-minated some of the genome dynamics potentially shared with other species in this genus.

In the Eukarya and Archaea, small RNA-guided pseudouridine modification is believed to be an esse... more In the Eukarya and Archaea, small RNA-guided pseudouridine modification is believed to be an essential step in ribosomal RNA maturation. While readily modeled and identified by computational methods in eukaryotic species, these guide RNAs have not been found in most archaeal genomes. Using high-throughput transcriptome sequencing and comparative genomics, we have identified ten novel small RNA families that appear to function as H/ACA pseudouridylation guide sRNAs, yet surprisingly lack several expected canonical features. The new RNA genes are transcribed and highly conserved across at least six species in the archaeal hyperthermophilic genus Pyrobaculum. The sRNAs exhibit a single hairpin structure interrupted by a conserved kinkturn motif, yet only two of ten families contain the complete canonical structure found in all other H/ACA sRNAs. Half of the sRNAs lack the conserved 39-terminal ACA sequence, and many contain only a single 39 guide region rather than the canonical 59 and...

The Limnospira genus is a recently established clade that is economically important due to its wo... more The Limnospira genus is a recently established clade that is economically important due to its worldwide use in biotechnology and agriculture. This genus includes organisms that were reclassified from Arthrospira, which are commercially marketed as “Spirulina.” Limnospira are photoautotrophic organisms that are widely used for research in nutrition, medicine, bioremediation, and biomanufacturing. Despite its widespread use, there is no closed genome for the Limnospira genus, and no reference genome for the type strain, Limnospira fusiformis. In this work, the L. fusiformis genome was sequenced using Oxford Nanopore Technologies MinION and assembled using only ultra-long reads (>35 kb). This assembly was polished with Illumina MiSeq reads sourced from an axenic L. fusiformis culture; axenicity was verified via microscopy and rDNA analysis. Ultra-long read sequencing resulted in a 6.42 Mb closed genome assembled as a single contig with no plasmid. Phylogenetic analysis placed L. fu...

Archaea use C/D box sno-like RNAs (sRNAs) to guide precise 2'- O -methyl modification of ribo... more Archaea use C/D box sno-like RNAs (sRNAs) to guide precise 2'- O -methyl modification of ribosomal and transfer RNAs. Although C/D box sRNAs are the most numerous archaeal small RNA class, most genomes have incomplete sRNA gene annotation because reliable, completely automated detection methods are unavailable. To study archaeal C/D box sRNA structure, function and evolution, we collected, predicted, and curated a comprehensive set of these sRNAs from six species within the crenarchaeal hyperthermophilic genus Pyrobaculum . We used high-throughput small RNA sequencing data, computational methods, and comparative genomics to curate 526 Pyrobaculum C/D box sRNAs and organized them into 110 families based on conservation of their guide sequences. Our comprehensive analysis provides a detailed narrative of C/D box sRNA evolutionary history, implications of target conservation on ribosome maturation, and sRNA impact on genome architecture. We find that in some cases the overlap of C/...

mBio, 2017

Many bacterial genomes are highly variable but nonetheless are typically published as a single as... more Many bacterial genomes are highly variable but nonetheless are typically published as a single assembled genome. Experiments tracking bacterial genome evolution have not looked at the variation present at a given point in time. Here, we analyzed the mouse-passaged Helicobacter pylori strain SS1 and its parent PMSS1 to assess intra- and intergenomic variability. Using high sequence coverage depth and experimental validation, we detected extensive genome plasticity within these H. pylori isolates, including movement of the transposable element IS 607 , large and small inversions, multiple single nucleotide polymorphisms, and variation in cagA copy number. The cagA gene was found as 1 to 4 tandem copies located off the cag island in both SS1 and PMSS1; this copy number variation correlated with protein expression. To gain insight into the changes that occurred during mouse adaptation, we also compared SS1 and PMSS1 and observed 46 differences that were distinct from the within-genome v...

Dedication x Acknowledgments xi 5 Conclusion 91 Appendices 92 A Paired-end mapping of Pyrobaculum... more Dedication x Acknowledgments xi 5 Conclusion 91 Appendices 92 A Paired-end mapping of Pyrobaculum oguniense 93 B Small RNA transcripts associated with protein coding regions 95 Bibliography 102

Frontiers in bioengineering and biotechnology, 2015

Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both t... more Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which ...

PLoS Pathogens, 2014

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not... more Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR inframe deletion mutant (DiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis DiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.

Medical & Biological Engineering & Computing, 1979