Menu

Auto Tag: Plasmid

April 21, 2020

Identification of plasmid encoded osmoregulatory genes from halophilic bacteria isolated from the rhizosphere of halophytes.

Bacterial plasmids carry genes that code for additional traits such as osmoregulation, CO2 fixation, antibiotic and heavy metal resistance, root nodulation and nitrogen fixation. The main objective of the current study was to identify plasmid-conferring osmoregulatory genes in bacteria isolated from rhizospheric and non-rhizospheric soils of halophytes (Salsola stocksii and Atriplex amnicola). More than 55% of halophilic bacteria from the rhizosphere and 70% from non-rhizospheric soils were able to grow at 3?M salt concentrations. All the strains showed optimum growth at 1.5-3.0?M NaCl. Bacterial strains from the Salsola rhizosphere showed maximum (31%) plasmid elimination during curing experiments as compared to bacterial strains from the Atriplex rhizosphere and non-rhizospheric soils. Two plasmid cured strains Bacillus HL2HP6 and Oceanobacillus HL2RP7 lost their ability to grow in halophilic medium, but they grew well on LB medium. The plasmid cured strains also showed a change in sensitivity to specific antibiotics. These plasmids were isolated and transformed into E. coli strains and growth response of wild-type and transformed E. coli strains was compared at 1.5-4?M NaCl concentrations. Chromosomal DNA and plasmids from Bacillus filamentosus HL2HP6 were sequenced by using high throughput sequencing approach. Results of functional analysis of plasmid sequences showed different proteins and enzymes involved in osmoregulation of bacteria, such as trehalose, ectoine synthetase, porins, proline, alanine, inorganic ion transporters, dehydrogenases and peptidases. Our results suggested that plasmid conferring osmoregulatory genes play a vital role to maintain internal osmotic balance of bacterial cells and these genes can be used to develop salt tolerant transgenic crops.Copyright © 2019 Elsevier GmbH. All rights reserved.

Read more
April 21, 2020

5’UTR-mediated regulation of Ataxin-1 expression.

Expression of mutant Ataxin-1 with an abnormally expanded polyglutamine domain is necessary for the onset and progression of spinocerebellar ataxia type 1 (SCA1). Understanding how Ataxin-1 expression is regulated in the human brain could inspire novel molecular therapies for this fatal, dominantly inherited neurodegenerative disease. Previous studies have shown that the ATXN1 3’UTR plays a key role in regulating the Ataxin-1 cellular pool via diverse post-transcriptional mechanisms. Here we show that elements within the ATXN1 5’UTR also participate in the regulation of Ataxin-1 expression. PCR and PacBio sequencing analysis of cDNA obtained from control and SCA1 human brain samples revealed the presence of three major, alternatively spliced ATXN1 5’UTR variants. In cell-based assays, fusion of these variants upstream of an EGFP reporter construct revealed significant and differential impacts on total EGFP protein output, uncovering a type of genetic rheostat-like function of the ATXN1 5’UTR. We identified ribosomal scanning of upstream AUG codons and increased transcript instability as potential mechanisms of regulation. Importantly, transcript-based analyses revealed significant differences in the expression pattern of ATXN1 5’UTR variants between control and SCA1 cerebellum. Together, the data presented here shed light into a previously unknown role for the ATXN1 5’UTR in the regulation of Ataxin-1 and provide new opportunities for the development of SCA1 therapeutics. Copyright © 2019. Published by Elsevier Inc.

Read more
April 21, 2020

Plasmid analysis of Escherichia coli isolates from South Korea co-producing NDM-5 and OXA-181 carbapenemases.

Recently, Escherichia coli isolates co-producing New Delhi metallo-ß-lactamase (NDM)-5 and oxacillinase (OXA)-181 were identified in a tertiary-care hospital of South Korea. Isolate CC1702-1 was collected from urine in January 2017 and isolate CC1706-1 was recovered from a transtracheal aspirate of a hospitalized patient in May 2017. Carbapenemase genes were identified by multiplex PCR and sequencing, and whole genome sequencing was performed subsequently using the PacBio RSII system. Both E. coli isolates belonged to the same clone (ST410) and were resistant to all ß-lactams including carbapenems. We obtained whole plasmid sequences of the isolates: pCC1702-NDM-5 from CC1702-1 and pCC1706-NDM-5 and pCC1706-OXA-181 from CC1706-1. The two E. coli isolates belonged to the same clone (ST410) and they were completely resistant to all ß-lactams, as well as carbapenems. Two blaNDM-5-harboring plasmids belonged to the same incompatibility group, IncFIA/B, and consisted of 79,613?bp and 111,890?bp with 87 and 130 coding sequences, respectively. The genetic structures of the two blaNDM-5-bearing plasmids, which were distinct from the blaNDM-5-bearing plasmids from the Klebsiella pneumoniae isolates previously transmitted from the United Arab Emirates (UAE) to South Korea, differed from each other. While pCC1702-NDM-5 showed high degree of identity with the plasmid from a multidrug-resistant isolate of Citrobacter fruendii P5571 found in China, pCC1706-NDM-5 was very similar to the plasmid from a multidrug-resistant isolate of E. coli AMA1176 found in Denmark. pCC1706-OXA-181, which was a 51?kb, self-transmissible IncX3 plasmid, was identical to the E. coli plasmids pAMA1167-OXA-181 from Denmark and pOXA-181-WCHEC14828 from China. Plasmids harboring blaNDM-5 in E. coli isolates might not be transferred from K. pneumoniae isolates co-producing NDM-5 and OXA-181. They probably originated from multiple sources.Copyright © 2019 Elsevier Inc. All rights reserved.

Read more
April 21, 2020

Complete genome sequence of Hahella sp. KA22, a prodigiosin-producing algicidal bacterium

Hahella sp. KA22 is a gamma-proteobacteria bacterium that belongs to the family Hahellaceae and order Oceanospirillales. Strain KA22 is capable of producing prodigiosin, which is a compound with algicidal activity. It is for this reason that further investigation of the genome of strain KA22 will help in revealing the prodigiosin producing mechanism and its ecological functions. In this study, we sequenced and annotated the complete genome of Hahella sp. KA22, the second complete genome sequence of prodigiosin-producing bacteria in the family Hahellacaeae. The genome of strain KA22 is 6,927,416 base pairs in size, contains one chrome with no plasmid and predicted to contain 6167 protein-coding genes and 86 RNA-only encoding genes. Genomic analysis of Hahella sp. KA22 reveals that this strain of bacteria can be used for biological elimination or control of harmful algal blooms (HABs).

Read more
April 21, 2020

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.

Read more
April 21, 2020

Genome analysis and genetic transformation of a water surface-floating microalga Chlorococcum sp. FFG039.

Microalgal harvesting and dewatering are the main bottlenecks that need to be overcome to tap the potential of microalgae for production of valuable compounds. Water surface-floating microalgae form robust biofilms, float on the water surface along with gas bubbles entrapped under the biofilms, and have great potential to overcome these bottlenecks. However, little is known about the molecular mechanisms involved in the water surface-floating phenotype. In the present study, we analysed the genome sequence of a water surface-floating microalga Chlorococcum sp. FFG039, with a next generation sequencing technique to elucidate the underlying mechanisms. Comparative genomics study with Chlorococcum sp. FFG039 and other non-floating green microalgae revealed some of the unique gene families belonging to this floating microalga, which may be involved in biofilm formation. Furthermore, genetic transformation of this microalga was achieved with an electroporation method. The genome information and transformation techniques presented in this study will be useful to obtain molecular insights into the water surface-floating phenotype of Chlorococcum sp. FFG039.

Read more
April 21, 2020

Genetic basis of functional variability in adhesion G protein-coupled receptors.

The enormous sizes of adhesion G protein-coupled receptors (aGPCRs) go along with complex genomic exon-intron architectures giving rise to multiple mRNA variants. There is a need for a comprehensive catalog of aGPCR variants for proper evaluation of the complex functions of aGPCRs found in structural, in vitro and animal model studies. We used an established bioinformatics pipeline to extract, quantify and visualize mRNA variants of aGPCRs from deeply sequenced transcriptomes. Data analysis showed that aGPCRs have multiple transcription start sites even within introns and that tissue-specific splicing is frequent. On average, 19 significantly expressed transcript variants are derived from a given aGPCR gene. The domain architecture of the N terminus encoded by transcript variants often differs and N termini without or with an incomplete seven-helix transmembrane anchor as well as separate seven-helix transmembrane domains are frequently derived from aGPCR genes. Experimental analyses of selected aGPCR transcript variants revealed marked functional differences. Our analysis has an impact on a rational design of aGPCR constructs for structural analyses and gene-deficient mouse lines and provides new support for independent functions of both, the large N terminus and the transmembrane domain of aGPCRs.

Read more
April 21, 2020

Genome and plasmid diversity of Extended-Spectrum ß-Lactamase-producing Escherichia coli ST131 – tracking phylogenetic trajectories with Bayesian inference.

Clonal lineages of ESBL (Extended-Spectrum ß-Lactamase)-producing E. coli belonging to sequence type 131 (ST131) have disseminated globally during the last 30 years, leading to an increased prevalence of resistance to fluoroquinolones and extended-spectrum cephalosporins in clinical isolates of E. coli. We aimed to study if Swedish ESBL-producing ST131 isolates originated from single or multiple introductions to the population by assessing the amount of genetic variation, on chromosomal and plasmid level, between Swedish and international E. coli ST131. Bayesian inference of Swedish E. coli ST131 isolates (n?=?29), sequenced using PacBio RSII, together with an international ST131 dataset showed that the Swedish isolates were part of the international ST131 A, C1 and C2 clades. Highly conserved plasmids were identified in three clusters although they were separated by several years, which indicates a strong co-evolution between some ST131 lineages and specific plasmids. In conclusion, the tight clonal relationship observed within the ST131 clades, together with highly conserved plasmids, challenges investigation of strain transmission events. A combination of few SNPs on a genome-wide scale and an epidemiological temporospatial link, are needed to track the spread of the ST131 subclones.

Read more
April 21, 2020

SMRT sequencing reveals differential patterns of methylation in two O111:H- STEC isolates from a hemolytic uremic syndrome outbreak in Australia.

In 1995 a severe haemolytic-uremic syndrome (HUS) outbreak in Adelaide occurred. A recent genomic analysis of Shiga toxigenic Escherichia coli (STEC) O111:H- strains 95JB1 and 95NR1 from this outbreak found that the more virulent isolate, 95NR1, harboured two additional copies of the Shiga toxin 2 (Stx2) genes encoded within prophage regions. The structure of the Stx2-converting prophages could not be fully resolved using short-read sequence data alone and it was not clear if there were other genomic differences between 95JB1 and 95NR1. In this study we have used Pacific Biosciences (PacBio) single molecule real-time (SMRT) sequencing to characterise the genome and methylome of 95JB1 and 95NR1. We completely resolved the structure of all prophages including two, tandemly inserted, Stx2-converting prophages in 95NR1 that were absent from 95JB1. Furthermore we defined all insertion sequences and found an additional IS1203 element in the chromosome of 95JB1. Our analysis of the methylome of 95NR1 and 95JB1 identified hemi-methylation of a novel motif (5′-CTGCm6AG-3′) in more than 4000 sites in the 95NR1 genome. These sites were entirely unmethylated in the 95JB1 genome, and included at least 177 potential promoter regions that could contribute to regulatory differences between the strains. IS1203 mediated deactivation of a novel type IIG methyltransferase in 95JB1 is the likely cause of the observed differential patterns of methylation between 95NR1 and 95JB1. This study demonstrates the capability of PacBio SMRT sequencing to resolve complex prophage regions and reveal the genetic and epigenetic heterogeneity within a clonal population of bacteria.

Read more
April 21, 2020

Extended insight into the Mycobacterium chelonae-abscessus complex through whole genome sequencing of Mycobacterium salmoniphilum outbreak and Mycobacterium salmoniphilum-like strains.

Members of the Mycobacterium chelonae-abscessus complex (MCAC) are close to the mycobacterial ancestor and includes both human, animal and fish pathogens. We present the genomes of 14 members of this complex: the complete genomes of Mycobacterium salmoniphilum and Mycobacterium chelonae type strains, seven M. salmoniphilum isolates, and five M. salmoniphilum-like strains including strains isolated during an outbreak in an animal facility at Uppsala University. Average nucleotide identity (ANI) analysis and core gene phylogeny revealed that the M. salmoniphilum-like strains are variants of the human pathogen Mycobacterium franklinii and phylogenetically close to Mycobacterium abscessus. Our data further suggested that M. salmoniphilum separates into three branches named group I, II and III with the M. salmoniphilum type strain belonging to group II. Among predicted virulence factors, the presence of phospholipase C (plcC), which is a major virulence factor that makes M. abscessus highly cytotoxic to mouse macrophages, and that M. franklinii originally was isolated from infected humans make it plausible that the outbreak in the animal facility was caused by a M. salmoniphilum-like strain. Interestingly, M. salmoniphilum-like was isolated from tap water suggesting that it can be present in the environment. Moreover, we predicted the presence of mutational hotspots in the M. salmoniphilum isolates and 26% of these hotspots overlap with genes categorized as having roles in virulence, disease and defense. We also provide data about key genes involved in transcription and translation such as sigma factor, ribosomal protein and tRNA genes.

Read more
April 21, 2020

An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation.

Bloodstream infections by Salmonella enterica serovar Typhimurium constitute a major health burden in sub-Saharan Africa (SSA). These invasive non-typhoidal (iNTS) infections are dominated by isolates of the antibiotic resistance-associated sequence type (ST) 313. Here, we report emergence of ST313 sublineage II.1 in the Democratic Republic of the Congo. Sublineage II.1 exhibits extensive drug resistance, involving a combination of multidrug resistance, extended spectrum ß-lactamase production and azithromycin resistance. ST313 lineage II.1 isolates harbour an IncHI2 plasmid we name pSTm-ST313-II.1, with one isolate also exhibiting decreased ciprofloxacin susceptibility. Whole genome sequencing reveals that ST313 II.1 isolates have accumulated genetic signatures potentially associated with altered pathogenicity and host adaptation, related to changes observed in biofilm formation and metabolic capacity. Sublineage II.1 emerged at the beginning of the 21st century and is involved in on-going outbreaks. Our data provide evidence of further evolution within the ST313 clade associated with iNTS in SSA.

Read more
April 21, 2020

Population dynamics of an Escherichia coli ST131 lineage during recurrent urinary tract infection.

Recurrent urinary tract infections (rUTIs) are extremely common, with ~?25% of all women experiencing a recurrence within 1 year of their original infection. Escherichia coli ST131 is a globally dominant multidrug resistant clone associated with high rates of rUTI. Here, we show the dynamics of an ST131 population over a 5-year period from one elderly woman with rUTI since the 1970s. Using whole genome sequencing, we identify an indigenous clonal lineage (P1A) linked to rUTI and persistence in the fecal flora, providing compelling evidence of an intestinal reservoir of rUTI. We also show that the P1A lineage possesses substantial plasmid diversity, resulting in the coexistence of antibiotic resistant and sensitive intestinal isolates despite frequent treatment. Our longitudinal study provides a unique comprehensive genomic analysis of a clonal lineage within a single individual and suggests a population-wide resistance mechanism enabling rapid adaptation to fluctuating antibiotic exposure.

Read more
April 21, 2020

CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.