April 21, 2020  |  

A unique methylation pattern by a type I HsdM methyltransferase prepares for DpnI rare cutting sites in the Pseudomonas aeruginosa PAO1 genome.

A physical genome map of Pseudomonas aeruginosa PAO1 had been constructed in the late 1980s by combinatorial SpeI/DpnI long-range restriction mapping. Whereas SpeI was known to cleave within the infrequent 5′-ACTAGT hexanucleotide, the rare recognition site 5′-Gm6ATC for the methylation-sensitive 4-cutter DpnI in the PAO1 genome remained elusive. Resequencing the PAO1 genome with the methylation-sensitive Single Molecule Real-Time (SMRT) sequencing technology identified DpnI to cleave the PAO1 genome at 15 palindromic GAC(N)6Gm6ATC(N)6GTC recognition sites with the central Gm6ATC palindrome to be methylated on both strands by the clone PAO1-specific type I HsdM superfamily methyltransferase M.PaePAOI (PA2735). © FEMS 2019.


April 21, 2020  |  

Genomic and transcriptomic characterization of Pseudomonas aeruginosa small colony variants derived from a chronic infection model.

Phenotypic change is a hallmark of bacterial adaptation during chronic infection. In the case of chronic Pseudomonas aeruginosa lung infection in patients with cystic fibrosis, well-characterized phenotypic variants include mucoid and small colony variants (SCVs). It has previously been shown that SCVs can be reproducibly isolated from the murine lung following the establishment of chronic infection with mucoid P. aeruginosa strain NH57388A. Using a combination of single-molecule real-time (PacBio) and Illumina sequencing we identify a large genomic inversion in the SCV through recombination between homologous regions of two rRNA operons and an associated truncation of one of the 16S rRNA genes and suggest this may be the genetic switch for conversion to the SCV phenotype. This phenotypic conversion is associated with large-scale transcriptional changes distributed throughout the genome. This global rewiring of the cellular transcriptomic output results in changes to normally differentially regulated genes that modulate resistance to oxidative stress, central metabolism and virulence. These changes are of clinical relevance because the appearance of SCVs during chronic infection is associated with declining lung function.


April 21, 2020  |  

Diversity of phytobeneficial traits revealed by whole-genome analysis of worldwide-isolated phenazine-producing Pseudomonas spp.

Plant-beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant-growth promotion and/or disease-suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine-1-carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil-dwelling plant pathogens and play a role in the ecological competence of phenazine-producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant-beneficial phenazine-producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein-coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant-beneficial phenazine-producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant-growth promotion and rhizosphere competence. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.


April 21, 2020  |  

Denitrifying Bacteria Active in Woodchip Bioreactors at Low-Temperature Conditions.

Woodchip bioreactor technology removes nitrate from agricultural subsurface drainage by using denitrifying microorganisms. Although woodchip bioreactors have demonstrated success in many field locations, low water temperature can significantly limit bioreactor efficiency and performance. To improve bioreactor performance, it is important to identify the microbes responsible for nitrate removal at low temperature conditions. Therefore, in this study, we identified and characterized denitrifiers active at low-temperature conditions by using culture-independent and -dependent approaches. By comparative 16S rRNA (gene) analysis and culture isolation technique, Pseudomonas spp., Polaromonas spp., and Cellulomonas spp. were identified as being important bacteria responsible for denitrification in woodchip bioreactor microcosms at relatively low temperature conditions (15°C). Genome analysis of Cellulomonas sp. strain WB94 confirmed the presence of nitrite reductase gene nirK. Transcription levels of this nirK were significantly higher in the denitrifying microcosms than in the non-denitrifying microcosms. Strain WB94 was also capable of degrading cellulose and other complex polysaccharides. Taken together, our results suggest that Cellulomonas sp. denitrifiers could degrade woodchips to provide carbon source and electron donors to themselves and other denitrifiers in woodchip bioreactors at low-temperature conditions. By inoculating these denitrifiers (i.e., bioaugmentation), it might be possible to increase the nitrate removal rate of woodchip bioreactors at low-temperature conditions.


April 21, 2020  |  

Single-molecule sequencing detection of N6-methyladenine in microbial reference materials.

The DNA base modification N6-methyladenine (m6A) is involved in many pathways related to the survival of bacteria and their interactions with hosts. Nanopore sequencing offers a new, portable method to detect base modifications. Here, we show that a neural network can improve m6A detection at trained sequence contexts compared to previously published methods using deviations between measured and expected current values as each adenine travels through a pore. The model, implemented as the mCaller software package, can be extended to detect known or confirm suspected methyltransferase target motifs based on predictions of methylation at untrained contexts. We use PacBio, Oxford Nanopore, methylated DNA immunoprecipitation sequencing (MeDIP-seq), and whole-genome bisulfite sequencing data to generate and orthogonally validate methylomes for eight microbial reference species. These well-characterized microbial references can serve as controls in the development and evaluation of future methods for the identification of base modifications from single-molecule sequencing data.


April 21, 2020  |  

Comparative Genomic Analyses Reveal Core-Genome-Wide Genes Under Positive Selection and Major Regulatory Hubs in Outlier Strains of Pseudomonas aeruginosa.

Genomic information for outlier strains of Pseudomonas aeruginosa is exiguous when compared with classical strains. We sequenced and constructed the complete genome of an environmental strain CR1 of P. aeruginosa and performed the comparative genomic analysis. It clustered with the outlier group, hence we scaled up the analyses to understand the differences in environmental and clinical outlier strains. We identified eight new regions of genomic plasticity and a plasmid pCR1 with a VirB/D4 complex followed by trimeric auto-transporter that can induce virulence phenotype in the genome of strain CR1. Virulence genotype analysis revealed that strain CR1 lacked hemolytic phospholipase C and D, three genes for LPS biosynthesis and had reduced antibiotic resistance genes when compared with clinical strains. Genes belonging to proteases, bacterial exporters and DNA stabilization were found to be under strong positive selection, thus facilitating pathogenicity and survival of the outliers. The outliers had the complete operon for the production of vibrioferrin, a siderophore present in plant growth promoting bacteria. The competence to acquire multidrug resistance and new virulence factors makes these strains a potential threat. However, we identified major regulatory hubs that can be used as drug targets against both the classical and outlier groups.


April 21, 2020  |  

Genome plasticity favours double chromosomal Tn4401b-blaKPC-2 transposon insertion in the Pseudomonas aeruginosa ST235 clone.

Pseudomonas aeruginosa Sequence Type 235 is a clone that possesses an extraordinary ability to acquire mobile genetic elements and has been associated with the spread of resistance genes, including genes that encode for carbapenemases. Here, we aim to characterize the genetic platforms involved in resistance dissemination in blaKPC-2-positive P. aeruginosa ST235 in Colombia.In a prospective surveillance study of infections in adult patients attended in five ICUs in five distant cities in Colombia, 58 isolates of P. aeruginosa were recovered, of which, 27 (46.6%) were resistant to carbapenems. The molecular analysis showed that 6 (22.2%) and 4 (14.8%) isolates harboured the blaVIM and blaKPC-2 genes, respectively. The four blaKPC-2-positive isolates showed a similar PFGE pulsotype and belonged to ST235. Complete genome sequencing of a representative ST235 isolate shows a unique chromosomal contig of 7097.241?bp with eight different resistance genes identified and five transposons: a Tn6162-like with ant(2?)-Ia, two Tn402-like with ant(3?)-Ia and blaOXA-2 and two Tn4401b with blaKPC-2. All transposons were inserted into the genomic islands. Interestingly, the two Tn4401b copies harbouring blaKPC-2 were adjacently inserted into a new genomic island (PAGI-17) with traces of a replicative transposition process. This double insertion was probably driven by several structural changes within the chromosomal region containing PAGI-17 in the ST235 background.This is the first report of a double Tn4401b chromosomal insertion in P. aeruginosa, just within a new genomic island (PAGI-17). This finding indicates once again the great genomic plasticity of this microorganism.


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