Properties of genes encoding transfer RNAs as integration sites for genomic islands and prophages in <i>Klebsiella pneumoniae</i>

ABSTRACTThe evolution of traits including antibiotic resistance, virulence, and increased fitness in Klebsiella pneumoniae and related species has been linked to the acquisition of mobile genetic elements through horizontal transfer. Among them, genomic islands (GIs) preferentially integrating at genes encoding tRNAs and the tmRNA (t(m)DNAs) would be significant in promoting chromosomal diversity. Here, we studied the whole set of t(m)DNAs present in 66 Klebsiella chromosomes, investigating their usage as integration sites and the properties of the integrated GIs. A total of 5,624 t(m)DNAs were classified based on their sequence conservation, genomic context, and prevalence. 161 different GIs and prophages were found at these sites, hosting 3,540 gene families including various related to virulence and drug resistance. Phylogenetic analyses supported the acquisition of several of these elements through horizontal gene transfer, likely mediated by a highly diverse set of encoded integrases targeting specific t(m)DNAs and sublocations inside them. Only a subset of the t(m)DNAs had integrated GIs and even identical tDNA copies showed dissimilar usage frequencies, suggesting that the genomic context would influence the integration site selection. This usage bias, likely towards avoiding disruption of polycistronic transcriptional units, would be conserved across Gammaproteobacteria. The systematic comparison of the t(m)DNAs across different strains allowed us to discover an unprecedented number of K. pneumoniae GIs and prophages and to raise important questions and clues regarding the fundamental properties of t(m)DNAs as targets for the integration of mobile genetic elements and drivers of bacterial genome evolution and pathogen emergence.</jats:p

In-Depth Genomic and Phenotypic Characterization of the Antarctic Psychrotolerant Strain Pseudomonas sp. MPC6 Reveals Unique Metabolic Features, Plasticity, and Biotechnological Potential

We obtained the complete genome sequence of the psychrotolerant extremophile Pseudomonas sp. MPC6, a natural Polyhydroxyalkanoates (PHAs) producing bacterium able to rapidly grow at low temperatures. Genomic and phenotypic analyses allowed us to situate this isolate inside the Pseudomonas fluorescens phylogroup of pseudomonads as well as to reveal its metabolic versatility and plasticity. The isolate possesses the gene machinery for metabolizing a variety of toxic aromatic compounds such as toluene, phenol, chloroaromatics, and TNT. In addition, it can use both C6- and C5-carbon sugars like xylose and arabinose as carbon substrates, an uncommon feature for bacteria of this genus. Furthermore, Pseudomonas sp. MPC6 exhibits a high-copy number of genes encoding for enzymes involved in oxidative and cold-stress response that allows it to cope with high concentrations of heavy metals (As, Cd, Cu) and low temperatures, a finding that was further validated experimentally. We then assessed the growth performance of MPC6 on glycerol using a temperature range from 0 to 45°C, the latter temperature corresponding to the limit at which this Antarctic isolate was no longer able to propagate. On the other hand, the MPC6 genome comprised considerably less virulence and drug resistance factors as compared to pathogenic Pseudomonas strains, thus supporting its safety. Unexpectedly, we found five PHA synthases within the genome of MPC6, one of which clustered separately from the other four. This PHA synthase shared only 40% sequence identity at the amino acid level against the only PHA polymerase described for Pseudomonas (63-1 strain) able to produce copolymers of short- and medium-chain length PHAs. Batch cultures for PHA synthesis in Pseudomonas sp. MPC6 using sugars, decanoate, ethylene glycol, and organic acids as carbon substrates result in biopolymers with different monomer compositions. This indicates that the PHA synthases play a critical role in defining not only the final chemical structure of the biosynthesized PHA, but also the employed biosynthetic pathways. Based on the results obtained, we conclude that Pseudomonas sp. MPC6 can be exploited as a bioremediator and biopolymer factory, as well as a model strain to unveil molecular mechanisms behind adaptation to cold and extreme environments

The Ferric uptake regulator (Fur) and iron availability control the production and maturation of the antibacterial peptide microcin E492

© 2018 Marcoleta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Microcin E492 is a pore-forming bacteriocin with toxic activity against Enterobacteriaceae, which undergoes amyloid aggregation as a mechanism to regulate its toxicity. To be active, it requires the posttranslational attachment to the C-terminus of a glycosylated enterochelin derivative (salmochelin), a process carried out by the proteins MceC, MceI and MceJ encoded in the MccE492 gene cluster. Both microcin E492 and salmochelin have a proposed role in the virulence of the bacterial pathogen Klebsiella pneumoniae. Besides, enterochelin is produced as a response to low iron availability and its synthesis is controlled by the global iron regulator Fur. Since the production of active microcin E492 depends on enterochelin biosynthesi

Live-cell imaging of Salmonella Typhimurium interaction with zebrafish larvae after injection and immersion delivery methods

© 2017The zebrafish model has been used to determine the role of vertebrate innate immunity during bacterial infections. Here, we compare the in vivo immune response induced by GFP-tagged Salmonella Typhimurium inoculated by immersion and microinjection in transgenic zebrafish larvae. Our novel infection protocols in zebrafish allow live-cell imaging of Salmonella colonization

MceX negatively regulates the expression of the microcin E492 structural gene.

<p>(A) <i>wt</i> and Δ<i>fur E</i>. <i>coli</i> cells transformed with the reporter p<i>mceBA</i>’-‘<i>lacZ</i> were grown in M9 medium supplemented with 100 μM 2,2’-Bipyridyl (low iron availability) or 10 μM FeSO₄ (high iron availability). β-galactosidase activity (expressed in Miller Units) was measured at different growth phases (Early exponential, OD₆₀₀ = 0.4–0.6; Late exponential, OD₆₀₀ = 0.9–1.1; Stationary, OD₆₀₀ = 1.9–2.1). In the construct scheme, the orange circle represents the previously determined transcription start site. (B) Wild type <i>E</i>. <i>coli</i> cells were transformed with pT5-<i>mceX</i> (allowing the IPTG-inducible expression of MceX), or with the backbone plasmid pUC57 as negative control. The activity was expressed as the percentage of β-galactosidase activity after induction respect to the negative control. Error bars represent the standard deviation between 6 independent experiments. ***<i>P</i><0.001, ****<i>P</i><0.0001, ns: not significant.</p

The expression of <i>mceX</i> and the microcin E492 maturation genes <i>mceJI</i> is regulated by Fur and iron availability.

<p><i>wt</i> and Δ<i>fur E</i>. <i>coli</i> cells transformed with the reporters p<i>mceX</i>’-‘<i>lacZ</i> (A) or p<i>mceJ17</i>’-‘<i>lacZ</i> (B), were grown in M9 medium supplemented with 100 0μM 2,2’-Bipyridyl (low iron availability) or 10 μM FeSO₄ (high iron availability). β-galactosidase activity (expressed in Miller Units) was measured at different phases of growth (Early exponential, OD₆₀₀ = 0.4–0.6; Late exponential, OD₆₀₀ = 0.9–1.1; Stationary, OD₆₀₀ = 1.9–2.1). In the construct schemes, the orange circle represents the experimentally determined transcription start site and the red square represents the functional Fur box. Error bars represent the standard deviation between 6 independent experiments. ***<i>P</i><0.001, ****<i>P</i><0.0001, ns: not significant.</p

<i>mceX</i>, <i>mceJ</i> and <i>mceI</i> genes are transcribed as a single mRNA from a promoter located upstream of the <i>mceX</i> Fur box.

<p>(A) RT-PCR detection of a polycistronic mRNA harboring <i>mceX</i>, <i>mceJ</i> and <i>mceI</i>. The cDNA was prepared from total RNA of <i>E</i>. <i>coli</i> cells transformed with pJAM229. The amplicons originated from each primer pair are represented in the scheme as blue dashed lines, and are marked with white arrowheads in the corresponding gel section. Total RNA non-subjected to reverse transcription was used as negative control (-), while pJAM229 plasmid was used as positive control (+). (B) 5’ RACE assay to determine the transcription start site of the <i>mceX/mceJI</i> unit. Two primers were used (blue arrowheads), one hybridizing to <i>mceX</i> and the other hybridizing to <i>mceJ</i>. A band of 147–190 bp was enriched after TAP treatment when using <i>mceX</i> primer (orange arrowhead). Bands not enriched after TAP treatment were also observed using <i>mceX</i> or <i>mceJ</i> primers (white arrowheads). Total RNA non-subjected to reverse transcription was used as negative control (-). (C) Two reporter constructs were obtained fusing <i>lacZ</i> to the last codon of <i>mceI</i> gene. The <i>mceJI’-‘lacZ</i> construct harbors <i>mceJ</i>, <i>mceX</i>, and its upstream region (including the TSS). The <i>mceJIΔTSS’-‘lacZ</i> construct is identical to <i>mceJI’-‘lacZ</i>, except for lacking a region (shaded in red) comprising the <i>mceX</i> promoter and part of its coding region. (D) β-galactosidase activity was measured in <i>E</i>. <i>coli</i> cells transformed with each of the reporter fusions and grown until different optical density. Error bars correspond to standard deviation of 3 independent experiments.</p

Fur overexpression reduces the antibacterial activity of <i>E</i>. <i>coli</i> cells carrying the gene cluster for microcin E492 production.

<p><i>E</i>. <i>coli</i> cells carrying pMccE492 (allowing the production of active MccE492) were transformed with p15A-<i>fur</i> (driving overexpression of Fur carrying a His-tag) or pACYC184 (control). (A) Two representative clones of p15A-<i>fur</i> containing cells were grown in M9 medium alone or supplemented with IPTG (1 mM), and total protein extracts were prepared after 3 and 24 h of growth. SDS-PAGE analysis of the extracts followed by Coomassie Blue staining revealed a ~17-kDa protein band largely enriched in cells grown in presence of IPTG (black arrow). Immunoblot using an anti-His antibody confirmed the induction of Fur expression in presence of IPTG, although a basal expression was detected after 24 h of growth in absence of inducer. (B) Antibacterial activity of <i>E</i>. <i>coli</i> cells producing MccE492, transformed with p15A-<i>fur</i> or a control plasmid (pACYC184). The antibacterial activity was measured as a function of the growth inhibition halo’s area over a sensitive strain layer, in M9 medium supplemented with IPTG, and then normalized to the value obtained in the control condition (plasmid backbone). Error bars indicate standard deviation from 40 measurements performed for each condition. ***<i>P</i><0.001.</p

Bacterial strains used in this work.

<p>Bacterial strains used in this work.</p

Diversity, Taxonomic Novelty, and Encoded Functions of Salar de Ascotán Microbiota, as Revealed by Metagenome-Assembled Genomes

Salar de Ascotán is a high-altitude arsenic-rich salt flat exposed to high ultraviolet radiation in the Atacama Desert, Chile. It hosts unique endemic flora and fauna and is an essential habitat for migratory birds, making it an important site for conservation and protection. However, there is limited information on the resident microbiota’s diversity, genomic features, metabolic potential, and molecular mechanisms that enable it to thrive in this extreme environment. We used long- and short-read metagenomics to investigate the microbial communities in Ascotán’s water, sediment, and soil. Bacteria predominated, mainly Pseudomonadota, Acidobacteriota, and Bacteroidota, with a remarkable diversity of archaea in the soil. Following hybrid assembly, we recovered high-quality bacterial (101) and archaeal (6) metagenome-assembled genomes (MAGs), including representatives of two putative novel families of Patescibacteria and Pseudomonadota and two novel orders from the archaeal classes Halobacteriota and Thermoplasmata. We found different metabolic capabilities across distinct lineages and a widespread presence of genes related to stress response, DNA repair, and resistance to arsenic and other metals. These results highlight the remarkable diversity and taxonomic novelty of the Salar de Ascotán microbiota and its rich functional repertoire, making it able to resist different harsh conditions. The highly complete MAGs described here could serve future studies and bioprospection efforts focused on salt flat extremophiles, and contribute to enriching databases with microbial genome data from underrepresented regions of our planet