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The Tn3family of transposons includes diverse elements that encode homologous transposases and contain conserved terminal inverted repeat sequences (IRs). The recent identification of non-autonomous elements, named TIMEs (Tn3-derived Inverted-repeat Miniature Elements), has shed new light on the diversity and evolution of this transposon family. A common feature of TIMEs and other members of this family is their ability to mobilize genomic DNA for transposition as part of composite transposons. These elements significantly influence the structure and properties of plasmids and other mobile genetic elements (MGEs). They may contain and move by transposition (i) plasmid replication systems, (ii) toxin-antitoxin systems and (iii) site-specific recombination modules that can resolve plasmid multimers. Some Tn3family elements may also transfer large segments of chromosomal DNA into plasmids, which increases the pool of mobile DNA that can take part in horizontal gene transfer. [ABSTRACT FROM PUBLISHER]

Functional transposable elements (TEs) of several Pseudomonas spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons – Tn5563a and Tn4662a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a res site typical for many Tn3-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements. [ABSTRACT FROM AUTHOR]

Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria. [ABSTRACT FROM AUTHOR]

Short modified oligonucleotides that bind in a sequence-specific way to messenger RNA essential for bacterial growth could be useful to fight bacterial infections. One such promising oligonucleotide is peptide nucleic acid (PNA), a synthetic DNA analog with a peptide-like backbone. However, the limitation precluding the use of oligonucleotides, including PNA, is that bacteria do not import them from the environment. We have shown that vitamin B 12 , which most bacteria need to take up for growth, delivers PNAs to Escherichia coli cells when covalently linked with PNAs. Vitamin B 12 enters E. coli via a TonB-dependent transport system and is recognized by the outer-membrane vitamin B 12 -specific BtuB receptor. We engineered the E. coli ΔbtuB mutant and found that transport of the vitamin B 12 -PNA conjugate requires BtuB. Thus, the conjugate follows the same route through the outer membrane as taken by free vitamin B 12 . From enhanced sampling all-atom molecular dynamics simulations, we determined the mechanism of conjugate permeation through BtuB. BtuB is a β-barrel occluded by its luminal domain. The potential of mean force shows that conjugate passage is unidirectional and its movement into the BtuB β-barrel is energetically favorable upon luminal domain unfolding. Inside BtuB, PNA extends making its permeation mechanically feasible. BtuB extracellular loops are actively involved in transport through an induced-fit mechanism. We prove that the vitamin B 12 transport system can be hijacked to enable PNA delivery to E. coli cells., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Paracoccus denitrificans Pd 1222 is a model methylotrophic bacterium. Its methylotrophy is based on autotrophic growth (enabled by the Calvin cycle) supported by energy from the oxidation of methanol or methylamine. The growing availability of genome sequence data has made it possible to investigate methylotrophy in other Paracoccus species . The examination of a large number of Paracoccus spp. genomes reveals great variability in C1 metabolism, which have been shaped by different evolutionary mechanisms. Surprisingly, the methylotrophy schemes of many Paracoccus strains appear to have quite different genetic and biochemical bases. Besides the expected 'autotrophic methylotrophs', many strains of this genus possess another C1 assimilatory pathway, the serine cycle, which seems to have at least three independent origins. Analysis of the co-occurrence of different methylotrophic pathways indicates, on the one hand, evolutionary linkage between the Calvin cycle and the serine cycle, and, on the other hand, that genes encoding some C1 substrate-oxidizing enzymes occur more frequently in association with one or the other. This suggests that some genetic module combinations form more harmonious enzymatic sets, which act with greater efficiency in the methylotrophic process and thus undergo positive selection.

Short modified oligonucleotides targeted at bacterial DNA or RNA could serve as antibacterial agents provided that they are efficiently taken up by bacterial cells. However, the uptake of such oligonucleotides is hindered by the bacterial cell wall. To overcome this problem, oligomers have been attached to cell-penetrating peptides, but the efficiency of delivery remains poor. Thus, we have investigated the ability of vitamin B 12 to transport peptide nucleic acid (PNA) oligomers into cells of Escherichia coli and Salmonella Typhimurium. Vitamin B 12 was covalently linked to a PNA oligomer targeted at the mRNA of a reporter gene expressing Red Fluorescent Protein. Cu-catalyzed 1,3-dipolar cycloaddition was employed for the synthesis of PNA-vitamin B 12 conjugates; namely the vitamin B 12 azide was reacted with PNA possessing the terminal alkyne group. Different types of linkers and spacers between vitamin B 12 and PNA were tested, including a disulfide bond. We found that vitamin B 12 transports antisense PNA into E. coli cells more efficiently than the most widely used cell-penetrating peptide (KFF) 3 K. We also determined that the structure of the linker impacts the antisense effect. The results of this study provide the foundation for developing vitamin B 12 as a carrier of PNA oligonucleotides into bacterial cells.

The Tn 3 family of transposons includes diverse elements that encode homologous transposases and contain conserved terminal inverted repeat sequences (IRs). The recent identification of non-autonomous elements, named TIMEs (Tn 3 -derived Inverted-repeat Miniature Elements), has shed new light on the diversity and evolution of this transposon family. A common feature of TIMEs and other members of this family is their ability to mobilize genomic DNA for transposition as part of composite transposons. These elements significantly influence the structure and properties of plasmids and other mobile genetic elements (MGEs). They may contain and move by transposition (i) plasmid replication systems, (ii) toxin-antitoxin systems and (iii) site-specific recombination modules that can resolve plasmid multimers. Some Tn 3 family elements may also transfer large segments of chromosomal DNA into plasmids, which increases the pool of mobile DNA that can take part in horizontal gene transfer.