The Type IV Secretion System of ICEAfe1: Formation of a Conjugative Pilus in Acidithiobacillus ferrooxidans

Abstract

The dispersal of mobile genetic elements and their gene cargo relies on type IV secretion systems (T4SS). In this work the ICEAfe1 Tra-type T4SS nanomachine, encoded in the publicly available genome of Acidithiobacillus ferrooxidans ATCC 23270TY, was characterized in terms of its organization, conservation, expression and mating bridge formation. Twenty-one conjugative genes grouped in four genetic clusters encode the ICEAfe1 T4SS, containing all the indispensable functions for the formation and stabilization of the pili and for DNA processing. The clusters’ organization resembles that of other mobile genetic elements (such as plasmids and integrative and conjugative elements–ICEs). Sequence conservation, genetic organization and distribution of the tra system in the genomes of other sequenced Acidithiobacillus spp. suggests that the ICEAfe1 T4SS could mediate the lateral gene transfer between related bacteria. All ICEAfe1 T4SS genes are transcriptionally active and expressed from four independent operons. The transcriptional levels of selected marker genes increase in response to Mitomycin C treatment, a DNA damage elicitor that has acknowledged stimulatory effects on excision rates and gene expression of other ICEs, including ICEAfe1. Using a tailor-made pilin-antiserum against ICEAfe1 T4SS TraA pilin and epifluorescence microscopy, the presence of the conjugative pili on the cell surface of A. ferrooxidans could be demonstrated. Additionally, immunodetection assays, by immunogold, allowed the identification of pili-like extracellular structures. Together, the results obtained in this work demonstrate that the ICEAfe1 T4SS is phylogenetically conserved within the taxon, is expressed at mRNA and protein levels in vivo in the A. ferrooxidans type strain, and produces a pili-like structure of extracellular and intercellular localization in this model acidophile, supporting its functionality. Additional efforts will be required to prove conjugation of the ICEAfe1 or parts of this element through the cognate T4SS.

CRISPR-Cas systems restrict horizontal gene transfer in Pseudomonas aeruginosa

“…4A). Crucially, spacers targeting conserved components of the conjugative machinery (tra genes, trb genes, type IV secretion system genes [84][85][86]) were found in 26/56 (46%) of STs that were associated with predicted functional CRISPR-Cas systems (Fig. 4B).…”

The Flexible Genome of Acidophilic Prokaryotes

“…Its genetic organization resembles that of other well-characterised integrative conjugative elements (ICEs), and includes integration/excision, conjugative DNA processing, transfer, maintenance and regulation proteins organized in a number of discrete gene modules. Excision of the element, expression of a complete set of genes encoding self-transfer functions under normal and DNA-damaging growth conditions and detection of ICEAfe1 T4SS TraA in a pilin pili-like structure of extracellular and intercellular localization, suggests this element is indeed functional (Bustamante et al, 2012;Flores-Ríos et al, 2019b). As in most mobile genetic elements, approximately 60% of its ORFs encode proteins with unknown or hypothetical functions, but additional genes encoding features that may confer selective advantages to the host strain have been identified, including a exopolysacharide biosinthesis cluster a CRISPR-Cas system and a cluster of 36 tRNA genes covering all 20 amino acids.…”

“…MGEs are mostly DNA-based platforms that encode enzymes and other proteins that mediate or facilitate self movement and that support different genetic cargos. The full set of MGEs of a microorganism and the genes they carry constitute the mobilome (Frost et al, 2005) or the flexible genomic compartment (Mira et al, 2014). Research in microbial MGEs has progressed rapidly over the last couple of decades.…”

Endogenous and Foreign Nucleoid-Associated Proteins of Bacteria: Occurrence, Interactions and Effects on Mobile Genetic Elements and Host's Biology

“…Many additional, as of yet uncharacterized, NAPs and NAP-like genes can be traced to reported annotations of integrated genomic islands (e.g. [51,156]) or available sequences in MGE data repositories (e.g. ICEBerg database [93]).…”