Hurst, M. R. H. | bioRxiv Science

bioRxiv2023

Lysis cassette-mediated exoprotein release in Yersinia entomophaga is controlled by a PhoB-like regulator

Secretion of exoproteins is a key component of bacterial virulence and is tightly regulated in response to environmental stimuli and host-dependent signals. The entomopathogenic bacterium Yersinia entomophaga MH96 produces a wide range of exoproteins including its main virulence factor, the 2.46 MDa insecticidal Yen-Tc toxin complex. Previously, a high-throughput transposon-based screening assay identified the region of exoprotein release (YeRER) as essential to exoprotein release in MH96. The current study defines the role of the YeRER-associated ambiguous holin/endolysin-based lysis cluster (ALC) and the novel RoeA regulator in the regulation and release of exoproteins in MH96. A mutation in the ALC region abolished exoprotein release and caused cell elongation, a phenotype able to be restored through trans-complementation with an intact ALC region. Endogenous ALC did not impact cell growth of the wild type, while artificial expression of an optimised ALC caused cell lysis. Using HolA-sfGFP and Rz1-sfGFP reporter, Rz1 expression was observed in all cells while HolA expression was limited to a small proportion of cells, which increased over time. Transcriptomic assessments found expression of the genes encoding the prominent exoproteins, including the Yen-Tc, was reduced in the roeA mutant and identified a 220 ncRNA of the YeRER intergenic that, when trans complemented in the wildtype, abolished exoprotein release. A model for Y. entomophaga mediated exoprotein regulation and release is proposed. ImportanceWhile theoretical models exist, there is not yet any empirical data that links ALC phage-like lysis cassettes with the release of large macro-molecular toxin complexes such as Yen-Tc in Gram-negative bacteria. In this study, we demonstrate that the novel Y. entomophaga RoeA activates the production of exoproteins (including Yen-Tc) and the ALC at the transcriptional level. The translation of the ALC holin is confined to a subpopulation of cells that then lyse over time, indicative of a complex hierarchical regulatory network. The presence of orthologous RoeA orthologue and a HolA like holin 5 of an eCIS Afp element in Pseudomonas chlororaphis combined with the presented data suggests a shared mechanism is required for the release of some large macromolecular protein assemblies such as the Yen-Tc, and further supports classification of phage-like lysis clusters as type 10 secretion systems.

microbiology↗

bioRxiv2022

Isolation, purification, and characterisation of a phage tail-like bacteriocin from the insect pathogenic bacterium Brevibacillus laterosporus

The gram-positive and spore-forming bacterium Brevibacillus laterosporus (Bl) belongs to the Brevibacillus brevis phylogenetic cluster. Isolates of the species have demonstrated pesticidal potency against a wide range of invertebrate pests and plant diseases. Two New Zealand isolates, Bl 1821L and Bl 1951, are under development as biopesticides for control of diamondback moth and other pests. However, due to often restricted growth of these endemic isolates, production can be an issue. During investigations of the cause of the disrupted growth, electron micrographs of crude lysate of Bl 1821L showed the presence of phages tail-like structures. PEG 8000 precipitated lysate harbouring the putative phage tail-like particles demonstrated broad-spectrum activity against several gram-positive bacteria. SDS-PAGE of purified and concentrated lysate showed a prominent protein band of [~]48 kD from where transmission electron microscopy revealed the presence of polysheath-like structures. N-terminal sequencing of the [~]48 kD protein mapped to a gene with weak predicted amino acid homology to a Bacillus PBSX phage-like element xkdK, the translated product of which shared >90% amino acid similarity to the phage tail-sheath protein of another Bl published genome, LMG15441. An xkdK homolog was also identified in the Bl 1951 genome. However, genome comparison of the region around the xkdK gene between Bl 1821L and Bl 1951 found differences including two glycine rich protein encoding genes which contain imperfect repeats (1700 bp) in Bl 1951, while a putative phage region resides in the analogous Bl 1821L region. Although comparative analysis of the genomic organisation of Bl 1821L and Bl 1951 PBSX-like region with the defective phages PBSX, PBSZ, and PBP 180 of Bacillus subtilis isolates 168 and W23, and Bacillus phage PBP180 revealed low amino acids similarity, the genes encode similar functional proteins in similar arrangements, including phage tail-sheath (XkdK), tail (XkdO), holin (XhlB), and N-acetylmuramoyl-L-alanine (XlyA). AMPA analysis identified a bactericidal stretch of 13 amino acids in the [~]48 kD sequenced protein of Bl 1821L. Assays of purified [~]48 kD protein of Bl 1821L caused a decrease of 34.2% in the number of viable cells of Bl 1951, 18 hours after treatment as compared to the control. Significance of the studyThis study for the first time isolated, purified, and characterised putative phage tail-like bacteriocins (PTLBs) from the insect pathogenic isolates of Brevibacillus laterosporus. Identified PTLBs caused a decrease in the number of viable cells of Bl 195, 18 hours after treatment as compared to control. Therefore, it is likely that the putative PTLBs might have implications in harnessing the insecticidal potential of this useful bacterium.

microbiology↗