An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi.

We report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted “bovine-type” allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species

The influence of extrachromosomal elements in the anthrax “cross-over” strain Bacillus cereus G9241

Bacillus cereus G9241 was isolated from a welder who survived a pulmonary anthrax-like disease. Strain G9241 carries two virulence plasmids, pBCX01 and pBC210, as well as an extrachromosomal prophage, pBFH_1. pBCX01 has 99.6% sequence identity to pXO1 carried by Bacillus anthracis and encodes the tripartite anthrax toxin genes and atxA, a mammalian virulence transcriptional regulator. This work looks at how the presence of pBCX01 and temperature may affect the lifestyle of B. cereus G9241 using a transcriptomic analysis and by studying spore formation, an important part of the B. anthracis lifecycle. Here we report that pBCX01 has a stronger effect on gene transcription at the mammalian infection relevant temperature of 37°C in comparison to 25°C. At 37°C, the presence of pBCX01 appears to have a negative effect on genes involved in cell metabolism, including biosynthesis of amino acids, whilst positively affecting the transcription of many transmembrane proteins. The study of spore formation showed B. cereus G9241 sporulated rapidly in comparison to the B. cereus sensu stricto type strain ATCC 14579, particularly at 37°C. The carriage of pBCX01 did not affect this phenotype suggesting that other genetic elements were driving rapid sporulation. An unexpected finding of this study was that pBFH_1 is highly expressed at 37°C in comparison to 25°C and pBFH_1 expression leads to the production of Siphoviridae-like phage particles in the supernatant of B. cereus G9241. This study provides an insight on how the extrachromosomal genetic elements in B. cereus G9241 has an influence in bacterial phenotypes

From cereus to anthrax and back again : the role of the PlcR regulator in the “cross-over” strain Bacillus cereus G9241

The plcR gene, which encodes the pleiotropic transcriptional regulator of secreted proteins found in most members of the Bacillus cereus group, is truncated in all Bacillus anthracis isolates. The current dogma suggests this truncation was evolved to accommodate the acquisition of the anthrax toxin regulator, AtxA. However, the B. cereus-B. anthracis “cross-over” strain Bacillus cereus G9241, isolated from a Louisiana welder suffering from an anthrax-like infection, appears to contradict the proposed dogma as it encodes intact copies of both regulators. Here we report that when cultured at 25 °C, cell free B. cereus G9241 culture supernatants are cytotoxic and haemolytic to various eukaryotic cells in addition to insect haemocytes from Manduca sexta. However, this cytotoxic and haemolytic activity of the culture supernatant is lost when the bacteria are grown at 37 °C, behaving much like the supernatants generated by B. anthracis. Using a combination of genetic and proteomic approaches, we identified several PlcR-regulated toxins secreted at 25 °C. We demonstrate that a limiting step for the production of these virulence factors at 37 °C exists within the PlcR-PapR regulation circuit in strain G9241, giving rise to the temperature-dependent haemolytic and cytotoxic activity of the culture supernatants. Differential expression of the protease responsible in processing the PlcR quorum sensing activator PapR appears to be responsible for this phenotype. This study confirms that B. cereus G9241 is able to ‘switch’ between B. cereus and B. anthracis–like phenotypes in a temperature-dependent manner, potentially accommodating the activities of both PlcR and AtxA

From cereus to anthrax and back again: Assessment of the temperature-dependent phenotypic switching in the "cross-over" strain Bacillus cereus G9241

Bacillus cereus G9241 was isolated from a Louisiana welder suffering from an anthrax-like infection. The organism carries two transcriptional regulators that have previously been proposed to be incompatible with each other in Bacillus anthracis: the pleiotropic transcriptional regulator PlcR found in most members of the Bacillus cereus group but truncated in all B. anthracis isolates, and the anthrax toxin regulator AtxA found in all B. anthracis strains and a few B. cereus sensu stricto strains. Here we report cytotoxic and hemolytic activity of cell free B. cereus G9241 culture supernatants cultured at 25°C to various eukaryotic cells. However, this is not observed at the mammalian infection relevant temperature 37°C, behaving much like the supernatants generated by B. anthracis. Using a combination of genetic and proteomic approaches to understand this unique phenotype, we identified several PlcR-regulated toxins to be secreted highly at 25°C compared to 37°C. Furthermore, results suggest that differential expression of the protease involved in processing the PlcR quorum sensing activator molecule PapR appears to be the limiting step for the production of PlcR-regulated toxins at 37°C, giving rise to the temperature-dependent hemolytic and cytotoxic activity of the culture supernatants. This study provides an insight on how B. cereus G9241 is able to “switch” between B. cereus and B. anthracis–like phenotypes in a temperature-dependent manner, potentially accommodating the activities of both PlcR and AtxA

A new family of globally distributed lytic roseophages with unusual deoxythymidine to deoxyuridine substitution

Marine bacterial viruses (bacteriophages) are abundant biological entities that are vital for shaping microbial diversity, impacting marine ecosystem function, and driving host evolution.1, 2, 3 The marine roseobacter clade (MRC) is a ubiquitous group of heterotrophic bacteria[4],[5] that are important in the elemental cycling of various nitrogen, sulfur, carbon, and phosphorus compounds.6, 7, 8, 9, 10 Bacteriophages infecting MRC (roseophages) have thus attracted much attention and more than 30 roseophages have been isolated,11, 12, 13 the majority of which belong to the N4-like group (Podoviridae family) or the Chi-like group (Siphoviridae family), although ssDNA-containing roseophages are also known.[14] In our attempts to isolate lytic roseophages, we obtained two new phages (DSS3_VP1 and DSS3_PM1) infecting the model MRC strain Ruegeria pomeroyi DSS-3. Here, we show that not only do these phages have unusual substitution of deoxythymidine with deoxyuridine (dU) in their DNA, but they are also phylogenetically distinct from any currently known double-stranded DNA bacteriophages, supporting the establishment of a novel family (“Naomiviridae”). These dU-containing phages possess DNA that is resistant to the commonly used library preparation method for metagenome sequencing, which may have caused significant underestimation of their presence in the environment. Nevertheless, our analysis of Tara Ocean metagenome datasets suggests that these unusual bacteriophages are of global importance and more diverse than other well-known bacteriophages, e.g., the Podoviridae in the oceans, pointing to an overlooked role for these novel phages in the environment

The Genome of a Pathogenic Rhodococcus: Cooptive Virulence Underpinned by Key Gene Acquisitions

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi

Photorhabdus asymbiotica as an insect and human pathogen

Photorhabdus asymbiotica is a species of bacterium that is pathogenic to humans whilst retaining the ability to infect insect hosts. Currently, there are two recognised subspecies, P. asymbiotica subsp. asymbiotica and P. asymbiotica subsp. australis with strains isolated from various locations in the USA, Australia, Thailand, Nepal and Europe. Like other species of Photorhabdus, P. asymbiotica subsp. australis was shown to form a symbiotic relationship with a Heterorhabditis nematode. In contrast to most strains of Photorhabdus luminescens, P. asymbiotica can grow at 37 °C and this is a defining factor in its ability to cause human disease. Insights into other adaptations it has undergone that have enabled host switching to occur have come from whole genome sequencing and transcriptomic studies. P. asymbiotica has a smaller genome compared to P. luminenscens with a lower diversity of insecticidal toxins. However, it has acquired plasmids and several pathogenicity islands in its genome. These encode genes with similarity to effectors or systems found in other known human pathogens such as Salmonella and Yersinia and are therefore likely to contribute to human pathogenicity. Of crucial importance to virulence is the fact that P. asymbiotica undergoes a large metabolic shift at the human host temperature