A eukaryotic-like ubiquitination system in bacterial antiviral defence.

Ubiquitination pathways have crucial roles in protein homeostasis, signalling and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2 and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6, but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here we demonstrate that a bacterial operon associated with phage defence islands encodes a complete ubiquitination pathway. Two structures of a bacterial E1-E2-Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 possesses an amino-terminal inactive adenylation domain and a carboxy-terminal active adenylation domain with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C terminus positioned for adenylation, and a second structure mimics an E1-to-E2 transthioesterification state with the E1 CYS domain adjacent to the bound E2. We show that a deubiquitinase in the same pathway preprocesses the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.

The phage defence island of a multidrug resistant plasmid uses both BREX and type IV restriction for complementary protection from viruses.

Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.

The complete genome sequence of Escherichia phage SRT7, a novel T7-like phage.

In this study, we isolated a novel virulent Escherichia phage, SRT7. Its genome is a double-stranded linear DNA molecule containing 39,883 bp. Direct terminal repeats with a length of 175 bp, are present at both ends of the genome. The G+C content is 50.54%. Forty-seven putative protein coding genes were identified. No tRNA or rRNA genes were identified. Comparative genomic analysis revealed that phage SRT7 is a novel member of the T7-like phage cluster, but it forms a singleton subcluster.

Giant viral genomic signatures in the previously reported gut metagenomes of pre-school children in rural India.

A recent study by Ghosh et al. compared the gut microbiomes of 20 preschool children from India and found an association between the gut microbiome and the nutritional status of the child. Here, we explored these metagenomes for the presence of genomic signatures of prokaryotic and eukaryotic viruses. Several of the viral signatures found in all 20 metagenomes belonged to giant viruses (GVs). In addition, we found hits for bacteriophages to several major human pathogens, including Shigella, Salmonella, Escherichia, and Enterobacter. Concurrently, we also detected several antibiotic resistance genes (ARGs) in the metagenomes. All of the ARGs detected in this study (beta-lactam, macrolide, metronidazole, and tetracycline) are associated with mobile genetic elements (MGEs) and have been reported to cause high levels of resistance to their respective antibiotics. Despite recent reports of giant viruses and their genomic signatures in gut microbiota, their role in human physiology remains poorly understood. The effect of cooccurrence of ARGs and GVs in the gut needs further investigation.

Isolation and characterization of a novel temperate bacteriophage from gut-associated Escherichia within black soldier fly larvae (Hermetia illucens L. [Diptera: Stratiomyidae]).

To gain insight into the presence and nature of prophages in the black soldier fly (BSF; Hermetia illucens L. [Diptera: Stratiomyidae]) gut, we isolated and characterized a novel, temperate Escherichia bacteriophage designated vB_EcoS_PHB10 (PHB10). Electron microscopy analysis revealed that phage PHB10 has a long, flexible, non-contractile tail and belongs to the family Siphoviridae. The phage was found to be stable over a wide range of temperatures (4-37 °C) and pH values (pH 5-9), and it lysed two out of 13 Escherichia strains tested. The genome of PHB10 contains genes encoding a putative transcriptional regulator and an integrase, and it shows a high degree of similarity to a region of the Enterobacter cloacae MBRL1077 genome. Induction experiments revealed that phage PHB10 could be induced by different gut substrates, suggesting that diet might be a potential regulator of lytic/lysogenic switches in commensal lysogens.

Alterations in the diversity and composition of mice gut microbiota by lytic or temperate gut phage treatment.

Phages, the most abundant species in the mammalian gut, have numerous advantages as biocontrol agent over antibiotics. In this study, mice were orally treated with the lytic gut phage PA13076 (group B), the temperate phage BP96115 (group C), no phage (group A), or streptomycin (group D) over 31 days. At the end of the experiment, fecal microbiota diversity and composition was determined and compared using high-throughput sequencing of the V3-V4 hyper-variable region of the 16S rRNA gene and virus-like particles (VLPs) were quantified in feces. There was high diversity and richness of microbiota in the lytic and temperate gut phage-treated mice, with the lytic gut phage causing an increased alpha diversity based on the Chao1 index (p < 0.01). However, the streptomycin treatment reduced the microbiota diversity and richness (p = 0.0299). Both phage and streptomycin treatments reduced the abundance of Bacteroidetes at the phylum level (p < 0.01) and increased the abundance of the phylum Firmicutes. Interestingly, two beneficial genera, Lactobacillus and Bifidobacterium, were enhanced by treatment with the lytic and temperate gut phage. The abundance of the genus Escherichia/Shigella was higher in mice after temperate phage administration than in the control group (p < 0.01), but lower than in the streptomycin group. Moreover, streptomycin treatment increased the abundance of the genera Klebsiella and Escherichia/Shigella (p < 0.01). In terms of the gut virome, fecal VLPs did not change significantly after phage treatment. This study showed that lytic and temperate gut phage treatment modulated the composition and diversity of gut microbiota and the lytic gut phage promoted a beneficial gut ecosystem, while the temperate phage may promote conditions enabling diseases to occur.

Three recently acknowledged Escherichia species strikingly differ in the incidence of bacteriocinogenic and lysogenic strains.

The incidence of bacteriocinogeny and lysogeny was followed in bacteria of 3 recently acknowledged species of the genus Escherichia: E. hermanii, E. vulneris and E. fergusonii. Almost all of the strains examined were of human origin. In 30 strains of E. hermanii no one was found bacteriocinogenic while 57% were lysogenic, in 30 strains of E. vulneris none was found to be bacteriocinogenic and only 10% were lysogenic, and in 50 strains E. fergusonii 12% were bacteriocinogenic and 40% lysogenic. From the 6 bacteriocinogenic strains of E. fergusonii, 3 were producers of colicin E1, 2 of colicin Ib and 1 of colicin Ia. In addition, 3 E. fergusonii strains produced aerobactin.

An active type IV secretion system encoded by the F plasmid sensitizes Escherichia coli to bile salts.

F(+) strains of Escherichia coli infected with donor-specific bacteriophage such as M13 are sensitive to bile salts. We show here that this sensitivity has two components. The first derives from secretion of bacteriophage particles through the cell envelope, but the second can be attributed to expression of the F genes required for the formation of conjugative (F) pili. The latter component was manifested as reduced or no growth of an F(+) strain in liquid medium containing bile salts at concentrations that had little or no effect on the isogenic F(-) strain or as a reduced plating efficiency of the F(+) strain on solid media; at 2% bile salts, plating efficiency was reduced 10(4)-fold. Strains with F or F-like R factors were consistently more sensitive to bile salts than isogenic, plasmid-free strains, but the quantitative effect of bile salts depended on both the plasmid and the strain. Sensitivity also depended on the bile salt, with conjugated bile salts (glycocholate and taurocholate) being less active than unconjugated bile salts (deoxycholate and cholate). F(+) cells were also more sensitive to sodium dodecyl sulfate than otherwise isogenic F(-) cells, suggesting a selectivity for amphipathic anions. A mutation in any but one F tra gene required for the assembly of F pili, including the traA gene encoding F pilin, substantially restored bile salt resistance, suggesting that bile salt sensitivity requires an active system for F pilin secretion. The exception was traW. A traW mutant was 100-fold more sensitive to cholate than the tra(+) strain but only marginally more sensitive to taurocholate or glycocholate. Bile salt sensitivity could not be attributed to a generalized change in the surface permeability of F(+) cells, as judged by the effects of hydrophilic and hydrophobic antibiotics and by leakage of periplasmic beta-lactamase into the medium.