A novel phage indirectly regulates diatom growth by infecting diatom-associated biofilm-forming bacterium

Algae and heterotrophic bacteria have close and intricate interactions, which are regulated by multiple factors in the natural environment. Phages are the major factor determining bacterial mortality. However, their impacts on the algae-associated bacteria and thus on the algae-bacteria interactions are poorly understood. Here, we obtained a diatom-associated bacterium Stappia indica SNL01 that could form biofilm and had an inhibitory effect on the growth of diatom Thalassiosira pseudonana . Meanwhile, the phage SI01 with a double-stranded circular DNA genome (44,247 bp) infecting S. indica SNL01 was isolated. Phylogenetic analysis revealed that phage SI01 represents a novel member of the Podoviridae family. The phage contained multiple lysis genes encoding for cell wall lysing muramidase and spore cortex lysing SleB, as well as depolymerase-like tail spike protein. By lysing the host bacterium and inhibiting the formation of biofilm, this phage could indirectly promote the growth of the diatom. Our results shed new insights into how phages indirectly regulate algal growth by infecting bacteria closely associated with algae or in the phycosphere. IMPORTANCE The impact of phage infection on the algae-bacteria relationship in the ocean is poorly understood. Here, a novel phage infecting the diatom-associated bacterium Stappia indica SNL01 was isolated. This bacterium could form biofilm and had a negative effect on diatom growth. We revealed that this phage contained multiple lysis genes and could inhibit the formation of bacterial biofilm, thus indirectly promoting diatom growth. This study implicates that phages are not only important regulators of bacteria but also have substantial indirect effects on algae as well as the algae-bacteria relationship.

Acquisition of the arginine deiminase system benefits epiparasitic Saccharibacteria and their host bacteria in a mammalian niche environment

Saccharibacteria are a group of widespread and genetically diverse ultrasmall bacteria with highly reduced genomes that belong to the Candidate Phyla Radiation. Comparative genomic analyses suggest convergent evolution of key functions enabling the adaptation of environmental Saccharibacteria to mammalian microbiomes. Currently, our understanding of this environment-to-mammal niche transition within Saccharibacteria and their obligate episymbiotic association with host bacteria is limited. Here, we identified a complete arginine deiminase system (ADS), found in further genome streamlined mammal-associated Saccharibacteria but missing in their environmental counterparts, suggesting acquisition during environment-to-mammal niche transition. Using TM7x, the first cultured Saccharibacteria strain from the human oral microbiome and its host bacterium Actinomyces odontolyticus, we experimentally tested the function and impact of the ADS. We demonstrated that by catabolizing arginine and generating adenosine triphosphate, the ADS allows metabolically restrained TM7x to maintain higher viability and infectivity when disassociated from the host bacterium. Furthermore, the ADS protects TM7x and its host bacterium from acid stress, a condition frequently encountered within the human oral cavity due to bacterial metabolism of dietary carbohydrates. Intriguingly, with a restricted host range, TM7x forms obligate associations with Actinomyces spp. lacking the ADS but not those carrying the ADS, suggesting the acquired ADS may also contribute to partner selection for cooperative episymbiosis within a mammalian microbiome. These data present experimental characterization of a mutualistic interaction between TM7x and their host bacteria, and illustrate the benefits of acquiring a novel pathway in the transition of Saccharibacteria to mammalian microbiomes.

A positive Selection Escherichia Coli Recombinant Protein Expression Vector for One-Step Cloning

We introduce OLIVAR (Orientation seLection of Insert in Vector through Antisense Reporter) as a novel selection strategy for the insertion of protein-coding genes into vector backbones. As a proof-of-concept, we have engineered a plasmid vector, pGRASS (Green fluorescent protein Reporter from Antisense promoter-based Screening System), for gene cloning in E. coli. With pGRASS, positive clones can be effortlessly distinguished from negative clones after blunt-end cloning. The vector not only screens clones with an insert but also for its correct orientation. The design further allows for the expression of recombinant protein from the T7 promoter in an appropriate host bacterium. With this vector, we are able to reduce the entire cloning workflow into a single step involving a 2-h reaction at room temperature. We believe that our cloning-cum-screening system presented here is extremely cost-effective and straightforward and can be applied to other vector systems and domains such as phage display and library construction.

Why are rhizobial symbiosis genes mobile?

Rhizobia are one of the most important and best studied groups of bacterial symbionts. They are defined by their ability to establish nitrogen-fixing intracellular infections within plant hosts. One surprising feature of this symbiosis is that the bacterial genes required for this complex trait are not fixed within the chromosome, but are encoded on mobile genetic elements (MGEs), namely plasmids or integrative and conjugative elements. Evidence suggests that many of these elements are actively mobilizing within rhizobial populations, suggesting that regular symbiosis gene transfer is part of the ecology of rhizobial symbionts. At first glance, this is counterintuitive. The symbiosis trait is highly complex, multipartite and tightly coevolved with the legume hosts, while transfer of genes can be costly and disrupt coadaptation between the chromosome and the symbiosis genes. However, horizontal gene transfer is a process driven not only by the interests of the host bacterium, but also, and perhaps predominantly, by the interests of the MGEs that facilitate it. Thus understanding the role of horizontal gene transfer in the rhizobium–legume symbiosis requires a ‘mobile genetic element's-eye view' on the ecology and evolution of this important symbiosis. This article is part of the theme issue ‘The secret lives of microbial mobile genetic elements’.

Are Bordetella bronchiseptica Siphoviruses (Genus Vojvodinavirus) Appropriate for Phage Therapy—Bacterial Allies or Foes?

Bordetella bronchiseptica is a respiratory animal pathogen that shows growing resistance to commonly used antibiotics, which has necessitated the examination of new antimicrobials, including bacteriophages. In this study, we examined the previously isolated and partially characterized B. bronchiseptica siphoviruses of the genus Vojvodinavirus (LK3, CN1, CN2, FP1 and MW2) for their ability to inhibit bacterial growth and biofilm, and we examined other therapeutically important properties through genomic analysis and lysogeny experiments. The phages inhibited bacterial growth at a low multiplicity of infection (MOI = 0.001) of up to 85% and at MOI = 1 for >99%. Similarly, depending on the phages and MOIs, biofilm formation inhibition ranged from 65 to 95%. The removal of biofilm by the phages was less efficient but still considerably high (40–75%). Complete genomic sequencing of Bordetella phage LK3 (59,831 bp; G + C 64.01%; 79 ORFs) showed integrase and repressor protein presence, indicating phage potential to lysogenize bacteria. Lysogeny experiments confirmed the presence of phage DNA in bacterial DNA upon infection using PCR, which showed that the LK3 phage forms more or less stable lysogens depending on the bacterial host. Bacterial infection with the LK3 phage enhanced biofilm production, sheep blood hemolysis, flagellar motility, and beta-lactam resistance. The examined phages showed considerable anti-B. bronchiseptica activity, but they are inappropriate for therapy because of their temperate nature and lysogenic conversion of the host bacterium.

Diverse Bacteriophages Infecting the Bacterial Striped Catfish Pathogen Edwardsiella ictaluri

Bacteriophages infecting Edwardsiella ictaluri have been less investigated, although the host bacterium is one of the most important fish pathogens causing enteric septicemia of catfish (ESC). We present here two distinctly novel bacteriophages vB_EiM_PVN06 and vB_EiA_PVN09 infecting Edwardsiella ictaluri E1, with their geographical origins from the Mekong Delta, Vietnam. Bacteriophage vB_EiM_PVN06 native to a mud sample reveals complete differences of biological properties with the phage vB_EiA_PVN09 originated from a viscus of a healthy catfish (Pangasianodon hypophthalmus) cultured in the same area. Morphological analyses combined with genomic data indicate that phage vB_EiM_PVN06 is classified to Myoviridae family and shares high similarity with E. ictaluri phage PEi21 genome, while vB_EiA_PVN09 is a member of Teseptimavirus genus, Autographiviridae family, and mostly closes to phage vB_EcoP_IME390. The vB_EiA_PVN09 is a T7-like bacteriophage, which has been firstly found infecting to E. ictaluri, and host range analysis also evidences for the cross-infection of this phage to Escherichia coli K12 and Escherichia coli DH5α. Together, our research highlights the diversity of bacteriophages infecting the pathogen E. ictaluri and suggests further explorations of lytic phages in environmental niches, to be exploited in feasible strategies of phage therapy in ESC disease control.

Adherent-Invasive and Non-Invasive Escherichia coli Isolates Differ in Their Effects on Caenorhabditis elegans’ Lifespan

The adherent-invasive Escherichia coli (AIEC) pathotype has been implicated in the pathogenesis of inflammatory bowel diseases in general and in Crohn’s disease (CD) in particular. AIEC strains are primarily characterized by their ability to adhere to and invade intestinal epithelial cells. However, the genetic and phenotypic features of AIEC isolates vary greatly as a function of the strain’s clonality, host factors, and the gut microenvironment. It is thus essential to identify the determinants of AIEC pathogenicity and understand their role in intestinal epithelial barrier dysfunction and inflammation. We reasoned that soil nematode Caenorhabditis elegans (a simple but powerful model of host-bacterium interactions) could be used to study the virulence of AIEC vs. non- AIEC E. coli strains. Indeed, we found that the colonization of C. elegans (strain N2) by E. coli impacted survival in a strain-specific manner. Moreover, the AIEC strains’ ability to invade cells in vitro was linked to the median lifespan in C. elegans (strain PX627). However, neither the E. coli intrinsic invasiveness (i.e., the fact for an individual strain to be characterized as invasive or not) nor AIEC’s virulence levels (i.e., the intensity of invasion, established in % from the infectious inoculum) in intestinal epithelial cells was correlated with C. elegans’ lifespan in the killing assay. Nevertheless, AIEC longevity of C. elegans might be a relevant model for screening anti-adhesion drugs and anti-invasive probiotics.

Micellar Antibiotics of Bacillus

Members of the Bacillus genus, particularly the “Bacillus subtilis group”, are known to produce amphipathic lipopeptides with biosurfactant activity. This includes the surfactins, fengycins and iturins that have been associated with antibacterial, antifungal, and anti-viral properties. We have screened a large collection of Bacillus, isolated from human, animal, estuarine water and soil samples and found that the most potent lipopeptide producers are members of the species Bacillus velezensis. B. velezensis lipopeptides exhibited anti-bacterial activity which was localised on the surface of both vegetative cells and spores. Interestingly, lipopeptide micelles (6–10 nm diameter) were detectable in strains exhibiting the highest levels of activity. Micelles were stable (heat and gastric stable) and shown to entrap other antimicrobials produced by the host bacterium (exampled here was the dipeptide antibiotic chlorotetaine). Commercially acquired lipopeptides did not exhibit similar levels of inhibitory activity and we suspect that micelle formation may relate to the particular isomeric forms produced by individual bacteria. Using naturally produced micelle formulations we demonstrated that they could entrap antimicrobial compounds (e.g., clindamycin, vancomycin and resveratrol). Micellar incorporation of antibiotics increased activity. Bacillus is a prolific producer of antimicrobials, and this phenomenon could be exploited naturally to augment antimicrobial activity. From an applied perspective, the ability to readily produce Bacillus micelles and formulate with drugs enables a possible strategy for enhanced drug delivery.

PixR, a novel activator of conjugative transfer of IncX4 resistance plasmids, mitigates the fitness cost of mcr-1 carriage in Escherichia coli

The emergence of the plasmid-borne colistin resistance gene mcr-1 threats public health. IncX4-type plasmids are one of the most epidemiologically successful vehicles for spreading mcr-1 worldwide. Since MCR-1 is known for imposing a fitness cost to its host bacterium, the successful spread of mcr-1-bearing plasmids might be linked to high conjugation frequency, which would enhance the maintenance of the plasmid in the host without antibiotic selection. However, the mechanism of IncX4 plasmids conjugation remains unclear. In this study, we used high-density transposon mutagenesis to identify factors required for IncX4 plasmid transfer and 18 genes were identified, including five with annotations unrelated to conjugation. The Cappable-seq and RNA-seq analysis confirmed that a novel transcriptional regulator gene, pixR, directly regulates the transfer of IncX4 plasmids by binding the promoter of 13 essential transfer genes to increase their transcription. Plasmid invasion and co-culture competition assays revealed that pixR is essential for the spread and persistence of mcr-1>-bearing IncX4 plasmids in bacterial populations, and effective conjugation is crucial for alleviating the fitness cost exerted by mcr-1 carriage. The existence of the IncX4-specific pixR gene increases plasmid transmissibility while promoting the invasion and persistence of mcr-1-bearing plasmids in bacterial populations, which helps explain their global prevalence.

Molecular Basis of Lysis–Lysogeny Decisions in Gram-Positive Phages

Temperate bacteriophages (phages) are viruses of bacteria. Upon infection of a susceptible host, a temperate phage can establish either a lytic cycle that kills the host or a lysogenic cycle as a stable prophage. The life cycle pursued by an infecting temperate phage can have a significant impact not only on the individual host bacterium at the cellular level but also on bacterial communities and evolution in the ecosystem. Thus, understanding the decision processes of temperate phages is crucial. This review delves into the molecular mechanisms behind lysis–lysogeny decision-making in Gram-positive phages. We discuss a variety of molecular mechanisms and the genetic organization of these well-understood systems. By elucidating the strategies used by phages to make lysis–lysogeny decisions, we can improve our understanding of phage–host interactions, which is crucial for a variety of studies including bacterial evolution, community and ecosystem diversification, and phage therapeutics. Expected final online publication date for the Annual Review of Microbiology, Volume 75 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.